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Ormsbee Golden BD, Gonzalez DV, Yochum GS, Coulter DW, Rizzino A. SOX2 represses c-MYC transcription by altering the co-activator landscape of the c-MYC super-enhancer and promoter regions. J Biol Chem 2024; 300:107642. [PMID: 39122009 PMCID: PMC11408076 DOI: 10.1016/j.jbc.2024.107642] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Revised: 07/05/2024] [Accepted: 07/22/2024] [Indexed: 08/12/2024] Open
Abstract
Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers.
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Affiliation(s)
- Briana D Ormsbee Golden
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Daisy V Gonzalez
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Gregory S Yochum
- Department of Surgery & Biochemistry & Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
| | - Donald W Coulter
- Hematology and Oncology Division, Department of Pediatrics, Nebraska Medical Center, Omaha, Nebraska, USA; Child Health Research Institute, University of Nebraska Medical Center, Omaha, Nebraska, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA
| | - Angie Rizzino
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska, USA; Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA.
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2
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Nikolouzakis TK, Chrysos E, Docea AO, Fragkiadaki P, Souglakos J, Tsiaoussis J, Tsatsakis A. Current and Future Trends of Colorectal Cancer Treatment: Exploring Advances in Immunotherapy. Cancers (Basel) 2024; 16:1995. [PMID: 38893120 PMCID: PMC11171065 DOI: 10.3390/cancers16111995] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2024] [Revised: 05/15/2024] [Accepted: 05/20/2024] [Indexed: 06/21/2024] Open
Abstract
Cancer of the colon and rectum (CRC) has been identified among the three most prevalent types of cancer and cancer-related deaths for both sexes. Even though significant progress in surgical and chemotherapeutic techniques has markedly improved disease-free and overall survival rates in contrast to those three decades ago, recent years have seen a stagnation in these improvements. This underscores the need for new therapies aiming to augment patient outcomes. A number of emerging strategies, such as immune checkpoint inhibitors (ICIs) and adoptive cell therapy (ACT), have exhibited promising outcomes not only in preclinical but also in clinical settings. Additionally, a thorough appreciation of the underlying biology has expanded the scope of research into potential therapeutic interventions. For instance, the pivotal role of altered telomere length in early CRC carcinogenesis, leading to chromosomal instability and telomere dysfunction, presents a promising avenue for future treatments. Thus, this review explores the advancements in CRC immunotherapy and telomere-targeted therapies, examining potential synergies and how these novel treatment modalities intersect to potentially enhance each other's efficacy, paving the way for promising future therapeutic advancements.
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Affiliation(s)
| | - Emmanuel Chrysos
- Department of General Surgery, University General Hospital of Heraklion, 71110 Heraklion, Greece; (T.K.N.); (E.C.)
| | - Anca Oana Docea
- Department of Toxicology, University of Medicine and Pharmacy of Craiova, 200349 Craiova, Romania
| | - Persefoni Fragkiadaki
- Department of Forensic Sciences and Toxicology, Faculty of Medicine, University of Crete, 71003 Heraklion, Greece; (P.F.); (A.T.)
| | - John Souglakos
- Laboratory of Translational Oncology, Medical School, University of Crete, 70013 Heraklion, Greece;
| | - John Tsiaoussis
- Department of Anatomy, Medical School, University of Crete, 70013 Heraklion, Greece;
| | - Aristidis Tsatsakis
- Department of Forensic Sciences and Toxicology, Faculty of Medicine, University of Crete, 71003 Heraklion, Greece; (P.F.); (A.T.)
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Bakiri L, Hasenfuss SC, Guío-Carrión A, Thomsen MK, Hasselblatt P, Wagner EF. Liver cancer development driven by the AP-1/c-Jun~Fra-2 dimer through c-Myc. Proc Natl Acad Sci U S A 2024; 121:e2404188121. [PMID: 38657045 PMCID: PMC11067056 DOI: 10.1073/pnas.2404188121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2024] [Accepted: 03/26/2024] [Indexed: 04/26/2024] Open
Abstract
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.
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Affiliation(s)
- Latifa Bakiri
- Laboratory Genes and Disease, Department of Laboratory Medicine, Medical University of Vienna, 1090, Vienna, Austria
- Genes, Development and Disease Group, National Cancer Research Centre, 28029, Madrid, Spain
| | - Sebastian C. Hasenfuss
- Genes, Development and Disease Group, National Cancer Research Centre, 28029, Madrid, Spain
| | - Ana Guío-Carrión
- Genes, Development and Disease Group, National Cancer Research Centre, 28029, Madrid, Spain
| | - Martin K. Thomsen
- Department of Biomedicine, University of Aarhus, 8000, Aarhus, Denmark
| | - Peter Hasselblatt
- Department of Medicine II, University Hospital and Faculty of Medicine, 79106, Freiburg, Germany
| | - Erwin F. Wagner
- Laboratory Genes and Disease, Department of Laboratory Medicine, Medical University of Vienna, 1090, Vienna, Austria
- Laboratory Genes and Disease, Department of Dermatology, Medical University of Vienna, 1090, Vienna, Austria
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4
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Yan L, Wu M, Wang T, Yuan H, Zhang X, Zhang H, Li T, Pandey V, Han X, Lobie PE, Zhu T. Breast Cancer Stem Cells Secrete MIF to Mediate Tumor Metabolic Reprogramming That Drives Immune Evasion. Cancer Res 2024; 84:1270-1285. [PMID: 38335272 DOI: 10.1158/0008-5472.can-23-2390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 11/29/2023] [Accepted: 02/06/2024] [Indexed: 02/12/2024]
Abstract
Reprogramming of energy metabolism exerts pivotal functions in cancer progression and immune surveillance. Identification of the mechanisms mediating metabolic changes in cancer may lead to improved strategies to suppress tumor growth and stimulate antitumor immunity. Here, it was observed that the secretomes of hypoxic breast cancer cells and breast cancer stem cells (BCSC) induced reprogramming of metabolic pathways, particularly glycolysis, in normoxic breast cancer cells. Screening of the BCSC secretome identified MIF as a pivotal factor potentiating glycolysis. Mechanistically, MIF increased c-MYC-mediated transcriptional upregulation of the glycolytic enzyme aldolase C by activating WNT/β-catenin signaling. Targeting MIF attenuated glycolysis and impaired xenograft growth and metastasis. MIF depletion in breast cancer cells also augmented intratumoral cytolytic CD8+ T cells and proinflammatory macrophages while decreasing regulatory T cells and tumor-associated neutrophils in the tumor microenvironment. Consequently, targeting MIF improved the therapeutic efficacy of immune checkpoint blockade in triple-negative breast cancer. Collectively, this study proposes MIF as an attractive therapeutic target to circumvent metabolic reprogramming and immunosuppression in breast cancer. SIGNIFICANCE MIF secreted by breast cancer stem cells induces metabolic reprogramming in bulk tumor cells and engenders an immunosuppressive microenvironment, identifying MIF targeting as a strategy to improve immunotherapy efficacy in breast cancer.
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Affiliation(s)
- Linlin Yan
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
| | - Mingming Wu
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
| | - Tianyu Wang
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
| | - Hui Yuan
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
| | - Xiao Zhang
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
| | - Huafeng Zhang
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
| | - Tao Li
- Department of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui, China
| | - Vijay Pandey
- Tsinghua-Berkeley Shenzhen Institute and Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Shenzhen, China
| | - Xinghua Han
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
| | - Peter E Lobie
- Tsinghua-Berkeley Shenzhen Institute and Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Shenzhen, China
- Shenzhen Bay Laboratory, Shenzhen, China
| | - Tao Zhu
- Division of Life Sciences and Medicine, Department of Oncology, The First Affiliated Hospital of USTC, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, University of Science and Technology of China, Hefei, Anhui, China
- Division of Life Sciences and Medicine, The CAS Key Laboratory of Innate Immunity and Chronic Disease, University of Science and Technology of China, Hefei, Anhui, China
- Shenzhen Bay Laboratory, Shenzhen, China
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Sanceau J, Poupel L, Joubel C, Lagoutte I, Caruso S, Pinto S, Desbois-Mouthon C, Godard C, Hamimi A, Montmory E, Dulary C, Chantalat S, Roehrig A, Muret K, Saint-Pierre B, Deleuze JF, Mouillet-Richard S, Forné T, Grosset CF, Zucman-Rossi J, Colnot S, Gougelet A. DLK1/DIO3 locus upregulation by a β-catenin-dependent enhancer drives cell proliferation and liver tumorigenesis. Mol Ther 2024; 32:1125-1143. [PMID: 38311851 PMCID: PMC11163201 DOI: 10.1016/j.ymthe.2024.01.036] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Revised: 01/05/2024] [Accepted: 01/31/2024] [Indexed: 02/06/2024] Open
Abstract
The CTNNB1 gene, encoding β-catenin, is frequently mutated in hepatocellular carcinoma (HCC, ∼30%) and in hepatoblastoma (HB, >80%), in which DLK1/DIO3 locus induction is correlated with CTNNB1 mutations. Here, we aim to decipher how sustained β-catenin activation regulates DLK1/DIO3 locus expression and the role this locus plays in HB and HCC development in mouse models deleted for Apc (ApcΔhep) or Ctnnb1-exon 3 (β-cateninΔExon3) and in human CTNNB1-mutated hepatic cancer cells. We identified an enhancer site bound by TCF-4/β-catenin complexes in an open conformation upon sustained β-catenin activation (DLK1-Wnt responsive element [WRE]) and increasing DLK1/DIO3 locus transcription in β-catenin-mutated human HB and mouse models. DLK1-WRE editing by CRISPR-Cas9 approach impaired DLK1/DIO3 locus expression and slowed tumor growth in subcutaneous CTNNB1-mutated tumor cell grafts, ApcΔhep HB and β-cateninΔExon3 HCC. Tumor growth inhibition resulted either from increased FADD expression and subsequent caspase-3 cleavage in the first case or from decreased expression of cell cycle actors regulated by FoxM1 in the others. Therefore, the DLK1/DIO3 locus is an essential determinant of FoxM1-dependent cell proliferation during β-catenin-driven liver tumorigenesis. Targeting the DLK1-WRE enhancer to silence the DLK1/DIO3 locus might thus represent an interesting therapeutic strategy to restrict tumor growth in primary liver cancers with CTNNB1 mutations.
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Affiliation(s)
- Julie Sanceau
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Lucie Poupel
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France; Inovarion, F-75005 Paris, France
| | - Camille Joubel
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Isabelle Lagoutte
- University Paris Cité, Institut Cochin, INSERM, CNRS, F-75014 Paris, France
| | - Stefano Caruso
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Sandra Pinto
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France
| | - Christèle Desbois-Mouthon
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Cécile Godard
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Akila Hamimi
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Enzo Montmory
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Cécile Dulary
- Centre National de Génotypage, Institut de Génomique, CEA, F-91057 Evry, France
| | - Sophie Chantalat
- Centre National de Génotypage, Institut de Génomique, CEA, F-91057 Evry, France
| | - Amélie Roehrig
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Kevin Muret
- Centre National de Génotypage, Institut de Génomique, CEA, F-91057 Evry, France
| | | | | | - Sophie Mouillet-Richard
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Thierry Forné
- IGMM, University Montpellier, CNRS, F-34293 Montpellier, France
| | - Christophe F Grosset
- University Bordeaux, INSERM, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancer, BMGIC, U1035, MIRCADE team, F-33076 Bordeaux, France; University Bordeaux, INSERM, Bordeaux Institute in Oncology, BRIC, U1312, MIRCADE team, F-33076 Bordeaux, France
| | - Jessica Zucman-Rossi
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Sabine Colnot
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France
| | - Angélique Gougelet
- Centre de Recherche des Cordeliers, Sorbonne Université, Inserm, Université Paris Cité, F-75006 Paris, France; Team « Oncogenic functions of beta-catenin signaling in the liver », Équipe labellisée par la Ligue Nationale contre le Cancer, F-75013 Paris, France; APHP, Institut du Cancer Paris CARPEM, F-75015 Paris, France.
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Voutsadakis IA. Targeting super-enhancer activity for colorectal cancer therapy. Am J Transl Res 2024; 16:700-719. [PMID: 38586095 PMCID: PMC10994804 DOI: 10.62347/qkhb5897] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Accepted: 02/28/2024] [Indexed: 04/09/2024]
Abstract
In addition to genetic variants and copy number alterations, epigenetic deregulation of oncogenes and tumor suppressors is a major contributor in cancer development and propagation. Regulatory elements for gene transcription regulation can be found in promoters which are located in the vicinity of transcription start sites but also at a distance, in enhancer sites, brought to interact with proximal sites when occupied by enhancer protein complexes. These sites provide most of the specific regulatory sequences recognized by transcription factors. A sub-set of enhancers characterized by a longer structure and stronger activity, called super-enhancers, are critical for the expression of specific genes, usually associated with individual cell type identity and function. Super-enhancers show deregulation in cancer, which may have profound repercussions for cancer cell survival and response to therapy. Dysfunction of super-enhancers may result from multiple mechanisms that include changes in their sequence, alterations in the topological neighborhoods where they belong, and alterations in the proteins that mediate their function, such as transcription factors and epigenetic modifiers. These can become potential targets for therapeutic interventions. Genes that are targets of super-enhancers are cell and cancer type specific and could also be of interest for therapeutic targeting. In colorectal cancer, a super-enhancer regulated and over-expressed oncogene is MYC, under the influence of the WNT/β-catenin pathway. Identification and targeting of additional oncogenes regulated by super-enhancers in colorectal cancer may pave the way for combination therapies targeting the super-enhancer machinery and signal transduction pathways that regulate the specific transcription factors operative on them.
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Affiliation(s)
- Ioannis A Voutsadakis
- Algoma District Cancer Program, Sault Area HospitalSault Ste. Marie, ON, Canada
- Division of Clinical Sciences, Section of Internal Medicine, Northern Ontario School of MedicineSudbury, ON, Canada
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Wang X, Tian Z, He L, Meng H, Zhu J, Li Y, Wang J, Hua X, Huang H, Huang C. DNMT3a-mediated upregulation of the stress inducible protein sestrin-2 contributes to malignant transformation of human bronchial epithelial cells following nickel exposure. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2024; 271:115954. [PMID: 38232523 DOI: 10.1016/j.ecoenv.2024.115954] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Revised: 12/07/2023] [Accepted: 01/06/2024] [Indexed: 01/19/2024]
Abstract
BACKGROUND Nickel is a confirmed human lung carcinogen. Nonetheless, the molecular mechanisms driving its carcinogenic impact on lung tissue remain poorly defined. In this study, we assessed SESN2 expression and the signaling pathways responsible for cellular transformation in human bronchial epithelial cells (HBECs) as a result of nickel exposure. METHODS We employed the Western blotting to determine the induction of SESN2 by nickel. To clarify the signaling pathways leading to cellular transformation following nickel exposure, we applied techniques such as gene knockdown, methylation-specific PCR, and chromatin immunoprecipitation. RESULT Exposure to nickel results in the upregulation of SESN2 and the initiation of autophagy in human bronchial epithelial cells (HBECs). This leads to degradation of HUR protein and consequently downregulation of USP28 mRNA, PP2AC protein, β-catenin protein, and diminished VHL transcription, culminating in the accumulation of hypoxia-inducible factor-1α (HIF-1α) and the malignant transformation of these cells. Mechanistic studies revealed that the increased expression of SESN2 is attributed to the demethylation of the SESN2 promoter induced by nickel, a process facilitated by decreased DNA methyl-transferase 3 A (DNMT3a) expression, while The downregulation of VHL transcription is linked to the suppression of the PP2A-C/GSK3β/β-Catenin/C-Myc pathway. Additionally, we discovered that SESN2-mediated autophagy triggers the degradation of HUR protein, which subsequently reduces the stability of USP28 mRNA and inhibits the PP2A-C/GSK3β/β-Catenin pathway and c-Myc transcription in HBECs post nickel exposure. CONCLUSION Our results reveal that nickel exposure leads to the downregulation of DNMT3a, resulting in the hypomethylation of the SESN2 promoter and its protein induction. This triggers autophagy-dependent suppression of the HUR/USP28/PP2A/β-Catenin/c-Myc pathway, subsequently leading to reduced VHL transcription, accumulation of HIF-1α protein, and the malignant transformation of human bronchial epithelial cells (HBECs). Our research offers novel insights into the molecular mechanisms that underlie the lung carcinogenic effects of nickel exposure. Specifically, nickel induces aberrant DNA methylation in the SESN2 promoter region through the decrease of DNMT3a levels, which ultimately leads to HIF-1α protein accumulation and the malignant transformation of HBECs. Specifically, nickel initiates DNA-methylation of the SESN2 promoter region by decreasing DNMT3a, ultimately resulting in HIF-1α protein accumulation and malignant transformation of HBECs. This study highlights DNMT3a as a potential prognostic biomarker or therapeutic target to improve clinical outcomes in lung cancer patients.
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Affiliation(s)
- Xinxing Wang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China; Tianjin Institute of Environmental and Operational Medicine, Tianjin 300050, China
| | - Zhongxian Tian
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Lijiong He
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Hao Meng
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Junlan Zhu
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Yang Li
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Jingjing Wang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Xiaohui Hua
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Haishan Huang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China
| | - Chuanshu Huang
- Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory, Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China.
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8
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King CM, Marx OM, Ding W, Koltun WA, Yochum GS. TCF7L1 Regulates LGR5 Expression in Colorectal Cancer Cells. Genes (Basel) 2023; 14:481. [PMID: 36833408 PMCID: PMC9956233 DOI: 10.3390/genes14020481] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2022] [Revised: 02/06/2023] [Accepted: 02/11/2023] [Indexed: 02/16/2023] Open
Abstract
Mutations in components of the Wnt/β-catenin signaling pathway drive colorectal cancer (CRC), in part, by deregulating expression of genes controlled by the T-cell factor (TCF) family of transcription factors. TCFs contain a conserved DNA binding domain that mediates association with TCF binding elements (TBEs) within Wnt-responsive DNA elements (WREs). Intestinal stem cell marker, leucine-rich-repeat containing G-protein-coupled receptor 5 (LGR5), is a Wnt target gene that has been implicated in CRC stem cell plasticity. However, the WREs at the LGR5 gene locus and how TCF factors directly regulate LGR5 gene expression in CRC have not been fully defined. Here, we report that TCF family member, TCF7L1, plays a significant role in regulating LGR5 expression in CRC cells. We demonstrate that TCF7L1 binds to a novel promoter-proximal WRE through association with a consensus TBE at the LGR5 locus to repress LGR5 expression. Using CRISPR activation and interference (CRISPRa/i) technologies to direct epigenetic modulation, we demonstrate that this WRE is a critical regulator of LGR5 expression and spheroid formation capacity of CRC cells. Furthermore, we found that restoring LGR5 expression rescues the TCF7L1-mediated reduction in spheroid formation efficiency. These results demonstrate a role for TCF7L1 in repressing LGR5 gene expression to govern the spheroid formation potential of CRC cells.
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Affiliation(s)
- Carli M. King
- Department of Biochemistry & Molecular Biology, College of Medicine, The Pennsylvania State University, Hershey, PA 17036, USA
- Department of Surgery, Division of Colon & Rectal Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17036, USA
| | - Olivia M. Marx
- Department of Biochemistry & Molecular Biology, College of Medicine, The Pennsylvania State University, Hershey, PA 17036, USA
- Department of Surgery, Division of Colon & Rectal Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17036, USA
| | - Wei Ding
- Department of Surgery, Division of Colon & Rectal Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17036, USA
| | - Walter A. Koltun
- Department of Surgery, Division of Colon & Rectal Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17036, USA
| | - Gregory S. Yochum
- Department of Biochemistry & Molecular Biology, College of Medicine, The Pennsylvania State University, Hershey, PA 17036, USA
- Department of Surgery, Division of Colon & Rectal Surgery, Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17036, USA
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9
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Ramakrishnan AB, Burby PE, Adiga K, Cadigan KM. SOX9 and TCF transcription factors associate to mediate Wnt/β-catenin target gene activation in colorectal cancer. J Biol Chem 2022; 299:102735. [PMID: 36423688 PMCID: PMC9771724 DOI: 10.1016/j.jbc.2022.102735] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Revised: 11/02/2022] [Accepted: 11/05/2022] [Indexed: 11/23/2022] Open
Abstract
Activation of the Wnt/β-catenin pathway regulates gene expression by promoting the formation of a β-catenin-T-cell factor (TCF) complex on target enhancers. In addition to TCFs, other transcription factors interact with the Wnt/β-catenin pathway at different levels to produce tissue-specific patterns of Wnt target gene expression. The transcription factor SOX9 potently represses many Wnt target genes by downregulating β-catenin protein levels. Here, we find using colony formation and cell growth assays that SOX9 surprisingly promotes the proliferation of Wnt-driven colorectal cancer (CRC) cells. In contrast to how it indirectly represses Wnt targets, SOX9 directly co-occupies and activates multiple Wnt-responsive enhancers in CRC cells. Our examination of the binding site grammar of these enhancers shows the presence of TCF and SOX9 binding sites that are necessary for transcriptional activation. In addition, we identify a physical interaction between the DNA-binding domains of TCFs and SOX9 and show that TCF-SOX9 interactions are important for target gene regulation and CRC cell growth. Our work demonstrates a highly context-dependent effect of SOX9 on Wnt targets, with the presence or absence of SOX9-binding sites on Wnt-regulated enhancers determining whether they are directly activated or indirectly repressed by SOX9.
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10
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Chun SK, Fortin BM, Fellows RC, Habowski AN, Verlande A, Song WA, Mahieu AL, Lefebvre AEYT, Sterrenberg JN, Velez LM, Digman MA, Edwards RA, Pannunzio NR, Seldin MM, Waterman ML, Masri S. Disruption of the circadian clock drives Apc loss of heterozygosity to accelerate colorectal cancer. SCIENCE ADVANCES 2022; 8:eabo2389. [PMID: 35947664 PMCID: PMC9365282 DOI: 10.1126/sciadv.abo2389] [Citation(s) in RCA: 42] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/21/2022] [Accepted: 06/23/2022] [Indexed: 05/12/2023]
Abstract
An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate Apc-driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc, a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset cancer prevention.
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Affiliation(s)
- Sung Kook Chun
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Bridget M. Fortin
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Rachel C. Fellows
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Amber N. Habowski
- Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA
| | - Amandine Verlande
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Wei A. Song
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Alisa L. Mahieu
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | | | | | - Leandro M. Velez
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Michelle A. Digman
- Department of Biomedical Engineering, University of California, Irvine, Irvine, CA 92697, USA
| | - Robert A. Edwards
- Department of Pathology and Laboratory Medicine, University of California, Irvine, Irvine, CA 92697, USA
| | | | - Marcus M. Seldin
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
| | - Marian L. Waterman
- Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA
| | - Selma Masri
- Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, USA
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11
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Kang X, Chen L, Yang S, Gong Z, Hu H, Zhang X, Liang C, Xu Y. Zuogui Wan slowed senescence of bone marrow mesenchymal stem cells by suppressing Wnt/β-catenin signaling. JOURNAL OF ETHNOPHARMACOLOGY 2022; 294:115323. [PMID: 35483559 DOI: 10.1016/j.jep.2022.115323] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/18/2022] [Revised: 04/11/2022] [Accepted: 04/21/2022] [Indexed: 06/14/2023]
Abstract
ETHNOPHARMACOLOGICAL RELEVANCE In traditional Chinese medicine (TCM), Zuogui Wan (ZGW) is a classical prescription for senile disorders and delay aging. Modern studies show that ZGW promotes central nerve cell regeneration, prevents and cures osteoporosis, enhances the body's antioxidant capacity, regulates the body's immune function, and promotes mesenchymal stem cells (MSCs) proliferation. AIM OF THE STUDY It has been shown that MSCs aging is closely associated with organism's aging and age-related disorders. The study aimed to define the effects of ZGW on the aging bone marrow mesenchymal stem cells (BMSCs) and to identify the mechanisms of ZGW delaying BMSCs senescence. MATERIALS AND METHODS Network pharmacology analysis combined with GEO data mining, molecular docking and experimental validation were used to evaluate the mechanisms by which ZGW delays MSCs senescence (MSCS). LC-MS was used for quality control analysis of ZGW. RESULTS PPI network analysis revealed that EGF, TNF, JUN, MMPs, IL-6, MAPK8, and MYC are components of the core PPI network. GO and KEGG analyses revealed that oxidative stress, regulation of response to DNA damage stimuli, and Wnt signaling were significantly enriched. GEO database validation also indicated that Wnt signaling closely correlated with MSCs aging. Molecular docking analysis of the top-13 active components in the "ZGW-Targets-MSCS" network indicated that most components have strong affinity for key proteins in Wnt signaling, suggesting that modulation of Wnt signaling is an important mechanism of ZGW activity against MSCS. Further experimental validation found that ZGW indeed regulates Wnt signaling and suppresses the expression of age-related factors to enhance cell proliferation, ameliorate DNA damage, and reduce senescence-related secretory phenotype (SASP) secretion, thereby maintaining multidirectional differentiation of rat BMSCs. Similar results were obtained using the Wnt inhibitor, XAV-939. CONCLUSIONS Together, our data show that ZGW slows BMSCs aging by suppressing Wnt signaling.
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Affiliation(s)
- Xiangping Kang
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Long Chen
- Experiment Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Shuchen Yang
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Zhangbin Gong
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Haiyan Hu
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Xueli Zhang
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Chao Liang
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
| | - Yanwu Xu
- College of Basic Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, China.
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12
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Horie C, Zhu C, Yamaguchi K, Nakagawa S, Isobe Y, Takane K, Ikenoue T, Ohta Y, Tanaka Y, Aikou S, Tsurita G, Ahiko Y, Shida D, Furukawa Y. Motile sperm domain containing 1 is upregulated by the Wnt/β‑catenin signaling pathway in colorectal cancer. Oncol Lett 2022; 24:282. [DOI: 10.3892/ol.2022.13402] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 05/17/2022] [Indexed: 11/05/2022] Open
Affiliation(s)
- Chiaki Horie
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Chi Zhu
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Kiyoshi Yamaguchi
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Saya Nakagawa
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Yumiko Isobe
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Kiyoko Takane
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Tsuneo Ikenoue
- Division of Clinical Genome Research, Advanced Clinical Research Center, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Yasunori Ohta
- Department of Pathology, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Yukihisa Tanaka
- Department of Pathology, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Susumu Aikou
- Department of Surgery, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Giichiro Tsurita
- Department of Surgery, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Yuka Ahiko
- Department of Surgery, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Dai Shida
- Department of Surgery, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
| | - Yoichi Furukawa
- Department of Surgery, Research Hospital, Institute of Medical Science, The University of Tokyo, Tokyo 108‑8639, Japan
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13
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Yu W, Liu W, Feng Y, Zhu C. Knockdown of GSG2 Suppresses the Progression of Colorectal Cancer Cells. Genet Test Mol Biomarkers 2022; 26:26-36. [PMID: 35089075 DOI: 10.1089/gtmb.2020.0298] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Introduction: As a serine/threonine kinase, Haspin (GSG2) has been reportedly associated with the development of malignant tumors. However, few studies have reported the role of GSG2 in colorectal cancer (CRC). Materials and Methods: Based on data from the Oncomine databases, GSG2 was found to be highly expressed in CRC patients' tissues. Therefore, the expression of GSG2 in CRC cell lines was subsequently evaluated. GSG2 loss-of-function experiments were conducted by infection with a lentivirus expressing shRNAs against GSG2. Colony-formation and cell viabilities were assessed using clonogenic and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, respectively. Migration was assessed using wound-healing and transwell assays. A GSG2 inhibitor experiment was used to investigate the key role of GSG2 in CRC. Immunoprecipitation was used to investigate the interaction between GSG2 and p-H3. In addition, apoptosis was evaluated by quantifying caspase 3/7 activities, and western blot analyses were used to investigate the underlying mechanisms of GSG2 in CRC. Results: GSG2 was found to be highly expressed in CRC tissues and cells. Furthermore, GSG2 knock-down suppressed proliferation, colony formation and invasion, and induced apoptosis in CRC cells. Mechanistically, GSG2 was revealed to regulate Myc, NF-κB, Snail-1, and β-catenin signaling. Conclusion: Collectively, we demonstrate that GSG2 is a potential biomarker of CRC, and that GSG2 interference suppresses the progression of CRC and promotes apoptosis in vitro. These data suggest GSG2 as a putative oncogene, but will require additional in vivo studies to confirm.
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Affiliation(s)
- Wenyan Yu
- Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Wei Liu
- Department of Pathology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Yuxin Feng
- Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Chunrong Zhu
- Department of Oncology, The First Affiliated Hospital of Soochow University, Suzhou, China
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14
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Chen WS, Liang Y, Zong M, Liu JJ, Kaneko K, Hanley KL, Zhang K, Feng GS. Single-cell transcriptomics reveals opposing roles of Shp2 in Myc-driven liver tumor cells and microenvironment. Cell Rep 2021; 37:109974. [PMID: 34758313 DOI: 10.1016/j.celrep.2021.109974] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 07/16/2021] [Accepted: 10/19/2021] [Indexed: 12/18/2022] Open
Abstract
The mechanisms of Myc-driven liver tumorigenesis are inadequately understood. Herein we show that Myc-driven hepatocellular carcinoma (HCC) is dramatically aggravated in mice with hepatocyte-specific Ptpn11/Shp2 deletion. However, Myc-induced tumors develop selectively from the rare Shp2-positive hepatocytes in Shp2-deficent liver, and Myc-driven oncogenesis depends on an intact Ras-Erk signaling promoted by Shp2 to sustain Myc stability. Despite a stringent requirement of Shp2 cell autonomously, Shp2 deletion induces an immunosuppressive environment, resulting in defective clearance of tumor-initiating cells and aggressive tumor progression. The basal Wnt/β-catenin signaling is upregulated in Shp2-deficient liver, which is further augmented by Myc transfection. Ablating Ctnnb1 suppresses Myc-induced HCC in Shp2-deficient livers, revealing an essential role of β-catenin. Consistently, Myc overexpression and CTNNB1 mutations are frequently co-detected in HCC patients with poor prognosis. These data elucidate complex mechanisms of liver tumorigenesis driven by cell-intrinsic oncogenic signaling in cooperation with a tumor-promoting microenvironment generated by disrupting the specific oncogenic pathway.
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MESH Headings
- Animals
- Biomarkers, Tumor
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Gene Expression Regulation, Neoplastic
- Hepatocytes/metabolism
- Hepatocytes/pathology
- Liver Neoplasms/genetics
- Liver Neoplasms/metabolism
- Liver Neoplasms/pathology
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Mutation
- Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics
- Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism
- Protein Tyrosine Phosphatase, Non-Receptor Type 11/physiology
- Proto-Oncogene Proteins c-myc/genetics
- Proto-Oncogene Proteins c-myc/metabolism
- Single-Cell Analysis/methods
- Transcriptome
- Tumor Microenvironment
- Wnt Signaling Pathway
- beta Catenin/genetics
- beta Catenin/metabolism
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Affiliation(s)
- Wendy S Chen
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Yan Liang
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Min Zong
- Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Jacey J Liu
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Kota Kaneko
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Kaisa L Hanley
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA
| | - Kun Zhang
- Department of Bioengineering, University of California at San Diego, La Jolla, CA 92093, USA
| | - Gen-Sheng Feng
- Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093, USA; Department of Pathology, University of California at San Diego, La Jolla, CA 92093, USA.
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15
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Ramakrishnan AB, Chen L, Burby PE, Cadigan KM. Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif. Nucleic Acids Res 2021; 49:8625-8641. [PMID: 34358319 PMCID: PMC8421206 DOI: 10.1093/nar/gkab657] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Revised: 07/10/2021] [Accepted: 07/23/2021] [Indexed: 01/01/2023] Open
Abstract
Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of β-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal type homeobox (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.
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Affiliation(s)
| | - Lisheng Chen
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109 USA
| | - Peter E Burby
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109 USA
| | - Ken M Cadigan
- Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109 USA
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16
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Waters AM, Khatib TO, Papke B, Goodwin CM, Hobbs GA, Diehl JN, Yang R, Edwards AC, Walsh KH, Sulahian R, McFarland JM, Kapner KS, Gilbert TSK, Stalnecker CA, Javaid S, Barkovskaya A, Grover KR, Hibshman PS, Blake DR, Schaefer A, Nowak KM, Klomp JE, Hayes TK, Kassner M, Tang N, Tanaseichuk O, Chen K, Zhou Y, Kalkat M, Herring LE, Graves LM, Penn LZ, Yin HH, Aguirre AJ, Hahn WC, Cox AD, Der CJ. Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition. Cell Rep 2021; 35:109291. [PMID: 34192548 PMCID: PMC8340308 DOI: 10.1016/j.celrep.2021.109291] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2020] [Revised: 01/31/2021] [Accepted: 06/03/2021] [Indexed: 12/28/2022] Open
Abstract
To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-β-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the βIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.
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Affiliation(s)
- Andrew M Waters
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Tala O Khatib
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Bjoern Papke
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Craig M Goodwin
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - G Aaron Hobbs
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - J Nathaniel Diehl
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Runying Yang
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - A Cole Edwards
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | | | - Rita Sulahian
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | | | - Kevin S Kapner
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
| | - Thomas S K Gilbert
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; UNC Michael Hooker Proteomics Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Clint A Stalnecker
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Sehrish Javaid
- Oral and Craniofacial Biomedicine PhD Program, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Anna Barkovskaya
- Institute for Cancer Research, Oslo University Hospital, Oslo 0379, Norway
| | - Kajal R Grover
- Department of Nutrition, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Priya S Hibshman
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Devon R Blake
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Antje Schaefer
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Katherine M Nowak
- Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Jennifer E Klomp
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Tikvah K Hayes
- Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Michelle Kassner
- Cancer and Cell Biology Division, Translational Genomic Research Institute, Phoenix, AZ 85004, USA
| | - Nanyun Tang
- Cancer and Cell Biology Division, Translational Genomic Research Institute, Phoenix, AZ 85004, USA
| | - Olga Tanaseichuk
- Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA
| | - Kaisheng Chen
- Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA
| | - Yingyao Zhou
- Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA
| | - Manpreet Kalkat
- Department of Medical Biophysics, University of Toronto, Toronto, ON M5S, Canada
| | - Laura E Herring
- UNC Michael Hooker Proteomics Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Lee M Graves
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Linda Z Penn
- Department of Medical Biophysics, University of Toronto, Toronto, ON M5S, Canada
| | - Hongwei H Yin
- Cancer and Cell Biology Division, Translational Genomic Research Institute, Phoenix, AZ 85004, USA
| | - Andrew J Aguirre
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02215, USA; Brigham and Women's Hospital, Boston, MA 02215, USA
| | - William C Hahn
- Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, Boston, MA 02215, USA; Brigham and Women's Hospital, Boston, MA 02215, USA
| | - Adrienne D Cox
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Oral and Craniofacial Biomedicine PhD Program, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Channing J Der
- Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Oral and Craniofacial Biomedicine PhD Program, School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
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17
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Kashyap T, Nath N, Mishra P, Jha A, Nagini S, Mishra R. Pluripotency transcription factor Nanog and its association with overall oral squamous cell carcinoma progression, cisplatin-resistance, invasion and stemness acquisition. Head Neck 2020; 42:3282-3294. [PMID: 32710593 DOI: 10.1002/hed.26373] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Revised: 05/28/2020] [Accepted: 06/23/2020] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Cisplatin-resistant oral squamous cell carcinoma (OSCC) cells acquire stem-like characteristics and are difficult to treat. Nanog is a transcription factor and needed for maintenance of pluripotency, but its transcription-promoting role in OSCC progression and cisplatin resistance is poorly understood. METHODS Here, 110 fresh human tissue specimens of various stages, including invasive (N1-3 )/chemoradiation-resistant OSCC samples, cisplatin-resistant (CisR-SCC-4/-9) OSCC cells/parental cells, photochemical ECGC, and siRNA (Nanog) were used. RESULTS Nanog overexpression was associated with overall progression, chemoresistance, and invasion of OSCC. Nanog recruitment to c-Myc, Slug, E-cadherin, and Oct-4 gene promoter was observed. Positive correlation of Nanog protein expression with c-Myc, Slug, cyclin D1, MMP-2/-9, and Oct-4 and negative correlation with E-cadherin gene expression were found. Knockdown of Nanog and treatment of epicatechin-3-gallate reversed cisplatin resistance and diminished invasion/migration potential. CONCLUSION Nanog directly participated in the regulation of Slug, E-cadherin, Oct-4, and c-Myc genes, causing cisplatin resistance/recurrence of OSCC.
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Affiliation(s)
- Tanushree Kashyap
- Department of Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ranchi, Jharkhand, India
| | - Nidhi Nath
- Department of Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ranchi, Jharkhand, India
| | - Prajna Mishra
- Centre for Applied Chemistry, School of Natural Sciences, Central University of Jharkhand, Ranchi, Jharkhand, India
| | - Arpita Jha
- Department of Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ranchi, Jharkhand, India
| | - Siddavaram Nagini
- Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India
| | - Rajakishore Mishra
- Department of Life Sciences, School of Natural Sciences, Central University of Jharkhand, Ranchi, Jharkhand, India
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18
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Wenzel J, Rose K, Haghighi EB, Lamprecht C, Rauen G, Freihen V, Kesselring R, Boerries M, Hecht A. Loss of the nuclear Wnt pathway effector TCF7L2 promotes migration and invasion of human colorectal cancer cells. Oncogene 2020; 39:3893-3909. [PMID: 32203164 PMCID: PMC7203011 DOI: 10.1038/s41388-020-1259-7] [Citation(s) in RCA: 43] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2019] [Revised: 03/03/2020] [Accepted: 03/04/2020] [Indexed: 12/25/2022]
Abstract
The transcription factor TCF7L2 is indispensable for intestinal tissue homeostasis where it transmits mitogenic Wnt/β-Catenin signals in stem and progenitor cells, from which intestinal tumors arise. Yet, TCF7L2 belongs to the most frequently mutated genes in colorectal cancer (CRC), and tumor-suppressive functions of TCF7L2 were proposed. This apparent paradox warrants to clarify the role of TCF7L2 in colorectal carcinogenesis. Here, we investigated TCF7L2 dependence/independence of CRC cells and the cellular and molecular consequences of TCF7L2 loss-of-function. By genome editing we achieved complete TCF7L2 inactivation in several CRC cell lines without loss of viability, showing that CRC cells have widely lost the strict requirement for TCF7L2. TCF7L2 deficiency impaired G1/S progression, reminiscent of the physiological role of TCF7L2. In addition, TCF7L2-negative cells exhibited morphological changes, enhanced migration, invasion, and collagen adhesion, albeit the severity of the phenotypic alterations manifested in a cell-line-specific fashion. To provide a molecular framework for the observed cellular changes, we performed global transcriptome profiling and identified gene-regulatory networks in which TCF7L2 positively regulates the proto-oncogene MYC, while repressing the cell cycle inhibitors CDKN2C/CDKN2D. Consistent with its function in curbing cell motility and invasion, TCF7L2 directly suppresses the pro-metastatic transcription factor RUNX2 and impinges on the expression of cell adhesion molecules. Altogether, we conclude that the proliferation-stimulating activity of TCF7L2 persists in CRC cells. In addition, TCF7L2 acts as invasion suppressor. Despite its negative impact on cell cycle progression, TCF7L2 loss-of-function may thereby increase malignancy, which could explain why TCF7L2 is mutated in a sizeable fraction of colorectal tumors.
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Affiliation(s)
- Janna Wenzel
- Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Stefan-Meier-Str. 17, 79104, Freiburg, Germany
- Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104, Freiburg, Germany
| | - Katja Rose
- Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Stefan-Meier-Str. 17, 79104, Freiburg, Germany
| | - Elham Bavafaye Haghighi
- Institute of Medical Bioinformatics and Systems Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Breisacherstr. 153, 79104, Freiburg, Germany
| | - Constanze Lamprecht
- Institute of Physics, University of Freiburg, Hermann-Herder-Str. 3a, 79104, Freiburg, Germany
- BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestraße 18, 79104, Freiburg, Germany
- Freiburg Center for Interactive Materials and Bioinspired Technology (FIT), University of Freiburg, Georges-Köhler-Allee 105, 79110, Freiburg, Germany
| | - Gilles Rauen
- Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104, Freiburg, Germany
- BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestraße 18, 79104, Freiburg, Germany
| | - Vivien Freihen
- Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Stefan-Meier-Str. 17, 79104, Freiburg, Germany
| | - Rebecca Kesselring
- Department of General and Visceral Surgery, Center for Surgery, Medical Center, Faculty of Medicine, University of Freiburg, Hugstetter Straße 55, 79106, Freiburg, Germany
| | - Melanie Boerries
- Institute of Medical Bioinformatics and Systems Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Breisacherstr. 153, 79104, Freiburg, Germany
- German Cancer Consortium (DKTK), Hugstetter Straße 55, 79106, Freiburg, Germany
- German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120, Heidelberg, Germany
| | - Andreas Hecht
- Institute of Molecular Medicine and Cell Research, Faculty of Medicine, University of Freiburg, Stefan-Meier-Str. 17, 79104, Freiburg, Germany.
- Faculty of Biology, University of Freiburg, Schänzlestraße 1, 79104, Freiburg, Germany.
- BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestraße 18, 79104, Freiburg, Germany.
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19
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Li Y, Li X, Qu J, Luo D, Hu Z. Cas9 Mediated Correction of β-catenin Mutation and Restoring the Expression of Protein Phosphorylation in Colon Cancer HCT-116 Cells Decrease Cell Proliferation in vitro and Hamper Tumor Growth in Mice in vivo. Onco Targets Ther 2020; 13:17-29. [PMID: 32021251 PMCID: PMC6954092 DOI: 10.2147/ott.s225556] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 12/17/2019] [Indexed: 12/14/2022] Open
Abstract
Purpose Colorectal cancer (CRC) is one of the major contributors to cancer mortality and morbidity. Finding strategies to fight against CRC is urgently required. Mutations in driver genes of APC or β-catenin play an important role in the occurrence and progression of CRC. In the present study, we jointly apply CRISPR/Cas9-sgRNA system and Single-stranded oligodeoxynucleotide (ssODN) as templates to correct a heterozygous ΔTCT deletion mutation of β-catenin present in a colon cancer cell line HCT-116. This method provides a potential strategy in gene therapy for cancer. Methods A Cas9/β-catenin-sgRNA-eGFP co-expression vector was constructed and co-transfected with ssODN into HCT-116 cells. Mutation-corrected single-cell clones were sorted by FACS and judged by TA cloning and DNA sequencing. Effects of CRISPR/Cas9-mediated correction were tested by real-time quantitative PCR, Western blotting, CCK8, EDU dyeing and cell-plated clones. Moreover, the growth of cell clones derived tumors was analyzed at nude mice xenografts. Results CRISPR/Cas9-mediated β-catenin mutation correction resulted in the presence of TCT sequence and the re-expression of phosphorylation β-catenin at Ser45, which restored the normal function of phosphorylation β-catenin including reduction of the transportation of nuclear β-catenin and the expression of downstream c-myc, survivin. Significantly reduced cell growth was observed in β-catenin mutation-corrected cells. Mice xenografted with mutation-corrected HCT-116 cells showed significantly smaller tumor size than uncorrected xenografts. Conclusion The data of this study documented that correction of the driven mutation by the combination of CRISPR/Cas9 and ssODN could greatly remedy the biological behavior of the cancer cell line, suggesting a potential application of this strategy in gene therapy of cancer.
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Affiliation(s)
- Yanlan Li
- Translational Medicine Institute, the First People's Hospital of Chenzhou Affiliated to University of South China, Hunan 432000, People's Republic of China.,Hunan Province Key Laboratory of Tumor Cellular and Molecular Pathology, Cancer Research Institute, University of South China, Hunan 421001, People's Republic of China
| | - Xiangning Li
- Translational Medicine Institute, the First People's Hospital of Chenzhou Affiliated to University of South China, Hunan 432000, People's Republic of China.,National & Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology, The First People's Hospital of Chenzhou, Hunan 432000, People's Republic of China
| | - Jiayao Qu
- Translational Medicine Institute, the First People's Hospital of Chenzhou Affiliated to University of South China, Hunan 432000, People's Republic of China.,National & Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology, The First People's Hospital of Chenzhou, Hunan 432000, People's Republic of China
| | - Dixian Luo
- Translational Medicine Institute, the First People's Hospital of Chenzhou Affiliated to University of South China, Hunan 432000, People's Republic of China.,National & Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology, The First People's Hospital of Chenzhou, Hunan 432000, People's Republic of China
| | - Zheng Hu
- Translational Medicine Institute, the First People's Hospital of Chenzhou Affiliated to University of South China, Hunan 432000, People's Republic of China.,National & Local Joint Engineering Laboratory for High-Through Molecular Diagnosis Technology, The First People's Hospital of Chenzhou, Hunan 432000, People's Republic of China
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20
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Kobayashi A, Waku T. New addiction to the NRF2-related factor NRF3 in cancer cells: Ubiquitin-independent proteolysis through the 20S proteasome. Cancer Sci 2020; 111:6-14. [PMID: 31742837 PMCID: PMC6942428 DOI: 10.1111/cas.14244] [Citation(s) in RCA: 28] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2019] [Revised: 11/04/2019] [Accepted: 11/07/2019] [Indexed: 12/18/2022] Open
Abstract
Accumulating evidence has revealed that human cancers develop by sequentially mutating pivotal genes, including driver genes, and acquiring cancer hallmarks. For instance, cancer cells are addicted to the transcription factor NRF2 (NFE2L2), which is a driver gene that utilizes the cellular cytoprotection system against oxidative stress and metabolic pathway reprogramming for sustaining high growth. Our group has recently discovered a new addiction to the NRF2-related factor NRF3 (NFE2L3) in cancer. For many years, the physiological function of NRF3 remained obscure, in part because Nrf3-deficient mice do not show apparent abnormalities. Nevertheless, human cancer genome databases suggest critical roles of NRF3 in cancer because of high NRF3 mRNA induction in several cancer types, such as colorectal cancer and pancreatic adenocarcinoma, with a poor prognosis. We found that NRF3 promotes tumor growth and malignancy by activating ubiquitin-independent 20S proteasome assembly through inducing the expression of the proteasome maturation protein (POMP) chaperone and thereby degrading the tumor suppressors p53 and Rb. The NRF3-POMP-20S proteasome axis has an entirely different effect on cancer than NRF2. In this review, we describe recent research advances regarding the new cancer effector NRF3, including unclarified ubiquitin-independent proteolysis by the NRF3-POMP-20S proteasome axis. The expected development of cancer therapeutic interventions for this axis is also discussed.
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Affiliation(s)
- Akira Kobayashi
- Laboratory for Genetic CodeGraduate School of Life and Medical SciencesDoshisha UniversityKyotanabeJapan
- Department of Life and Medical SciencesDoshisha UniversityKyotanabeJapan
| | - Tsuyoshi Waku
- Department of Life and Medical SciencesDoshisha UniversityKyotanabeJapan
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21
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Prieto I, Alarcón CR, García-Gómez R, Berdún R, Urgel T, Portero M, Pamplona R, Martínez-Ruiz A, Ruiz-Sanz JI, Ruiz-Larrea MB, Jove M, Cerdán S, Monsalve M. Metabolic adaptations in spontaneously immortalized PGC-1α knock-out mouse embryonic fibroblasts increase their oncogenic potential. Redox Biol 2019; 29:101396. [PMID: 31926622 PMCID: PMC6921228 DOI: 10.1016/j.redox.2019.101396] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2019] [Revised: 11/20/2019] [Accepted: 11/26/2019] [Indexed: 12/14/2022] Open
Abstract
PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear β-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer.
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Affiliation(s)
- Ignacio Prieto
- Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain.
| | - Carmen Rubio Alarcón
- Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain.
| | - Raquel García-Gómez
- Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain.
| | - Rebeca Berdún
- Institut de Recerca Biomédica Lleida, Avda, Alcalde Rovira Roure 80, 25198, Lleida, Spain.
| | - Tamara Urgel
- Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain.
| | - Manuel Portero
- Institut de Recerca Biomédica Lleida, Avda, Alcalde Rovira Roure 80, 25198, Lleida, Spain.
| | - Reinald Pamplona
- Institut de Recerca Biomédica Lleida, Avda, Alcalde Rovira Roure 80, 25198, Lleida, Spain.
| | - Antonio Martínez-Ruiz
- Unidad de Ivestigación, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa (IIS-IP). Maestro Vives 3, 28009, Madrid, Spain; Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares (CIBERCV), Spain.
| | - José Ignacio Ruiz-Sanz
- Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco, Euskal Herriko Unibertsitea, Barrio Sarriena s/n, 48940, Leioa, Spain.
| | - M Begoña Ruiz-Larrea
- Departamento de Fisiología, Facultad de Medicina y Enfermería, Universidad del País Vasco, Euskal Herriko Unibertsitea, Barrio Sarriena s/n, 48940, Leioa, Spain.
| | - Mariona Jove
- Institut de Recerca Biomédica Lleida, Avda, Alcalde Rovira Roure 80, 25198, Lleida, Spain.
| | - Sebastián Cerdán
- Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain.
| | - María Monsalve
- Instituto de Investigaciones Biomédicas "Alberto Sols" (CSIC-UAM), Arturo Duperier 4, 28029, Madrid, Spain.
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22
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Ahn K, Ji H, Kim HE, Cho H, Sun Q, Shi S, He Y, Kim BG, Kim O. Raphanus sativus L. seed extracts induce apoptosis and reduce migration of oral squamous cell carcinoma KB and KBCD133+cells by downregulation of β-catenin. Nutr Cancer 2019; 72:1378-1389. [DOI: 10.1080/01635581.2019.1684527] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Affiliation(s)
- Kyuhyeon Ahn
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Hyeongjoon Ji
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Hye-Eun Kim
- Department of Orthodontics and Divisions of Pediatric Dentistry and Community of Oral Health, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Hyejoung Cho
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Qiaochu Sun
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Shuhan Shi
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Yuzhu He
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
| | - Byung-Gook Kim
- Department of Oral Medicine, School of Dentistry, Chonnam National University, Gwangju, Korea
| | - Okjoon Kim
- Department of Oral Pathology, School of Dentistry, Chonnam National University, Gwangju, Republic of Korea
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23
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β-Catenin/TCF4 Complex-Mediated Induction of the NRF3 ( NFE2L3) Gene in Cancer Cells. Int J Mol Sci 2019; 20:ijms20133344. [PMID: 31288376 PMCID: PMC6651286 DOI: 10.3390/ijms20133344] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2019] [Revised: 07/02/2019] [Accepted: 07/03/2019] [Indexed: 01/13/2023] Open
Abstract
Remarkable upregulation of the NRF2 (NFE2L2)-related transcription factor NRF3 (NFE2L3) in several cancer tissues and its correlation with poor prognosis strongly suggest the physiological function of NRF3 in tumors. Indeed, we had recently uncovered the function of NRF3, which promotes cancer cell proliferation by p53 degradation via the 20S proteasome. Nevertheless, the molecular mechanism underlying the induction of NRF3 gene expression in cancer cells is highly elusive. We herein describe that NRF3 upregulation is induced by the β-catenin/TCF4 complex in colon cancer cells. We first confirmed high NRF3 mRNA expression in human colon cancer specimens. The genome database indicated that the human NRF3 gene possesses a species-conserved WRE sequence (TCF/LEF consensus element), implying that the β-catenin/TCF complex activates NRF3 expression in colon cancer. Consistently, we observed that the β-catenin/TCF4 complex mediates NRF3 expression by binding directly to the WRE site. Furthermore, inducing NRF3 activates cell proliferation and the expression of the glucose transporter GLUT1. The existence of the β-catenin/TCF4-NRF3 axis was also validated in the intestine and organoids of Apc-deficient mice. Finally, the positive correlation between NRF3 and β-catenin target gene expression strongly supports our conclusion. Our findings clearly demonstrate that NRF3 induction in cancer cells is controlled by the Wnt/β-catenin pathway.
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24
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Luong-Gardiol N, Siddiqui I, Pizzitola I, Jeevan-Raj B, Charmoy M, Huang Y, Irmisch A, Curtet S, Angelov GS, Danilo M, Juilland M, Bornhauser B, Thome M, Hantschel O, Chalandon Y, Cazzaniga G, Bourquin JP, Huelsken J, Held W. γ-Catenin-Dependent Signals Maintain BCR-ABL1 + B Cell Acute Lymphoblastic Leukemia. Cancer Cell 2019; 35:649-663.e10. [PMID: 30991025 DOI: 10.1016/j.ccell.2019.03.005] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/30/2015] [Revised: 01/29/2019] [Accepted: 03/14/2019] [Indexed: 11/23/2022]
Abstract
The BCR-ABL1 fusion protein is the cause of chronic myeloid leukemia (CML) and of a significant fraction of adult-onset B cell acute lymphoblastic leukemia (B-ALL) cases. Using mouse models and patient-derived samples, we identified an essential role for γ-catenin in the initiation and maintenance of BCR-ABL1+ B-ALL but not CML. The selectivity was explained by a partial γ-catenin dependence of MYC expression together with the susceptibility of B-ALL, but not CML, to reduced MYC levels. MYC and γ-catenin enabled B-ALL maintenance by augmenting BIRC5 and enforced BIRC5 expression overcame γ-catenin loss. Since γ-catenin was dispensable for normal hematopoiesis, these lineage- and disease-specific features of canonical Wnt signaling identified a potential therapeutic target for the treatment of BCR-ABL1+ B-ALL.
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Affiliation(s)
- Noemie Luong-Gardiol
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Imran Siddiqui
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Irene Pizzitola
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Beena Jeevan-Raj
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Mélanie Charmoy
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Yun Huang
- Department of Pediatric Oncology and Children's Research Centre, University Children's Hospital Zürich, Zürich, Switzerland
| | - Anja Irmisch
- Swiss Institute for Experimental Cancer Research (ISREC), Federal University of Technology Lausanne (EPFL), Lausanne, Switzerland
| | - Sara Curtet
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Georgi S Angelov
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Maxime Danilo
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland
| | - Mélanie Juilland
- Department of Biochemistry, University of Lausanne, Epalinges, Switzerland
| | - Beat Bornhauser
- Department of Pediatric Oncology and Children's Research Centre, University Children's Hospital Zürich, Zürich, Switzerland
| | - Margot Thome
- Department of Biochemistry, University of Lausanne, Epalinges, Switzerland
| | - Oliver Hantschel
- Swiss Institute for Experimental Cancer Research (ISREC), Federal University of Technology Lausanne (EPFL), Lausanne, Switzerland
| | - Yves Chalandon
- Service d'Hématologie, Hôpitaux Universitaire de Genève, Geneva, Switzerland
| | - Gianni Cazzaniga
- Centro Ricerca Tettamanti, Pediatric Clinic University of Milano-Bicocca, Monza, Italy
| | - Jean-Pierre Bourquin
- Department of Pediatric Oncology and Children's Research Centre, University Children's Hospital Zürich, Zürich, Switzerland
| | - Joerg Huelsken
- Swiss Institute for Experimental Cancer Research (ISREC), Federal University of Technology Lausanne (EPFL), Lausanne, Switzerland
| | - Werner Held
- Department of Oncology UNIL CHUV, University of Lausanne, Epalinges, Switzerland.
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25
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Wei X, Zhang L, Zhou Z, Kwon OJ, Zhang Y, Nguyen H, Dumpit R, True L, Nelson P, Dong B, Xue W, Birchmeier W, Taketo MM, Xu F, Creighton CJ, Ittmann MM, Xin L. Spatially Restricted Stromal Wnt Signaling Restrains Prostate Epithelial Progenitor Growth through Direct and Indirect Mechanisms. Cell Stem Cell 2019; 24:753-768.e6. [PMID: 30982770 DOI: 10.1016/j.stem.2019.03.010] [Citation(s) in RCA: 40] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2018] [Revised: 12/11/2018] [Accepted: 03/10/2019] [Indexed: 12/31/2022]
Abstract
Cell-autonomous Wnt signaling has well-characterized functions in controlling stem cell activity, including in the prostate. While niche cells secrete Wnt ligands, the effects of Wnt signaling in niche cells per se are less understood. Here, we show that stromal cells in the proximal prostatic duct near the urethra, a mouse prostate stem cell niche, not only produce multiple Wnt ligands but also exhibit strong Wnt/β-catenin activity. The non-canonical Wnt ligand Wnt5a, secreted by proximal stromal cells, directly inhibits proliefration of prostate epithelial stem or progenitor cells whereas stromal cell-autonomous canonical Wnt/β-catenin signaling indirectly suppresses prostate stem or progenitor activity via the transforming growth factor β (TGFβ) pathway. Collectively, these pathways restrain the proliferative potential of epithelial cells in the proximal prostatic ducts. Human prostate likewise exhibits spatially restricted distribution of stromal Wnt/β-catenin activity, suggesting a conserved mechanism for tissue patterning. Thus, this study shows how distinct stromal signaling mechanisms within the prostate cooperate to regulate tissue homeostasis.
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Affiliation(s)
- Xing Wei
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Graduate Program in Integrative Molecular and Biomedical Sciences, Baylor College of Medicine, Houston, TX 77030, USA; Department of Urology, University of Washington, Seattle, WA 98109, USA
| | - Li Zhang
- Department of Urology, University of Washington, Seattle, WA 98109, USA
| | - Zhicheng Zhou
- Department of Urology, University of Washington, Seattle, WA 98109, USA
| | - Oh-Joon Kwon
- Department of Urology, University of Washington, Seattle, WA 98109, USA
| | - Yiqun Zhang
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA
| | - Hoang Nguyen
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Center of Stem Cell and Regenerative Medicine, Baylor College of Medicine, Houston, TX 77030, USA
| | - Ruth Dumpit
- Human Biology Division, Fred Hutch Cancer Research Center, Seattle, WA 98109, USA
| | - Lawrence True
- Department of Pathology, University of Washington, Seattle, WA 98109, USA
| | - Peter Nelson
- Human Biology Division, Fred Hutch Cancer Research Center, Seattle, WA 98109, USA
| | - Baijun Dong
- Department of Urology, RenJi Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Wei Xue
- Department of Urology, RenJi Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Walter Birchmeier
- Max Delbrück Center for Molecular Medicine (MDC), Robert-Rössle-Str. 10, 13092 Berlin, Germany
| | - Makoto M Taketo
- Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Kyoto, Japan
| | - Feng Xu
- Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore, Singapore
| | - Chad J Creighton
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA; Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA
| | - Michael M Ittmann
- Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA; Michael E. DeBakey Department of Veterans Affairs Medical Center, Houston, TX 77030, USA
| | - Li Xin
- Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA; Department of Urology, University of Washington, Seattle, WA 98109, USA; Institute of Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA.
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Matthews SM, Eshelman MA, Berg AS, Koltun WA, Yochum GS. The Crohn's disease associated SNP rs6651252 impacts MYC gene expression in human colonic epithelial cells. PLoS One 2019; 14:e0212850. [PMID: 30794691 PMCID: PMC6386311 DOI: 10.1371/journal.pone.0212850] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2018] [Accepted: 02/11/2019] [Indexed: 12/11/2022] Open
Abstract
Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that arises from chronic inflammation in the gastrointestinal tract. Genome-wide association studies (GWAS) have identified over 200 single nucleotide polymorphisms (SNPs) that are associated with a predisposition for developing IBD. For the majority, the causal variant and target genes affected are unknown. Here, we investigated the CD-associated SNP rs6651252 that maps to a gene desert region on chromosome 8. We demonstrate that rs6651252 resides within a Wnt responsive DNA enhancer element (WRE) and that the disease associated allele augments binding of the TCF7L2 transcription factor to this region. Using CRISPR/Cas9 directed gene editing and epigenetic modulation, we find that the rs6651252 enhancer regulates expression of the c-MYC proto-oncogene (MYC). Furthermore, we found MYC transcript levels are elevated in patient-derived colonic segments harboring the disease-associated allele in comparison to those containing the ancestral allele. These results suggest that Wnt/MYC signaling contributes to CD pathogenesis and that patients harboring the disease-associated allele may benefit from therapies that target MYC or MYC-regulated genes.
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Affiliation(s)
- Stephen M. Matthews
- Department of Biochemistry & Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
- Department of Surgery, Division of Colon and Rectal Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Melanie A. Eshelman
- Department of Biochemistry & Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
- Department of Surgery, Division of Colon and Rectal Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Arthur S. Berg
- Department of Public Health Sciences, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Walter A. Koltun
- Department of Surgery, Division of Colon and Rectal Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
| | - Gregory S. Yochum
- Department of Biochemistry & Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
- Department of Surgery, Division of Colon and Rectal Surgery, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, United States of America
- * E-mail:
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27
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Li Z, Lim SK, Liang X, Lim YP. The transcriptional coactivator WBP2 primes triple-negative breast cancer cells for responses to Wnt signaling via the JNK/Jun kinase pathway. J Biol Chem 2018; 293:20014-20028. [PMID: 30442712 PMCID: PMC6311518 DOI: 10.1074/jbc.ra118.005796] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2018] [Revised: 10/29/2018] [Indexed: 12/17/2022] Open
Abstract
The transcriptional coactivator WW domain-binding protein 2 (WBP2) is an emerging oncogene and serves as a node between the signaling protein Wnt and other signaling molecules and pathways, including epidermal growth factor receptor, estrogen receptor/progesterone receptor, and the Hippo pathway. The upstream regulation of WBP2 is well-studied, but its downstream activity remains unclear. Here, we elucidated WBP2's role in triple-negative breast cancer (TNBC), in which Wnt signaling is predominantly activated. Using RNAi coupled with RNA-Seq and MS analyses to identify Wnt/WBP2- and WBP2-dependent targets in MDA-MB-231 TNBC cells, we found that WBP2 is required for the expression of a core set of genes in Wnt signaling. These included AXIN2, which was essential for Wnt/WBP2-mediated breast cancer growth and migration. WBP2 also regulated a much larger set of genes and proteins independently of Wnt, revealing that WBP2 primes cells to Wnt activity by up-regulating G protein pathway suppressor 1 (GPS1) and TRAF2- and NCK-interacting kinase (TNIK). GPS1 activated the c-Jun N-terminal kinase (JNK)/Jun pathway, resulting in a positive feedback loop with TNIK that mediated Wnt-induced AXIN2 expression. WBP2 promoted TNBC growth by integrating JNK with Wnt signaling, and its expression profoundly influenced the sensitivity of TNBC to JNK/TNIK inhibitors. In conclusion, WBP2 links JNK to Wnt signaling in TNBC. GPS1 and TNIK are constituents of a WBP2-initiated cascade that primes responses to Wnt ligands and are also important for TNBC biology. We propose that WBP2 is a potential drug target for JNK/TNIK-based precision medicine for managing TNBC.
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Affiliation(s)
- Zilin Li
- From the Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545
| | - Shen Kiat Lim
- From the Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545
| | - Xu Liang
- From the Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545
| | - Yoon Pin Lim
- From the Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117545,; the National University Cancer Institute, Singapore 119082, and; the NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 117456.
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28
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MiRNA-target interactions in osteogenic signaling pathways involving zinc via the metal regulatory element. Biometals 2018; 32:111-121. [PMID: 30564968 DOI: 10.1007/s10534-018-00162-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2018] [Accepted: 12/04/2018] [Indexed: 01/11/2023]
Abstract
Adequate zinc nutriture is necessary for normal bone growth and development, though the precise mechanisms for zinc-mediated bone growth remain poorly defined. A key transcription factor activated by zinc is metal response element-binding transcription factor 1 (MTF-1), which binds to the metal regulatory element (MRE). We hypothesize that MREs will be found upstream of miRNA genes as well as miRNA target genes in the following bone growth and development signaling pathways: TGF-β, MAPK, and Wnt. A Bioconductor-based workflow in R was designed to identify interactions between MREs, miRNAs, and target genes. MRE sequences were found upstream from 64 mature miRNAs that interact with 213 genes which have MRE sequences in their own promoter regions. MAPK1 exhibited the most miRNA-target interactions (MTIs) in the TGF-β and MAPK signaling pathways; CCND2 exhibited the most interactions in the Wnt signaling pathway. Hsa-miR-124-3p exhibited the most MTIs in the TGF-β and MAPK signaling pathways; hsa-miR-20b-5p exhibited the most MTIs in the Wnt signaling pathway. MYC and hsa-miR-34a-5p were shared between all three signaling pathways, also forming an MTI unit. JUN exhibited the most protein-protein interactions, followed by MAPK8. These in silico data support the hypothesis that intracellular zinc status plays a role in osteogenesis through the transcriptional regulation of miRNA genes via the zinc/MTF-1/MRE complex.
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29
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Suzuki A, Minamide R, Iwata J. WNT/β-catenin signaling plays a crucial role in myoblast fusion through regulation of nephrin expression during development. Development 2018; 145:dev.168351. [PMID: 30389854 DOI: 10.1242/dev.168351] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2018] [Accepted: 10/24/2018] [Indexed: 01/06/2023]
Abstract
Skeletal muscle development is controlled by a series of multiple orchestrated regulatory pathways. WNT/β-catenin is one of the most important pathways for myogenesis; however, it remains unclear how this signaling pathway regulates myogenesis in a temporal- and spatial-specific manner. Here, we show that WNT/β-catenin signaling is crucial for myoblast fusion through regulation of the nephrin (Nphs1) gene in the Myog-Cre-expressing myoblast population. Mice deficient for the β-catenin gene in Myog-Cre-expressing myoblasts (Ctnnb1F/F;Myog-Cre mice) displayed myoblast fusion defects, but not migration or cell proliferation defects. The promoter region of Nphs1 contains the conserved β-catenin-binding element, and Nphs1 expression was induced by the activation of WNT/β-catenin signaling. The induction of Nphs1 in cultured myoblasts from Ctnnb1F/F;Myog-Cre mice restored the myoblast fusion defect, indicating that nephrin is functionally relevant in WNT/β-catenin-dependent myoblast fusion. Taken together, our results indicate that WNT/β-catenin signaling is crucial for myoblast fusion through the regulation of the Nphs1 gene.
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Affiliation(s)
- Akiko Suzuki
- Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center at Houston (UT Health) School of Dentistry, Houston, TX 77054, USA.,Center for Craniofacial Research, UT Health School of Dentistry, Houston, TX 77054, USA
| | - Ryohei Minamide
- Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center at Houston (UT Health) School of Dentistry, Houston, TX 77054, USA.,Center for Craniofacial Research, UT Health School of Dentistry, Houston, TX 77054, USA
| | - Junichi Iwata
- Department of Diagnostic and Biomedical Sciences, The University of Texas Health Science Center at Houston (UT Health) School of Dentistry, Houston, TX 77054, USA .,Center for Craniofacial Research, UT Health School of Dentistry, Houston, TX 77054, USA.,MD Anderson Cancer Center, UT Health Graduate School of Biomedical Sciences, Houston, TX 77054, USA
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30
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Bioinformatics-based interaction analysis of miR-92a-3p and key genes in tamoxifen-resistant breast cancer cells. Biomed Pharmacother 2018; 107:117-128. [PMID: 30086458 DOI: 10.1016/j.biopha.2018.07.158] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Revised: 07/19/2018] [Accepted: 07/31/2018] [Indexed: 12/15/2022] Open
Abstract
The abnormal expression of miR-92a-3p was detected in multiple cancers. However, the biological role and underlying mechanism of miR-92a-3p in tamoxifen-resistant cells are still unknown. The main objective of our study was to find potential miR-92a-3p regulating pathways involved in tamoxifen resistance and to construct their regulatory network using bioinformatics. Four gene expression profiles were retrieved from GEO database and the GEO2R tool was used for analysis. GSE41922 and GSE42072 were applied to investigate aberrant miR-92a-3p expression in breast cancer serum and tissue. We found that miR-92a-3p expression was higher in breast cancer serum or tissue than in healthy volunteer serum or adjacent normal tissue, and high expression of miR-92a-3p could predict poor prognosis of breast cancer patients. In our qRT-PCR validation, we found that miR-92a-3p was upregulated in tamoxifen-resistant cells. MiR-92a-3p might play a role in tamoxifen resistance. In order to find the relationship between miR-92a-3p and some key genes and their potential molecular mechanisms in tamoxifen-resistant cells. The microarray data GSE26459 and GSE28267 were analyzed to determine the differentially expressed genes (DEGs) or miRNAs (DEMs). Furthermore, the related long non-coding RNAs (lncRNAs) were screened with starBase v2.0. Finally,microRNA.org,miRDB, targetminer and targetscan were applied to predict the targets of miR-92a-3p. Through analysis, we find that miR-92a-3p may be used as a potential biomarker for early detection of cancer and monitoring the efficacy of endocrine therapy.
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31
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Nakajima T, Uehara T, Kobayashi Y, Kinugawa Y, Yamanoi K, Maruyama Y, Suga T, Ota H. Leucine-rich repeat-containing G-protein-coupled receptor 5 expression and clinicopathological features of colorectal neuroendocrine neoplasms. Pathol Int 2018; 68:467-472. [PMID: 30043418 DOI: 10.1111/pin.12707] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2018] [Accepted: 06/30/2018] [Indexed: 01/27/2023]
Abstract
LGR5 is expressed in various tumors and has been identified as a putative intestinal stem cell marker. Here we investigated LGR5 expression in colorectal neuroendocrine neoplasms and analyzed the correlation with pathological characteristics. We evaluated the clinicopathological features of 8 neuroendocrine tumor (NET) grade 1 (NET G1), 4 NET Grade 2 (NET G2), and 8 NET Grade 3 (NET G3; also termed neuroendocrine carcinoma, or NEC) cases. We examined LGR5 expression using an RNAscope, a newly developed RNA in situ hybridization technique, with a tissue microarray of the neuroendocrine neoplasm samples. LGR5 staining in individual tumor cells was semi-quantitatively scored using an H-score scale. We also performed a combination of LGR5 RNA in situ hybridization and synaptophysin immunohistochemistry. All cases contained tumor cells with some LGR5-positive dots. For all cases, H-scores showed a positive correlation with nuclear beta-catenin expression. In the NEC group, there was a strong positive correlation between H-score and beta-catenin expression. Our findings suggest that LGR5 may serve as a stem cell marker in NEC, as is the case in colon adenocarcinoma. The positive correlation between H-score and beta-catenin expression suggests that LGR5 expression might be affected by beta-catenin expression in neuroendocrine neoplasms and especially in NEC.
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Affiliation(s)
- Tomoyuki Nakajima
- Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
| | - Takeshi Uehara
- Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
| | - Yukihiro Kobayashi
- Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
| | - Yasuhiro Kinugawa
- Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
| | - Kazuhiro Yamanoi
- Interdisciplinary Cluster for Cutting Edge Research, Institute for Biomedical Sciences, Department of Advanced Medicine for Health Promotion, Shinshu University, Matsumoto, Japan
| | - Yasuhiro Maruyama
- Department of Gastroenterology, Suwa Red Cross Hospital, Suwa, Japan
| | - Tomoaki Suga
- Department of Gastroenterology, Shinshu University School of Medicine, Matsumoto, Japan
| | - Hiroyoshi Ota
- Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
- Department of Biomedical Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan
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32
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Nayak G, Odaka Y, Prasad V, Solano AF, Yeo EJ, Vemaraju S, Molkentin JD, Trumpp A, Williams B, Rao S, Lang RA. Developmental vascular regression is regulated by a Wnt/β-catenin, MYC and CDKN1A pathway that controls cell proliferation and cell death. Development 2018; 145:dev154898. [PMID: 29777010 PMCID: PMC6031408 DOI: 10.1242/dev.154898] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2017] [Accepted: 05/08/2018] [Indexed: 12/12/2022]
Abstract
Normal development requires tight regulation of cell proliferation and cell death. Here, we have investigated these control mechanisms in the hyaloid vessels, a temporary vascular network in the mammalian eye that requires a Wnt/β-catenin response for scheduled regression. We investigated whether the hyaloid Wnt response was linked to the oncogene Myc, and the cyclin-dependent kinase inhibitor CDKN1A (P21), both established regulators of cell cycle progression and cell death. Our analysis showed that the Wnt pathway co-receptors LRP5 and LRP6 have overlapping activities that mediate the Wnt/β-catenin signaling in hyaloid vascular endothelial cells (VECs). We also showed that both Myc and Cdkn1a are downstream of the Wnt response and are required for hyaloid regression but for different reasons. Conditional deletion of Myc in VECs suppressed both proliferation and cell death. By contrast, conditional deletion of Cdkn1a resulted in VEC overproliferation that countered the effects of cell death on regression. When combined with analysis of MYC and CDKN1A protein levels, this analysis suggests that a Wnt/β-catenin and MYC-CDKN1A pathway regulates scheduled hyaloid vessel regression.
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Affiliation(s)
- Gowri Nayak
- The Visual Systems Group, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Chronobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Yoshinobu Odaka
- The Visual Systems Group, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Chronobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Vikram Prasad
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Alyssa F Solano
- The Visual Systems Group, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Chronobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Eun-Jin Yeo
- The Visual Systems Group, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Chronobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Shruti Vemaraju
- The Visual Systems Group, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Chronobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Jeffery D Molkentin
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Howard Hughes Medical Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
| | - Andreas Trumpp
- Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), 69120 Heidelberg, Germany; Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), 69120 Heidelberg, Germany; German Cancer Consortium (DKTK), 69120 Heidelberg, Germany
| | - Bart Williams
- Center for Skeletal Disease Research and Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, MI 49503, USA
| | - Sujata Rao
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- The Cleveland Clinic, Ophthalmic Research, 9500 Euclid Avenue, OH 44195, USA
| | - Richard A Lang
- The Visual Systems Group, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Center for Chronobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Abrahamson Pediatric Eye Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Divisions of Pediatric Ophthalmology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA
- Department of Ophthalmology, College of Medicine, University of Cincinnati, Cincinnati, OH 45229, USA
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33
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Alaee M, Nool K, Pasdar M. Plakoglobin restores tumor suppressor activity of p53 R175H mutant by sequestering the oncogenic potential of β-catenin. Cancer Sci 2018; 109:1876-1888. [PMID: 29660231 PMCID: PMC5989865 DOI: 10.1111/cas.13612] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2018] [Revised: 04/03/2018] [Accepted: 04/06/2018] [Indexed: 12/16/2022] Open
Abstract
Tumor suppressor/transcription factor p53 is mutated in over 50% of all cancers. Some mutant p53 proteins have not only lost tumor suppressor activities but they also gain oncogenic functions (GOF). One of the most frequently expressed GOF p53 mutants is Arg175His (p53R175H ) with well-documented roles in cancer development and progression. Plakoglobin is a cell adhesion and signaling protein and a paralog of β-catenin. Unlike β-catenin that has oncogenic function through its role in the Wnt pathway, plakoglobin generally acts as a tumor/metastasis suppressor. We have shown that plakoglobin interacted with wild type and a number of p53 mutants in various carcinoma cell lines. Plakoglobin and mutant p53 interacted with the promoter and regulated the expression of several p53 target genes. Furthermore, plakoglobin interactions with p53 mutants restored their tumor suppressor/metastasis activities in vitro. GOF p53 mutants induce accumulation and oncogenic activation of β-catenin. Previously, we showed that one mechanism by which plakoglobin may suppress tumorigenesis is by sequestering β-catenin's oncogenic activity. Here, we examined the effects of p53R175H expression on β-catenin accumulation and transcriptional activation and their modifications by plakoglobin coexpression. We showed that p53R175H expression in plakoglobin null cells increased total and nuclear levels of β-catenin and its transcriptional activity. Coexpression of plakoglobin in these cells promoted β-catenin's proteasomal degradation, and decreased its nuclear levels and transactivation. Wnt/β-catenin targets, c-MYC and S100A4 were upregulated in p53R175H cells and were downregulated when plakoglobin was coexpressed. Plakoglobin-p53R175H cells also showed significant reduction in their migration and invasion in vitro.
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Affiliation(s)
- Mahsa Alaee
- Department of OncologyUniversity of AlbertaEdmontonCanada
| | - Kristina Nool
- Department of OncologyUniversity of AlbertaEdmontonCanada
| | - Manijeh Pasdar
- Department of OncologyUniversity of AlbertaEdmontonCanada
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34
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Bardales JA, Wieser E, Kawaji H, Murakawa Y, Darzacq X. Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers. Genes (Basel) 2018; 9:genes9060270. [PMID: 29882899 PMCID: PMC6027352 DOI: 10.3390/genes9060270] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2018] [Revised: 05/14/2018] [Accepted: 05/15/2018] [Indexed: 11/16/2022] Open
Abstract
In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation.
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Affiliation(s)
- Jorge A Bardales
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
- Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
| | - Evin Wieser
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
| | - Hideya Kawaji
- Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
- RIKEN Preventive Medicine and Diagnosis Innovation Program, Yokohama 230-0045, Japan.
- Preventive Medicine and Applied Genomics Unit, RIKEN Advanced Center for Computing and Communication, Yokohama 230-0045, Japan.
| | - Yasuhiro Murakawa
- Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama 230-0045, Japan.
- RIKEN Preventive Medicine and Diagnosis Innovation Program, Yokohama 230-0045, Japan.
| | - Xavier Darzacq
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
- Li Ka Shing Center for Biomedical and Health Sciences, CIRM Center of Excellence, University of California, Berkeley, CA 94720, USA.
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35
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Somasagara RR, Spencer SM, Tripathi K, Clark DW, Mani C, da Silva LM, Scalici J, Kothayer H, Westwell AD, Rocconi RP, Palle K. RAD6 promotes DNA repair and stem cell signaling in ovarian cancer and is a promising therapeutic target to prevent and treat acquired chemoresistance. Oncogene 2017; 36:6680-6690. [PMID: 28806395 PMCID: PMC5709226 DOI: 10.1038/onc.2017.279] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2017] [Revised: 06/14/2017] [Accepted: 07/07/2017] [Indexed: 12/28/2022]
Abstract
Ovarian cancer (OC) is the most deadly gynecological cancer and unlike most other neoplasms, survival rates for OC have not significantly improved in recent decades. We show that RAD6, an ubiquitin-conjugating enzyme, is significantly overexpressed in ovarian tumors and its expression increases in response to carboplatin chemotherapy. RAD6 expression correlated strongly with acquired chemoresistance and malignant behavior of OC cells, expression of stem cell genes and poor prognosis of OC patients, suggesting an important role for RAD6 in ovarian tumor progression. Upregulated RAD6 enhances DNA damage tolerance and repair efficiency of OC cells and promotes their survival. Increased RAD6 levels cause histone 2B ubiquitination-mediated epigenetic changes that stimulate transcription of stem cell genes, including ALDH1A1 and SOX2, leading to a cancer stem cell phenotype, which is implicated in disease recurrence and metastasis. Downregulation of RAD6 or its inhibition using a small molecule inhibitor attenuated DNA repair signaling and expression of cancer stem cells markers and sensitized chemoresistant OC cells to carboplatin. Together, these results suggest that RAD6 could be a therapeutic target to prevent and treat acquired chemoresistance and disease recurrence in OC and enhance the efficacy of standard chemotherapy.
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Affiliation(s)
- Ranganatha R. Somasagara
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Sebastian M. Spencer
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Kaushlendra Tripathi
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - David W. Clark
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Chinnadurai Mani
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Luciana Madeira da Silva
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Jennifer Scalici
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Hend Kothayer
- Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Egypt
| | - Andrew D. Westwell
- School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, Wales, UK
| | - Rodney P. Rocconi
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
| | - Komaraiah Palle
- Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, 1660 Springhill Avenue, Mobile, Alabama 36604, USA
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Eshelman MA, Shah M, Raup-Konsavage WM, Rennoll SA, Yochum GS. TCF7L1 recruits CtBP and HDAC1 to repress DICKKOPF4 gene expression in human colorectal cancer cells. Biochem Biophys Res Commun 2017; 487:716-722. [PMID: 28450117 DOI: 10.1016/j.bbrc.2017.04.123] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2017] [Accepted: 04/22/2017] [Indexed: 12/11/2022]
Abstract
The T-cell factor/Lymphoid enhancer factor (TCF/LEF; hereafter TCF) family of transcription factors are critical regulators of colorectal cancer (CRC) cell growth. Of the four TCF family members, TCF7L1 functions predominantly as a repressor of gene expression. Few studies have addressed the role of TCF7L1 in CRC and only a handful of target genes regulated by this repressor are known. By silencing TCF7L1 expression in HCT116 cells, we show that it promotes cell proliferation and tumorigenesis in vivo by driving cell cycle progression. Microarray analysis of transcripts differentially expressed in control and TCF7L1-silenced CRC cells identified genes that control cell cycle kinetics and cancer pathways. Among these, expression of the Wnt antagonist DICKKOPF4 (DKK4) was upregulated when TCF7L1 levels were reduced. We found that TCF7L1 recruits the C-terminal binding protein (CtBP) and histone deacetylase 1 (HDAC1) to the DKK4 promoter to repress DKK4 gene expression. In the absence of TCF7L1, TCF7L2 and β-catenin occupancy at the DKK4 promoter is stimulated and DKK4 expression is increased. These findings uncover a critical role for TCF7L1 in repressing DKK4 gene expression to promote the oncogenic potential of CRCs.
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Affiliation(s)
- Melanie A Eshelman
- Department of Biochemistry & Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA
| | - Meera Shah
- Department of Biochemistry & Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA
| | - Wesley M Raup-Konsavage
- Department of Biochemistry & Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA
| | - Sherri A Rennoll
- Department of Biochemistry & Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA
| | - Gregory S Yochum
- Department of Biochemistry & Molecular Biology, The Pennsylvania State University College of Medicine, Hershey, PA, USA; Department of Surgery, The Pennsylvania State University College of Medicine, Hershey, PA, USA.
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Abstract
New nephrons are induced by the interaction between mesenchymal progenitor cells and collecting duct tips, both of which are located at the outer edge of the kidney. This leading edge of active nephron induction is known as the nephrogenic zone. Cell populations found within this zone include collecting duct tips, cap mesenchyme cells, pretubular aggregates, nephrogenic zone interstitium, hemoendothelial progenitor cells, and macrophages. The close association of these dynamic progenitor cell compartments enables the intricate and synchronized patterning of the epithelial and the vascular components of the nephron. Understanding signaling interactions between the distinct progenitor cells of the nephrogenic zone are essential to determining the basis for new nephron formation, an important goal in regenerative medicine. A variety of technologies have been applied to define essential signaling pathways, including organ culture, mouse genetics, and primary cell culture. This chapter provides an overview of essential signaling pathways and discusses how these may be integrated.
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Zhu J, Wang P, Yu Z, Lai W, Cao Y, Huang P, Xu Q, Yu M, Xu J, Huang Z, Zeng B. Advanced glycosylation end product promotes forkhead box O1 and inhibits Wnt pathway to suppress capacities of epidermal stem cells. Am J Transl Res 2016; 8:5569-5579. [PMID: 28078027 PMCID: PMC5209507] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2016] [Accepted: 11/08/2016] [Indexed: 06/06/2023]
Abstract
Diabetes mellitus is frequently accompanied by chronic complications like delayed wound healing, which is consider to be attributed to the accumulation of advanced glycosylation end product (AGE). However, the impacts of AGE on epidermal stem cells (ESCs) are largely unknown. This study aims to address the influence and mechanism of AGE on ESCs. ESCs isolated from rats were cultured in AGE-modified bovine serum albumin and transfected with small interfering RNA to knock down AGE-specific receptor (AGER). Expression of stem cell markers integrin β1 (ITGB1) and keratin 19 (KRT19), cell viability, apoptosis and reactive oxygen species (ROS) were examined. Wnt pathway-related factors Wnt family member 1 (WNT1), WNT3A, β-catenin, v-myc avian myelocytomatosis viral oncogene homolog (MYC), cyclin D1 (CCND1) and matrix metallopeptidase 7 (MMP7) were quantified. The interaction between forkhead box O1 (FOXO1) and β-catenin was assessed by co-immunoprecipitation. Results indicated that AGE down-regulated ITGB1 and KRT19 expression, suppressed ESC viability and promoted apoptosis, and ROS level (P < 0.01), implying decreased capacities of ESCs. AGE also promoted AGER and FOXO1, while AGER knockdown had the opposite effects. Moreover, AGER knockdown elevated the level of WNT1, WNT3A, MYC, CCND1 and MMP7 that were suppressed by AGE (P < 0.01). Immunoprecipitation analysis showed that FOXO1 could compete with lymphoid enhancer binding factor 1 to interact with β-catenin, which might help to elucidate the mechanism of AGE repressing ESCs. This study helps to understand the mechanism of accumulated AGE in affecting ESC capacities, and provides potential therapeutic targets to meliorate diabetic wound healing.
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Affiliation(s)
- Jie Zhu
- Department of Emergency, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Peng Wang
- Institute of Cardiopulmonary Cerebral Resuscitation, Sun Yat-sen UniversityGuangzhou 510275, Guangdong, China
| | - Zhimin Yu
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Wei Lai
- Department of Gastrointestinal Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Yi Cao
- Department of Emergency, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Pinbo Huang
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Qiaodong Xu
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Menglei Yu
- Department of Emergency, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Junyao Xu
- Department of Hepatobiliary Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen UniversityGuangzhou 510288, Guangdong, China
| | - Zitong Huang
- Institute of Cardiopulmonary Cerebral Resuscitation, Sun Yat-sen UniversityGuangzhou 510275, Guangdong, China
| | - Bing Zeng
- Department of Gastrointestinal Surgery, The Sixth Affiliated Hospital of Guangzhou Medical UniversityQingyuan 511518, Guangdong, China
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Rezzoug F, Thomas SD, Rouchka EC, Miller DM. Discovery of a Family of Genomic Sequences Which Interact Specifically with the c-MYC Promoter to Regulate c-MYC Expression. PLoS One 2016; 11:e0161588. [PMID: 27551915 PMCID: PMC4995011 DOI: 10.1371/journal.pone.0161588] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2016] [Accepted: 08/08/2016] [Indexed: 12/14/2022] Open
Abstract
G-quadruplex forming sequences are particularly enriched in the promoter regions of eukaryotic genes, especially of oncogenes. One of the most well studied G-quadruplex forming sequences is located in the nuclease hypersensitive element (NHE) III1 of the c-MYC promoter region. The oncoprotein c-MYC regulates a large array of genes which play important roles in growth regulation and metabolism. It is dysregulated in >70% of human cancers. The silencer NHEIII1 located upstream of the P1 promoter regulates up-to 80% of c-MYC transcription and includes a G-quadruplex structure (Pu27) that is required for promoter inhibition. We have identified, for the first time, a family of seventeen G-quadruplex-forming motifs with >90% identity with Pu27, located on different chromosomes throughout the human genome, some found near or within genes involved in stem cell maintenance or neural cell development. Notably, all members of the Pu27 family interact specifically with NHEIII1 sequence, in vitro. Crosslinking studies demonstrate that Pu27 oligonucleotide binds specifically to the C-rich strand of the NHEIII1 resulting in the G-quadruplex structure stabilization. Pu27 homologous sequences (Pu27-HS) significantly inhibit leukemic cell lines proliferation in culture. Exposure of U937 cells to the Pu27-HS induces cell growth inhibition associated with cell cycle arrest that is most likely due to downregulation of c-MYC expression at the RNA and/or protein levels. Expression of SOX2, another gene containing a Pu27-HS, was affected by Pu27-HS treatment as well. Our data suggest that the oligonucleotides encoding the Pu27 family target complementary DNA sequences in the genome, including those of the c-MYC and SOX2 promoters. This effect is most likely cell type and cell growth condition dependent. The presence of genomic G-quadruplex-forming sequences homologous to Pu27 of c-MYC silencer and the fact that they interact specifically with the parent sequence suggest a common regulatory mechanism for genes whose promoters contain these sequences.
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Affiliation(s)
- Francine Rezzoug
- James Graham Brown Cancer Center, Department of Medicine, University of Louisville, Louisville Kentucky, United States of America
- * E-mail: (FR); (DMM)
| | - Shelia D. Thomas
- James Graham Brown Cancer Center, Department of Medicine, University of Louisville, Louisville Kentucky, United States of America
| | - Eric C. Rouchka
- Department of Computer Engineering and Computer Science, Speed School of Engineering, University of Louisville, Kentucky, United States of America
| | - Donald M. Miller
- James Graham Brown Cancer Center, Department of Medicine, University of Louisville, Louisville Kentucky, United States of America
- * E-mail: (FR); (DMM)
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The MYC 3' Wnt-Responsive Element Drives Oncogenic MYC Expression in Human Colorectal Cancer Cells. Cancers (Basel) 2016; 8:cancers8050052. [PMID: 27223305 PMCID: PMC4880869 DOI: 10.3390/cancers8050052] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Revised: 05/11/2016] [Accepted: 05/18/2016] [Indexed: 12/11/2022] Open
Abstract
Mutations in components of the Wnt/β-catenin signaling pathway drive colorectal cancer (CRC) by deregulating expression of downstream target genes including the c-MYC proto-oncogene (MYC). The critical regulatory DNA enhancer elements that control oncogenic MYC expression in CRC have yet to be fully elucidated. In previous reports, we correlated T-cell factor (TCF) and β-catenin binding to the MYC 3′ Wnt responsive DNA element (MYC 3′ WRE) with MYC expression in HCT116 cells. Here we used CRISPR/Cas9 to determine whether this element is a critical driver of MYC. We isolated a clonal population of cells that contained a deletion of a single TCF binding element (TBE) within the MYC 3′ WRE. This deletion reduced TCF/β-catenin binding to this regulatory element and decreased MYC expression. Using RNA-Seq analysis, we found altered expression of genes that regulate metabolic processes, many of which are known MYC target genes. We found that 3′ WRE-Mut cells displayed a reduced proliferative capacity, diminished clonogenic growth, and a decreased potential to form tumors in vivo. These findings indicate that the MYC 3′ WRE is a critical driver of oncogenic MYC expression and suggest that this element may serve as a therapeutic target for CRC.
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Raup-Konsavage WM, Cooper TK, Yochum GS. A Role for MYC in Lithium-Stimulated Repair of the Colonic Epithelium After DSS-Induced Damage in Mice. Dig Dis Sci 2016; 61:410-22. [PMID: 26320084 DOI: 10.1007/s10620-015-3852-0] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/02/2015] [Accepted: 08/17/2015] [Indexed: 02/07/2023]
Abstract
BACKGROUND Chronic inflammation disrupts the colonic epithelial layer in patients afflicted by ulcerative colitis (UC). The use of inhibitors of glycogen synthase kinase three beta (GSK3β) has proven efficacious to mitigate disease symptoms in rodent models of UC by reducing the pro-inflammatory response. Less is known about whether these inhibitors promote colonic regeneration by stimulating proliferation of colonic epithelial cells. AIMS We investigated whether delivery of the GSK3β inhibitor, lithium chloride (LiCl), during the recovery period from acute DSS-induced colitis in mice promoted colonic regeneration and ameliorated disease symptoms. We also tested whether the c-MYC transcription factor (MYC) was involved in this response. METHODS Acute colitis was induced by administration of 2.5 % dextran sodium sulfate (DSS) to wild-type C57BL/6 mice for 5 days. During the recovery period, mice received a daily intraperitoneal (IP) injection of LiCl or 1X PBS as a control. Mice were weighed, colon lengths measured, disease activity index (DAI) scores were assessed, and histological analyses were performed on colonic sections. We analyzed transcripts and proteins in purified preparations of the colonic epithelium. We delivered the MYC inhibitor 10058-F4 via IP injection to assess the role of MYC in colonic regeneration. RESULTS Lithium treatments promoted recovery from acute DSS-induced damage by increasing expression of Myc transcripts, MYC proteins, and expression of a subset of Wnt/MYC target genes in the colonic epithelium. Inhibiting MYC function with 10058-F4 blunted the lithium response. CONCLUSIONS By inducing Myc expression in the colonic epithelium, lithium promotes colonic regeneration after DSS-induced colitis. Therefore, the use of lithium may be of therapeutic value to manage individuals afflicted by UC.
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Affiliation(s)
- Wesley M Raup-Konsavage
- Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Dr., H171, Hershey, PA, 17033, USA.
| | - Timothy K Cooper
- Department of Comparative Medicine, The Pennsylvania State University College of Medicine, 500 University Dr., H171, Hershey, PA, 17033, USA. .,Department of Pathology, The Pennsylvania State University College of Medicine, 500 University Dr., H054, Hershey, PA, 17033, USA.
| | - Gregory S Yochum
- Department of Biochemistry and Molecular Biology, The Pennsylvania State University College of Medicine, 500 University Dr., H171, Hershey, PA, 17033, USA.
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Shah M, Rennoll SA, Raup-Konsavage WM, Yochum GS. A dynamic exchange of TCF3 and TCF4 transcription factors controls MYC expression in colorectal cancer cells. Cell Cycle 2015; 14:323-32. [PMID: 25659031 DOI: 10.4161/15384101.2014.980643] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Deregulated Wnt/β-catenin signaling promotes colorectal cancer (CRC) by activating expression of the c-MYC proto-oncogene (MYC). In the nucleus, the β-catenin transcriptional co-activator binds T-cell factor (TCF) transcription factors, and together TCF/β-catenin complexes activate MYC expression through Wnt responsive DNA regulatory elements (WREs). The MYC 3' WRE maps 1.4-kb downstream from the MYC transcription stop site and binds TCF4/β-catenin transcription complexes to activate MYC. However, the underlying mechanisms for how this element operates are not fully understood. Here, we report that the TCF family member, TCF3, plays an important role in regulating MYC expression in CRCs. We demonstrate that TCF3 binds the MYC 3' WRE to repress MYC. When TCF3 is depleted using shRNAs, the MYC 3' WRE is more available to bind TCF4/β-catenin complexes. Stimulating downstream Wnt/β-catenin signaling by inhibiting GSK3β causes an exchange of TCF3 with TCF4/β-catenin complexes to activate MYC. Finally, this transcription factor switch at the MYC 3' WRE controls MYC expression as quiescent cells re-enter the cell cycle and progress to S phase. These results indicate that a dynamic interplay of TCF transcription factors governs MYC gene expression in CRCs.
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Key Words
- APC, adenomatous polyposis coli
- CRC, colorectal cancer
- ChIP, chromatin immunoprecipitation
- GSK3β, glycogen synthase kinase 3 β
- HDAC, histone deacetylase
- Lef, Lymphoid enhancer-binding factor
- LiCl, lithium chloride
- MYC
- MYC, myelocytomatosis
- RT, reverse transcription
- TCF, T-cell factor
- TCF3
- TCF4
- TLE, Transducin-like enhancer of split
- WRE
- WRE, Wnt responsive DNA element
- colorectal cancer
- qPCR, quantitative PCR
- transcription
- β-catenin
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Affiliation(s)
- Meera Shah
- a Department of Biochemistry and Molecular Biology ; The Pennsylvania State University College of Medicine ; Hershey , PA USA
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Rennoll S, Yochum G. Regulation of MYC gene expression by aberrant Wnt/β-catenin signaling in colorectal cancer. World J Biol Chem 2015; 6:290-300. [PMID: 26629312 PMCID: PMC4657124 DOI: 10.4331/wjbc.v6.i4.290] [Citation(s) in RCA: 113] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/10/2015] [Revised: 08/26/2015] [Accepted: 10/13/2015] [Indexed: 02/05/2023] Open
Abstract
The Wnt/β-catenin signaling pathway controls intestinal homeostasis and mutations in components of this pathway are prevalent in human colorectal cancers (CRCs). These mutations lead to inappropriate expression of genes controlled by Wnt responsive DNA elements (WREs). T-cell factor/Lymphoid enhancer factor transcription factors bind WREs and recruit the β-catenin transcriptional co-activator to activate target gene expression. Deregulated expression of the c-MYC proto-oncogene (MYC) by aberrant Wnt/β-catenin signaling drives colorectal carcinogenesis. In this review, we discuss the current literature pertaining to the identification and characterization of WREs that control oncogenic MYC expression in CRCs. A common theme has emerged whereby these WREs often map distally to the MYC genomic locus and control MYC gene expression through long-range chromatin loops with the MYC proximal promoter. We propose that by determining which of these WREs is critical for CRC pathogenesis, novel strategies can be developed to treat individuals suffering from this disease.
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Inferring regulatory element landscapes and transcription factor networks from cancer methylomes. Genome Biol 2015; 16:105. [PMID: 25994056 PMCID: PMC4460959 DOI: 10.1186/s13059-015-0668-3] [Citation(s) in RCA: 144] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2015] [Accepted: 05/07/2015] [Indexed: 12/13/2022] Open
Abstract
Recent studies indicate that DNA methylation can be used to identify transcriptional enhancers, but no systematic approach has been developed for genome-wide identification and analysis of enhancers based on DNA methylation. We describe ELMER (Enhancer Linking by Methylation/Expression Relationships), an R-based tool that uses DNA methylation to identify enhancers and correlates enhancer state with expression of nearby genes to identify transcriptional targets. Transcription factor motif analysis of enhancers is coupled with expression analysis of transcription factors to infer upstream regulators. Using ELMER, we investigated more than 2,000 tumor samples from The Cancer Genome Atlas. We identified networks regulated by known cancer drivers such as GATA3 and FOXA1 (breast cancer), SOX17 and FOXA2 (endometrial cancer), and NFE2L2, SOX2, and TP63 (squamous cell lung cancer). We also identified novel networks with prognostic associations, including RUNX1 in kidney cancer. We propose ELMER as a powerful new paradigm for understanding the cis-regulatory interface between cancer-associated transcription factors and their functional target genes.
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Zhu HH, Zhu XY, Zhou MH, Cheng GY, Lou WH. Effect of WNT5A on epithelial-mesenchymal transition and its correlation with tumor invasion and metastasis in nasopharyngeal carcinoma. ASIAN PAC J TROP MED 2015; 7:488-91. [PMID: 25066400 DOI: 10.1016/s1995-7645(14)60080-8] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Revised: 01/15/2014] [Accepted: 03/15/2014] [Indexed: 10/25/2022] Open
Abstract
OBJECTIVE To investigate the correlation between nasopharyngeal carcinoma cell WNT5A and epithelial-mesenchymal transition (emt)/metastasis, and investigate its possible mechanisms. METHODS RT-PCR and gene transfection were used to detect the expression of nasopharyngeal carcinoma cell strains WNT5A and EMT related factor 5-8F. Transient transfection of NPC cell line 5-8F was determined by liposome of plasmid with WNT5A gene. The differential expressions of WNT5A and EMT-related factors in cells before and after transfection were detected by RT-PCR. Cell scratch assay and Transwell assay were used to detect the motility abilities of cells before and after 5-8F transfection. RESULTS The expressions of WNT5A and EMT related factors matrix metalloproteinase-2 of the WNT5A transferred group in the nasopharyngeal carcinoma cell line 5-8F were higher than the blank control group and the empty vector transferred group, and the transfer ability of the WNT5A transferred group was higher than that in the blank control group and the empty vector transferred group, while the expressions of EMT related factors E-cadherin were lower than that in the blank control group and the empty vector transferred group, and the transfer ability of the WNT5A transferred group was higher than that in the blank control group and the empty vector transferred group. CONCLUSIONS In nasopharyngeal carcinoma cells, WNT5A can regulate the epithelial-mesenchymal transition and affect the ability of tumor invasion and metastasis of nasopharyngeal carcinoma.
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Affiliation(s)
- Hong-Hai Zhu
- Rhinology of the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
| | - Xiao-Yuan Zhu
- Department of Otolaryngology-Head and Neck Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
| | - Ming-Hui Zhou
- Rhinology of the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China.
| | - Gen-Yang Cheng
- Department of Nephrology of the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
| | - Wei-Hua Lou
- Department of Otolaryngology-Head and Neck Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, Henan, China
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WNT/β-Catenin Signaling Regulates Multiple Steps of Myogenesis by Regulating Step-Specific Targets. Mol Cell Biol 2015; 35:1763-76. [PMID: 25755281 DOI: 10.1128/mcb.01180-14] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2014] [Accepted: 02/27/2015] [Indexed: 12/23/2022] Open
Abstract
Molecules involved in WNT/β-catenin signaling show specific spatiotemporal expression and play vital roles in myogenesis; however, it is still largely unknown how WNT/β-catenin signaling regulates each step of myogenesis. Here, we show that WNT/β-catenin signaling can control diverse biological processes of myogenesis by regulating step-specific molecules. In order to identify the temporally specific roles of WNT/β-catenin signaling molecules in muscle development and homeostasis, we used in vitro culture systems for both primary mouse myoblasts and C2C12 cells, which can differentiate into myofibers. We found that a blockade of WNT/β-catenin signaling in the proliferating cells decreases proliferation activity, but does not induce cell death, through the regulation of genes cyclin A2 (Ccna2) and cell division cycle 25C (Cdc25c). During muscle differentiation, the inhibition of WNT/β-catenin signaling blocks myoblast fusion through the inhibition of the Fermitin family homolog 2 (Fermt2) gene. Blocking WNT/β-catenin signaling in the well-differentiated myofibers results in the failure of maintenance of their structure by disruption of cadherin/β-catenin/actin complex formation, which plays a crucial role in connecting a myofiber's cytoskeleton to the surrounding extracellular matrix. Thus, our results indicate that WNT/β-catenin signaling can regulate multiple steps of myogenesis, including cell proliferation, myoblast fusion, and homeostasis, by targeting step-specific molecules.
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de la Parra C, Borrero-Garcia LD, Cruz-Collazo A, Schneider RJ, Dharmawardhane S. Equol, an isoflavone metabolite, regulates cancer cell viability and protein synthesis initiation via c-Myc and eIF4G. J Biol Chem 2015; 290:6047-57. [PMID: 25593313 PMCID: PMC4358247 DOI: 10.1074/jbc.m114.617415] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2014] [Revised: 12/30/2014] [Indexed: 12/28/2022] Open
Abstract
Epidemiological studies implicate dietary soy isoflavones as breast cancer preventives, especially due to their anti-estrogenic properties. However, soy isoflavones may also have a role in promoting breast cancer, which has yet to be clarified. We previously reported that equol, a metabolite of the soy isoflavone daidzein, may advance breast cancer potential via up-regulation of the eukaryotic initiation factor 4GI (eIF4GI). In estrogen receptor negative (ER-) metastatic breast cancer cells, equol induced elevated levels of eIF4G, which were associated with increased cell viability and the selective translation of mRNAs that use non-canonical means of initiation, including internal ribosome entry site (IRES), ribosome shunting, and eIF4G enhancers. These mRNAs typically code for oncogenic, survival, and cell stress molecules. Among those mRNAs translationally increased by equol was the oncogene and eIF4G enhancer, c-Myc. Here we report that siRNA-mediated knockdown of c-Myc abrogates the increase in cancer cell viability and mammosphere formation by equol, and results in a significant down-regulation of eIF4GI (the major eIF4G isoform), as well as reduces levels of some, but not all, proteins encoded by mRNAs that are translationally stimulated by equol treatment. Knockdown of eIF4GI also markedly reduces an equol-mediated increase in IRES-dependent mRNA translation and the expression of specific oncogenic proteins. However, eIF4GI knockdown did not reciprocally affect c-Myc levels or cell viability. This study therefore implicates c-Myc as a potential regulator of the cancer-promoting effects of equol via up-regulation of eIF4GI and selective initiation of translation on mRNAs that utilize non-canonical initiation, including certain oncogenes.
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Affiliation(s)
- Columba de la Parra
- From the Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936 and Department of Microbiology and Radiation Oncology, NYU Cancer Institute, New York University School of Medicine, New York, New York 10016
| | - Luis D Borrero-Garcia
- From the Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936 and
| | - Ailed Cruz-Collazo
- From the Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936 and
| | - Robert J Schneider
- Department of Microbiology and Radiation Oncology, NYU Cancer Institute, New York University School of Medicine, New York, New York 10016
| | - Suranganie Dharmawardhane
- From the Department of Biochemistry, School of Medicine, University of Puerto Rico, Medical Sciences Campus, San Juan, Puerto Rico 00936 and
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Park MS, Kausar R, Kim MW, Cho SY, Lee YS, Lee MA. Tcf7l1-mediated transcriptional regulation of Krüppel-like factor 4 gene. Anim Cells Syst (Seoul) 2015. [DOI: 10.1080/19768354.2014.991351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022] Open
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Oncogenic KRAS signalling promotes the Wnt/β-catenin pathway through LRP6 in colorectal cancer. Oncogene 2014; 34:4914-27. [PMID: 25500543 PMCID: PMC4687460 DOI: 10.1038/onc.2014.416] [Citation(s) in RCA: 114] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2014] [Revised: 11/04/2014] [Accepted: 11/08/2014] [Indexed: 12/14/2022]
Abstract
Aberrant regulation of the Wnt/β-catenin signaling pathway is one of the major causes of colorectal cancer (CRC). Loss-of-function mutations in APC are commonly found in CRC, leading to inappropriate activation of canonical Wnt signaling. Conversely, gain-of-function mutations in KRAS and BRAF genes are detected in up to 60% of CRCs. Whereas KRAS/mitogen-activated protein kinase (MAPK) and canonical Wnt/β-catenin pathways are critical for intestinal tumorigenesis, mechanisms integrating these two important signaling pathways during CRC development are unknown. Results herein demonstrate that transformation of normal intestinal epithelial cells (IECs) by oncogenic forms of KRAS, BRAF or MEK1 was associated with a marked increase in β-catenin/TCF4 and c-MYC promoter transcriptional activities and mRNA levels of c-Myc, Axin2 and Lef1. Notably, expression of a dominant-negative mutant of T-Cell Factor 4 (ΔNTCF4) severely attenuated IEC transformation induced by oncogenic MEK1 and markedly reduced their tumorigenic and metastatic potential in immunocompromised mice. Interestingly, the Frizzled co-receptor LRP6 was phosphorylated in a MEK-dependent manner in transformed IECs and in human CRC cell lines. Expression of LRP6 mutant in which serine/threonine residues in each particular ProlineProlineProlineSerine/ThreonineProline motif were mutated to alanines (LRP6-5A) significantly reduced β-catenin/TCF4 transcriptional activity. Accordingly, MEK inhibition in human CRC cells significantly diminished β-catenin/TCF4 transcriptional activity and c-MYC mRNA and protein levels without affecting β-catenin expression or stability. Lastly, LRP6 phosphorylation was also increased in human colorectal tumors, including adenomas, in comparison with healthy adjacent normal tissues. Our data indicate that oncogenic activation of KRAS/BRAF/MEK signaling stimulates the canonical Wnt/β-catenin pathway, which in turn promotes intestinal tumor growth and invasion. Moreover, LRP6 phosphorylation by ERK1/2 may provide a unique point of convergence between KRAS/MAPK and Wnt/β-catenin signalings during oncogenesis.
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Hara K, Kageji T, Mizobuchi Y, Kitazato KT, Okazaki T, Fujihara T, Nakajima K, Mure H, Kuwayama K, Hara T, Nagahiro S. Blocking of the interaction between Wnt proteins and their co-receptors contributes to the anti-tumor effects of adenovirus-mediated DKK3 in glioblastoma. Cancer Lett 2014; 356:496-505. [PMID: 25301448 DOI: 10.1016/j.canlet.2014.09.045] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2014] [Revised: 09/19/2014] [Accepted: 09/25/2014] [Indexed: 11/15/2022]
Abstract
The effect of the third member of the Dickkopf family (DKK3) in the Wnt pathway in glioblastoma remains unclear. We first demonstrated the non-specific interaction of Wnt3a and Wnt5a with the receptors LRP6 and ROR2 and the up-regulation of the Wnt pathway in glioblastoma cells. We used an adenovirus vector and found that an increase in DKK3 protein attenuated the expression of Wnt3a, Wnt5a and LRP6, but not of ROR2, and their interaction, thereby affecting both canonical- and non-canonical Wnt downstream cascades. This produced anti-tumor effects in GBM xenograft models. The suppression of Wnt pathways upstream by DKK3 may have promise for the treatment of glioblastoma.
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Affiliation(s)
- Keijiro Hara
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan.
| | - Teruyoshi Kageji
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Yoshifumi Mizobuchi
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Keiko T Kitazato
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Toshiyuki Okazaki
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Toshitaka Fujihara
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Kohei Nakajima
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Hideo Mure
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Kazuyuki Kuwayama
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
| | - Tomoyo Hara
- Faculty of Medicine, The University of Tokushima Graduate School, Tokushima, Japan
| | - Shinji Nagahiro
- Department of Neurosurgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
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