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Bosch PS, Cho B, Axelrod JD. Flamingo participates in multiple models of cell competition. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.09.24.559197. [PMID: 37790459 PMCID: PMC10542155 DOI: 10.1101/2023.09.24.559197] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/05/2023]
Abstract
The growth and survival of cells with different fitness, such as those with a proliferative advantage or a deleterious mutation, is controlled through cell competition. During development, cell competition enables healthy cells to eliminate less fit cells that could jeopardize tissue integrity, and facilitates the elimination of pre-malignant cells by healthy cells as a surveillance mechanism to prevent oncogenesis. Malignant cells also benefit from cell competition to promote their expansion. Despite its ubiquitous presence, the mechanisms governing cell competition, particularly those common to developmental competition and tumorigenesis, are poorly understood. Here, we show that in Drosophila, the planar cell polarity (PCP) protein Flamingo (Fmi) is required by winners to maintain their status during cell competition in malignant tumors to overtake healthy tissue, in early pre-malignant cells when they overproliferate among wildtype cells, in healthy cells when they later eliminate pre-malignant cells, and by supercompetitors as they compete to occupy excessive territory within wildtype tissues. "Would-be" winners that lack Fmi are unable to over-proliferate, and instead become losers. We demonstrate that the role of Fmi in cell competition is independent of PCP, and that it uses a distinct mechanism that may more closely resemble one used in other less well-defined functions of Fmi.
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Affiliation(s)
- Pablo Sanchez Bosch
- Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford CA, 94305, USA
| | - Bomsoo Cho
- Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford CA, 94305, USA
| | - Jeffrey D Axelrod
- Department of Pathology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford CA, 94305, USA
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2
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Zhang J, Zhang S, Dörflein I, Ren X, Pfeffer S, Britzen-Laurent N, Grützmann R, Duan X, Pilarsky C. Impact of CRISPR/Cas9-Mediated CD73 Knockout in Pancreatic Cancer. Cancers (Basel) 2023; 15:4842. [PMID: 37835536 PMCID: PMC10572021 DOI: 10.3390/cancers15194842] [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: 08/18/2023] [Revised: 09/22/2023] [Accepted: 09/26/2023] [Indexed: 10/15/2023] Open
Abstract
Pancreatic cancer is among the cancers with the highest mortality rates. Most of the patients are found to have advanced cancer, losing the chance of surgical treatment, and there is an urgent need to find new treatment methods. Targeted therapy for specific genes that play a key role in cancer is now an important means to improve the survival rate of patients. We determined that CD73 is highly expressed in pancreatic cancer by flow cytometry and qRT-PCR assays combined with bioinformatics techniques. Application of CRISPR/Cas9 technology to knockout CD73 in human and murine cell lines, respectively, revealed that CD73 inactivation inhibited cell growth and migration and induced G1 cell cycle arrest. We also found that CD73 deletion inhibited the ERK/STAT3 pathway and activated the E-cadherin pathway. In addition, a CRISPR/Cas9 protein kinase library screen was performed and identified Pbk, Fastk, Cdk19, Adck5, Trim28, and Pfkp as possible genes regulating CD73.
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Affiliation(s)
- Jinping Zhang
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
- Second Department of General Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068, China;
- Second Department of General Surgery, Third Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710068, China
| | - Shuman Zhang
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
| | - Isabella Dörflein
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
| | - Xiaofan Ren
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
| | - Susanne Pfeffer
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
| | - Nathalie Britzen-Laurent
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
| | - Robert Grützmann
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
| | - Xianglong Duan
- Second Department of General Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068, China;
- Second Department of General Surgery, Third Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710068, China
- Institute of Medical Research, Northwestern Polytechnical University, Xi’an 710072, China
| | - Christian Pilarsky
- Department of Surgery, Universitätsklinikum Erlangen, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany; (J.Z.); (S.Z.); (I.D.); (X.R.); (S.P.); (N.B.-L.); (R.G.)
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Kuburich NA, Sabapathy T, Demestichas BR, Maddela JJ, den Hollander P, Mani SA. Proactive and reactive roles of TGF-β in cancer. Semin Cancer Biol 2023; 95:120-139. [PMID: 37572731 PMCID: PMC10530624 DOI: 10.1016/j.semcancer.2023.08.002] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2023] [Revised: 08/04/2023] [Accepted: 08/05/2023] [Indexed: 08/14/2023]
Abstract
Cancer cells adapt to varying stress conditions to survive through plasticity. Stem cells exhibit a high degree of plasticity, allowing them to generate more stem cells or differentiate them into specialized cell types to contribute to tissue development, growth, and repair. Cancer cells can also exhibit plasticity and acquire properties that enhance their survival. TGF-β is an unrivaled growth factor exploited by cancer cells to gain plasticity. TGF-β-mediated signaling enables carcinoma cells to alter their epithelial and mesenchymal properties through epithelial-mesenchymal plasticity (EMP). However, TGF-β is a multifunctional cytokine; thus, the signaling by TGF-β can be detrimental or beneficial to cancer cells depending on the cellular context. Those cells that overcome the anti-tumor effect of TGF-β can induce epithelial-mesenchymal transition (EMT) to gain EMP benefits. EMP allows cancer cells to alter their cell properties and the tumor immune microenvironment (TIME), facilitating their survival. Due to the significant roles of TGF-β and EMP in carcinoma progression, it is essential to understand how TGF-β enables EMP and how cancer cells exploit this plasticity. This understanding will guide the development of effective TGF-β-targeting therapies that eliminate cancer cell plasticity.
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Affiliation(s)
- Nick A Kuburich
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA; Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA
| | - Thiru Sabapathy
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA; Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA
| | - Breanna R Demestichas
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA; Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA
| | - Joanna Joyce Maddela
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA; Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA
| | - Petra den Hollander
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA; Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA
| | - Sendurai A Mani
- Legorreta Cancer Center, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA; Department of Pathology and Lab Medicine, The Warren Alpert Medical School, Brown University, Providence, RI 02912, USA.
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Indoloquinoline-Mediated Targeted Downregulation of KRAS through Selective Stabilization of the Mid-Promoter G-Quadruplex Structure. Genes (Basel) 2022; 13:genes13081440. [PMID: 36011352 PMCID: PMC9408018 DOI: 10.3390/genes13081440] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/26/2022] [Accepted: 08/10/2022] [Indexed: 11/27/2022] Open
Abstract
KRAS is a well-validated anti-cancer therapeutic target, whose transcriptional downregulation has been demonstrated to be lethal to tumor cells with aberrant KRAS signaling. G-quadruplexes (G4s) are non-canonical nucleic acid structures that mediate central dogmatic events, such as DNA repair, telomere elongation, transcription and splicing events. G4s are attractive drug targets, as they are more globular than B-DNA, enabling more selective gene interactions. Moreover, their genomic prevalence is increased in oncogenic promoters, their formation is increased in human cancers, and they can be modulated with small molecules or targeted nucleic acids. The putative formation of multiple G4s has been described in the literature, but compounds with selectivity among these structures have not yet been able to distinguish between the biological contribution of the predominant structures. Using cell free screening techniques, synthesis of novel indoloquinoline compounds and cellular models of KRAS-dependent cancer cells, we describe compounds that choose between KRAS promoter G4near and G4mid, correlate compound cytotoxic activity with KRAS regulation, and highlight G4mid as the lead molecular non-canonical structure for further targeting efforts.
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Shai A, Galouk E, Miari R, Tareef H, Sammar M, Zeidan M, Rayan A, Falah M. Inhibiting mutant KRAS G12D gene expression using novel peptide nucleic acid‑based antisense: A potential new drug candidate for pancreatic cancer. Oncol Lett 2022; 23:130. [PMID: 35251350 PMCID: PMC8895471 DOI: 10.3892/ol.2022.13250] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2021] [Accepted: 02/02/2022] [Indexed: 11/22/2022] Open
Abstract
KRAS mutations, which are the main cause of the pathogenesis of lethal pancreatic adenocarcinomas, impair the functioning of the GTPase subunit, thus rendering it constitutively active and signaling intracellular pathways that end with cell transformation. In the present study, the AsPC-1 cell line, which has a G12D-mutated KRAS gene sequence, was utilized as a cellular model to test peptide nucleic acid-based antisense technology. The use of peptide nucleic acids (PNAs) that are built to exhibit improved hybridization specificity and have an affinity for complementary RNA and DNA sequences, as well as a simple chemical structure and high biological stability that affords resistance to nucleases and proteases, enabled targeting of the KRAS-mutated gene to inhibit its expression at the translation level. Because PNA-based antisense molecules should be capable of binding to KRAS mRNA sequences, PNAs were utilized to target the mRNA of the mutated KRAS gene, a strategy that could lead to the development of a novel drug for pancreatic cancer. Moreover, it was demonstrated that introducing new PNA to cells inhibited the growth of cancer cells and induced apoptotic death and, notably, that it can inhibit G12D-mutated KRAS gene expression, as demonstrated by RT-PCR and western blotting. Altogether, these data strongly suggest that the use of PNA-based antisense agents is an attractive therapeutic approach to treating KRAS-driven cancers and may lead to the development of novel drugs that target the expression of other mutated genes.
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Affiliation(s)
- Ayelet Shai
- Oncology Department, Galilee Medical Center, Nahariya 2210001, Israel
| | - Evleen Galouk
- Oncology Department, Galilee Medical Center, Nahariya 2210001, Israel
| | - Reem Miari
- Oncology Department, Galilee Medical Center, Nahariya 2210001, Israel
| | - Hala Tareef
- Oncology Department, Galilee Medical Center, Nahariya 2210001, Israel
| | - Marei Sammar
- Ephraim Katzir Department of Biotechnology Engineering, ORT Braude College, Karmiel 2161002, Israel
| | - Mouhammad Zeidan
- Molecular Genetics and Virology Laboratory, Al‑Qasemi Center of Research Excellence, Baka EL‑Garbiah 30100, Israel
| | - Anwar Rayan
- Faculty of Science, Al‑Qasemi Academic College, Baka EL‑Garbiah 30100, Israel
| | - Mizied Falah
- Oncology Department, Galilee Medical Center, Nahariya 2210001, Israel
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Gunda V, Genaro-Mattos TC, Kaushal JB, Chirravuri-Venkata R, Natarajan G, Mallya K, Grandgenett PM, Mirnics K, Batra SK, Korade Z, Rachagani S. Ubiquitous Aberration in Cholesterol Metabolism across Pancreatic Ductal Adenocarcinoma. Metabolites 2022; 12:metabo12010047. [PMID: 35050168 PMCID: PMC8779872 DOI: 10.3390/metabo12010047] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 12/24/2021] [Accepted: 12/25/2021] [Indexed: 12/12/2022] Open
Abstract
Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens.
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Affiliation(s)
- Venugopal Gunda
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
| | - Thiago C. Genaro-Mattos
- Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE 68106, USA; (T.C.G.-M.); (K.M.)
| | - Jyoti B. Kaushal
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
| | - Ramakanth Chirravuri-Venkata
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
| | - Gopalakrishnan Natarajan
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
| | - Kavita Mallya
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
| | - Paul M. Grandgenett
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA;
| | - Karoly Mirnics
- Munroe-Meyer Institute for Genetics and Rehabilitation, University of Nebraska Medical Center, Omaha, NE 68106, USA; (T.C.G.-M.); (K.M.)
| | - Surinder K. Batra
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA
- Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, USA
| | - Zeljka Korade
- Department of Pediatrics, Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, USA;
| | - Satyanarayana Rachagani
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198, USA; (V.G.); (J.B.K.); (R.C.-V.); (G.N.); (K.M.); (S.K.B.)
- Correspondence: ; Tel.: +1-(402)559-3312; Fax: +1-(402)559-6650
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Ju J, Wismans LV, Mustafa DAM, Reinders MJT, van Eijck CHJ, Stubbs AP, Li Y. Robust deep learning model for prognostic stratification of pancreatic ductal adenocarcinoma patients. iScience 2021; 24:103415. [PMID: 34901786 PMCID: PMC8637475 DOI: 10.1016/j.isci.2021.103415] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2021] [Revised: 09/27/2021] [Accepted: 11/05/2021] [Indexed: 02/07/2023] Open
Abstract
A major challenge for treating patients with pancreatic ductal adenocarcinoma (PDAC) is the unpredictability of their prognoses due to high heterogeneity. We present Multi-Omics DEep Learning for Prognosis-correlated subtyping (MODEL-P) to identify PDAC subtypes and to predict prognoses of new patients. MODEL-P was trained on autoencoder integrated multi-omics of 146 patients with PDAC together with their survival outcome. Using MODEL-P, we identified two PDAC subtypes with distinct survival outcomes (median survival 10.1 and 22.7 months, respectively, log rank p = 1 × 10−6), which correspond to DNA damage repair and immune response. We rigorously validated MODEL-P by stratifying patients in five independent datasets into these two survival groups and achieved significant survival difference, which is superior to current practice and other subtyping schemas. We believe the subtype-specific signatures would facilitate PDAC pathogenesis discovery, and MODEL-P can provide clinicians the prognoses information in the treatment decision-making to better gauge the benefits versus the risks.
We developed DL-based MODEL-P to identify prognosis-correlated PDAC subtypes The identified subtypes related to DNA damage repair and immune response processes MODEL-P stratified patients from independent datasets into distinct survival groups MODEL-P could be used in clinics to aid treatment decision-making
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Affiliation(s)
- Jie Ju
- Department of Pathology & Clinical Bioinformatics, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands
| | - Leonoor V Wismans
- Department of Surgery, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands
| | - Dana A M Mustafa
- Department of Pathology & Clinical Bioinformatics, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands
| | - Marcel J T Reinders
- The Delft Bioinformatics Lab, Delft University of Technology, Rotterdam, the Netherlands
| | - Casper H J van Eijck
- Department of Surgery, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands
| | - Andrew P Stubbs
- Department of Pathology & Clinical Bioinformatics, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands
| | - Yunlei Li
- Department of Pathology & Clinical Bioinformatics, Erasmus MC Cancer Institute, University Medical Center Rotterdam, Rotterdam, the Netherlands
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Krishnan T, Roberts-Thomson R, Broadbridge V, Price T. Targeting Mutated KRAS Genes to Treat Solid Tumours. Mol Diagn Ther 2021; 26:39-49. [PMID: 34914038 DOI: 10.1007/s40291-021-00564-0] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/17/2021] [Indexed: 02/06/2023]
Abstract
Kirsten rat sarcoma (KRAS) is one of the most frequently mutated oncogenes in solid tumours. It encodes an important signalling pathway that drives cellular proliferation and growth. It is frequently mutated in aggressive advanced solid tumours, particularly colorectal, lung and pancreatic cancer. Since the first mutated KRAS was discovered in the 1980s, decades of research to develop targeted inhibitors of mutant KRAS have fallen short of the task, until recently. Multiple agents are now in clinical trials, including specific mutant KRAS inhibitors, pan-KRAS inhibitors, therapeutic vaccines and other targeted inhibitors. Mutant-specific KRAS G12C inhibitors are the most advanced, with two inhibitors, adagrasib and sotorasib, achieving approval in 2021 for the second-line treatment of patients with KRAS G12C mutant lung cancer. In this review, we summarise the importance of mutant KRAS in solid tumours, prior attempts at inhibiting mutant KRAS, and the current promising targeted agents being investigated in clinical trials, along with future challenges.
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Affiliation(s)
- Tharani Krishnan
- Medical Oncology Department, Calvary North Adelaide Hospital, North Adelaide, SA, Australia
| | - Rachel Roberts-Thomson
- Medical Oncology Department, Calvary North Adelaide Hospital, North Adelaide, SA, Australia.,Medical Oncology Department, The Queen Elizabeth Hospital, Woodville South, SA, Australia
| | - Vy Broadbridge
- Medical Oncology Department, Calvary North Adelaide Hospital, North Adelaide, SA, Australia.,Medical Oncology Department, The Queen Elizabeth Hospital, Woodville South, SA, Australia
| | - Timothy Price
- Medical Oncology Department, Calvary North Adelaide Hospital, North Adelaide, SA, Australia. .,Medical Oncology Department, The Queen Elizabeth Hospital, Woodville South, SA, Australia.
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Hossain MM, Nakayama K, Shanta K, Razia S, Ishikawa M, Ishibashi T, Yamashita H, Sato S, Iida K, Kanno K, Ishikawa N, Kiyono T, Kyo S. Establishment of a Novel In Vitro Model of Endometriosis with Oncogenic KRAS and PIK3CA Mutations for Understanding the Underlying Biology and Molecular Pathogenesis. Cancers (Basel) 2021; 13:cancers13133174. [PMID: 34202354 PMCID: PMC8269352 DOI: 10.3390/cancers13133174] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Revised: 06/17/2021] [Accepted: 06/22/2021] [Indexed: 12/26/2022] Open
Abstract
Simple Summary Endometriosis is a common gynecological condition that causes pelvic pain and infertility. Despite having normal histological features, several cells bear cancer-associated somatic mutations that result in local tissue invasion but rarely metastasize. Several cancer-associated genes, such as KRAS and PIK3CA, are frequently mutated in the endometriotic epithelium. However, the functional behavior and molecular pathogenesis of this disorder remain unclear. In this study, we developed an immortalized endometriotic epithelial cell line with mutations in KRAS and PIK3CA, which are genes associated with aggressive behaviors, such as increased cell migration, invasion, and proliferation. Through microarray analysis, the KRAS- and PIK3CA-specific gene signatures were identified; LOX and PTX3 were found to be responsible for this metastatic behavior. Knockdown of these two genes by siRNA markedly reduced the metastatic ability of the cells. Our findings suggest that inhibition of LOX and PTX3 may be an alternative therapeutic strategy to reduce the incidence of endometriosis. Abstract Endometriosis-harboring cancer-associated somatic mutations of PIK3CA and KRAS provides new opportunities for studying the multistep processes responsible for the functional and molecular changes in this disease. We aimed to establish a novel in vitro endometriosis model to clarify the functional behavior and molecular pathogenesis of this disorder. Immortalized HMOsisEC10 human ovarian endometriotic epithelial cell line was used in which KRAS and PIK3CA mutations were introduced. Migration, invasion, proliferation, and microarray analyses were performed using KRAS and PIK3CA mutant cell lines. In vitro assays showed that migration, invasion, and proliferation were significantly increased in KRAS and PIK3CA mutant cell lines, indicating that these mutations played causative roles in the aggressive behavior of endometriosis. Microarray analysis identified a cluster of gene signatures; among them, two significantly upregulated cancer-related genes, lysyl oxidase (LOX) and pentraxin3 (PTX3), were associated with cell proliferation, invasion, and migration capabilities. Furthermore, siRNA knockdown of the two genes markedly reduced the metastatic ability of the cells. These results suggest that endometriosis with KRAS or PIK3CA mutations can significantly enhance cell migration, invasion, and proliferation by upregulating LOX and PTX3. We propose that LOX and PTX3 silencing using small molecules could be an alternative therapeutic regimen for severe endometriosis.
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Affiliation(s)
- Mohammad Mahmud Hossain
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Kentaro Nakayama
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
- Correspondence: (K.N.); (T.K.)
| | - Kamrunnahar Shanta
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Sultana Razia
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Masako Ishikawa
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Tomoka Ishibashi
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Hitomi Yamashita
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Seiya Sato
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Kouji Iida
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Kosuke Kanno
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
| | - Noriyoshi Ishikawa
- Department of Organ Pathology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan;
| | - Tohru Kiyono
- Project for Prevention of HPV-Related Cancer, Exploratory Oncology Research and Clinical Trial Center (EPOC), National Cancer Center, Kashiwa 277-8577, Japan
- Correspondence: (K.N.); (T.K.)
| | - Satoru Kyo
- Department of Obstetrics and Gynecology, Shimane University Faculty of Medicine, Izumo 693-8501, Japan; (M.M.H.); (K.S.); (S.R.); (M.I.); (T.I.); (H.Y.); (S.S.); (K.I.); (K.K.); (S.K.)
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10
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Wu N, Huang Y, Liu F, Xu X, Liu B, Wei J. KRAS gene status in gastric signet-ring cell carcinoma patients and acts as biomarker of MEK inhibitor. J Gastrointest Oncol 2021; 12:1020-1030. [PMID: 34295553 DOI: 10.21037/jgo-20-617] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/27/2020] [Accepted: 03/28/2021] [Indexed: 12/12/2022] Open
Abstract
Background Signet-ring cell carcinoma (SRCC) is a specific subtype of stomach cancer with unique epidemiology. Here, we sought to explore the role of KRAS in SRCC. Methods KRAS status was studied both in The Cancer Genome Atlas (TCGA) and internal cohorts. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed in formalin-fixed and paraffin-embedded (FFPE) samples. We explored patients' survival and clinicopathological characteristics in terms of KRAS mutation and expression. We also explored KRAS status and drug response curve of MEK/mTOR inhibitors in SRCC cell lines. Results Patients with KRAS mutations and copy number variation (CNV) showed higher mRNA level compared to non-mutant cases (P=0.003 and P<0.001). In internal cohort, 15 samples harbored KRAS mutations. Survival analysis showed that these patients had significantly lower overall survival (OS) (P=0.048). We further analyzed 75 patients with sufficient FFPE samples. Eight patients showed KRAS mutations and one patient showed KRAS amplification. The median OS was 12.5 months for patients with KRAS mutation, and 19.5 months for patients without KRAS mutation (P=0.005). Positive expression of KRAS as shown by IHC was detected in majority of SRCC samples, which was higher than our intestinal cohort (28% vs. 12.6%, P=0.033). We further explored the correlation between KRAS status and drug sensitivity in 4 SRCC cell lines. SNU601 and SNU668, which harbored KRAS mutation, were hypersensitive to MEK and mTOR inhibitors than KRAS wide type cell lines KATO-III and NUGC-4. Conclusions Our findings demonstrate that KRAS gene plays an important role in SRCC and reveals therapeutic potential of targeting tumors by inhibiting MEK and mTOR pathways.
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Affiliation(s)
- Nandie Wu
- The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China
| | - Ying Huang
- The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China
| | - Fangcen Liu
- Department of Pathology, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China
| | - Xingyun Xu
- Department of Pathology, Drum Tower Hospital, Medical School of Nanjing University, Nanjing, China
| | - Baorui Liu
- The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China
| | - Jia Wei
- The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University & Clinical Cancer Institute of Nanjing University, Nanjing, China
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11
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Ma H, Song B, Guo S, Li G, Jin G. Identification of germline and somatic mutations in pancreatic adenosquamous carcinoma using whole exome sequencing. Cancer Biomark 2020; 27:389-397. [PMID: 31958074 DOI: 10.3233/cbm-190236] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
OBJECTIVES Pancreatic cancer is one of the most lethal malignancies worldwide. Pancreatic adenosquamous carcinoma (PASC) is a rare histological type of pancreatic carcinoma with a poor prognosis. The median survival time after diagnosis is less than one year. It is believed that the pathogenesis of PASC is different from pancreatic adenocarcinoma. In this study, we tried to reveal the intrinsic gene mutations associated with PASC through whole exome sequencing. METHODS Both cancerous and paracancerous tissues were collected from 12 pathologically diagnosed PASC patients. Their clinical characteristics were collected, and patient survival information was obtained through follow-up. The correlations between the mutations and clinical characteristics were analysed. RESULTS Germline mutations were identified in MAP3K1 (9 cases), PDE4DIP (7), BCR (7), ALK (6), USP6 (5), AR (4), HLA-A (4), SPEN (4), KMT2D (3), NUTM2B (3), ZFHX3 (3), and MN1 (3), while somatic mutations were found in TP53 (5), KRAS (3), HRNR (3), and OBSCN (3). Peripheral tissue invasion was associated with somatic mutations in KRAS (P= 0.0339). Additionally, there were significant correlations between lymphatic metastasis and germline mutations in USP6 (P= 0.0228) and somatic mutations in OBSCN and HRNR (P= 0.0339). CONCLUSION In conclusion, susceptibility genes including MAP3K1, PDE4DIP, and BCR are frequently found to be mutated in the germlines of PASC patients. Somatic mutations in KRAS, OBSCN, and HRNR and germline mutations in USP6 are related to tumour invasion and metastasis, reinforcing the necessity of translating these potential biomarkers into clinical practice.
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Affiliation(s)
- Hongyun Ma
- Department of Pancreatic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China
| | - Bin Song
- Department of Pancreatic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China
| | - Shiwei Guo
- Department of Pancreatic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China
| | - Gang Li
- Department of Pancreatic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China
| | - Gang Jin
- Department of Pancreatic Surgery, Changhai Hospital, Second Military Medical University, Shanghai, China.,Shanghai Institute for Advanced Communication and Data Science, Shanghai University, Shanghai, China
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12
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Mallya K, Haridas D, Seshacharyulu P, Pothuraju R, Junker WM, Krishn SR, Muniyan S, Vengoji R, Batra SK, Rachagani S. Acinar transformed ductal cells exhibit differential mucin expression in a tamoxifen-induced pancreatic ductal adenocarcinoma mouse model. Biol Open 2020; 9:bio052878. [PMID: 32709695 PMCID: PMC7502593 DOI: 10.1242/bio.052878] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2020] [Accepted: 07/16/2020] [Indexed: 11/24/2022] Open
Abstract
Pancreatic cancer (PC) is acquired postnatally; to mimic this scenario, we developed an inducible KrasG12D; Ptf1a-CreER™ (iKC) mouse model, in which Kras is activated postnatally at week 16 upon tamoxifen (TAM) administration. Upon TAM treatment, iKC mice develop pancreatic intraepithelial neoplasia (PanIN) lesions and PC with metastasis at the fourth and fortieth weeks, respectively, and exhibited acinar-to-ductal metaplasia (ADM) and transdifferentiation. Kras activation upregulated the transcription factors Ncoa3, p-cJun and FoxM1, which in turn upregulated expression of transmembrane mucins (Muc1, Muc4 and Muc16) and secretory mucin (Muc5Ac). Interestingly, knockdown of KrasG12D in multiple PC cell lines resulted in downregulation of MUC1, MUC4, MUC5AC and MUC16. In addition, iKC mice exhibited ADM and transdifferentiation. Our results show that the iKC mouse more closely mimics human PC development and can be used to investigate pancreatic ductal adenocarcinoma (PDAC) biomarkers, early onset of PDAC, and ADM. The iKC model can also be used for preclinical strategies such as targeting mucin axis alone or in combination with neo-adjuvant, immunotherapeutic approaches and to monitor chemotherapy response.
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Affiliation(s)
- Kavita Mallya
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Dhanya Haridas
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Parthasarathy Seshacharyulu
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Ramesh Pothuraju
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Wade M Junker
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
- Sanguine Diagnostics and Therapeutics, Inc., Omaha, NE 68106-1423, USA
| | - Shiv Ram Krishn
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Sakthivel Muniyan
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Raghupathy Vengoji
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
| | - Surinder K Batra
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
- Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68106, USA
- Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198-5950, USA
| | - Satyanarayana Rachagani
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE 68198-5870, USA
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13
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Awaji M, Saxena S, Wu L, Prajapati DR, Purohit A, Varney ML, Kumar S, Rachagani S, Ly QP, Jain M, Batra SK, Singh RK. CXCR2 signaling promotes secretory cancer-associated fibroblasts in pancreatic ductal adenocarcinoma. FASEB J 2020; 34:9405-9418. [PMID: 32453916 PMCID: PMC7501205 DOI: 10.1096/fj.201902990r] [Citation(s) in RCA: 54] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2019] [Revised: 04/21/2020] [Accepted: 05/04/2020] [Indexed: 12/21/2022]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging malignancies. Desmoplasia and tumor-supporting inflammation are hallmarks of PDAC. The tumor microenvironment contributes significantly to tumor progression and spread. Cancer-associated fibroblasts (CAFs) facilitate therapy resistance and metastasis. Recent reports emphasized the concurrence of multiple subtypes of CAFs with diverse roles, fibrogenic, and secretory. C-X-C motif chemokine receptor 2 (CXCR2) is a chemokine receptor known for its role during inflammation and its adverse role in PDAC. Oncogenic Kras upregulates CXCR2 and its ligands and, thus, contribute to tumor proliferation and immunosuppression. CXCR2 deletion in a PDAC syngeneic mouse model produced increased fibrosis revealing a potential undescribed role of CXCR2 in CAFs. In this study, we demonstrate that the oncogenic Kras-CXCR2 axis regulates the CAFs function in PDAC and contributes to CAFs heterogeneity. We observed that oncogenic Kras and CXCR2 signaling alter CAFs, producing a secretory CAF phenotype with low fibrogenic features; and increased secretion of pro-tumor cytokines and CXCR2 ligands, utilizing the NF-κB activity. Finally, using syngeneic mouse models, we demonstrate that oncogenic Kras is associated with secretory CAFs and that CXCR2 inhibition promotes activation of fibrotic cells (myofibroblasts) and impact tumors in a mutation-dependent manner.
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MESH Headings
- Animals
- Apoptosis
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Cancer-Associated Fibroblasts/metabolism
- Cancer-Associated Fibroblasts/pathology
- Carcinoma, Pancreatic Ductal/genetics
- Carcinoma, Pancreatic Ductal/metabolism
- Carcinoma, Pancreatic Ductal/pathology
- Cell Proliferation
- Gene Expression Regulation, Neoplastic
- Mice
- Mice, Knockout
- Mutation
- Pancreatic Neoplasms/genetics
- Pancreatic Neoplasms/metabolism
- Pancreatic Neoplasms/pathology
- Receptors, Interleukin-8B/genetics
- Receptors, Interleukin-8B/metabolism
- Signal Transduction
- Tumor Cells, Cultured
- Tumor Microenvironment
- Pancreatic Neoplasms
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Affiliation(s)
- Mohammad Awaji
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
- Department of Pathology and Laboratory Medicine, King Fahad Specialist Hospital, Dammam, Saudi Arabia 31444
| | - Sugandha Saxena
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
| | - Lingyun Wu
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
| | - Dipakkumar R. Prajapati
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
| | - Abhilasha Purohit
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
| | - Michelle L. Varney
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
| | - Sushil Kumar
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha NE 68198-5870
| | - Satyanarayana Rachagani
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha NE 68198-5870
| | - Quan P. Ly
- Department of Surgical Oncology, University of Nebraska Medical Center, Omaha NE 68198-6880
| | - Maneesh Jain
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha NE 68198-5870
| | - Surinder K. Batra
- Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha NE 68198-5870
| | - Rakesh K. Singh
- Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha NE 68198-5900
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14
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Prognostic Value of Poorly Differentiated Clusters in Liver Metastatic Lesions of Colorectal Carcinoma. Am J Surg Pathol 2020; 43:1341-1348. [PMID: 31318710 DOI: 10.1097/pas.0000000000001329] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Colorectal liver metastasis (CRLM) is the most common pattern of metastases or recurrence in colorectal carcinoma; however, no robust pathologic prognostic factors have been identified. This study aimed to verify the prognostic value of poorly differentiated clusters (PDC) in liver metastatic lesions and to clarify the correlation between PDC in liver metastatic lesions (PDC) and the primary tumor histology. Consecutive patients who underwent resection for CRLM were pathologically reviewed. PDC was defined as cancer clusters comprising ≥5 cancer cells and lacking glandular formation and was quantifiably graded as G1 (<5 clusters), G2 (5 to 9 clusters), and G3 (≥10 clusters) based on the highest number of clusters observed under ×20 magnification. The cohort comprised 204 patients. PDC was classified as G1, G2, and G3 for 68, 69, and 67 patients, respectively, and it was significantly associated with PDC grade in the primary tumor (P<0.001). Among the potential prognostic factors, tumor budding in the primary tumor, PDC in the primary tumor, the number of liver metastases, extrahepatic metastasis, and PDC significantly influenced overall survival (OS) after CRLM resection. According to the PDC grade, the 5-year OS rates were 68.9%, 48.3%, and 39.5% for G1, G2, and G3 (P<0.001), respectively. Multivariate analysis for OS showed that PDC grade, tumor budding in the primary tumor, the number of liver metastasis and extrahepatic metastasis were independent prognostic factors. In conclusion, there is a correlation in the PDC grade between the primary tumor and liver metastatic lesion, and PDC grade could be a promising new prognostic factor after CRLM resection.
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15
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Overgaard NH, Fan TM, Schachtschneider KM, Principe DR, Schook LB, Jungersen G. Of Mice, Dogs, Pigs, and Men: Choosing the Appropriate Model for Immuno-Oncology Research. ILAR J 2019; 59:247-262. [PMID: 30476148 DOI: 10.1093/ilar/ily014] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Revised: 07/30/2018] [Indexed: 02/06/2023] Open
Abstract
The immune system plays dual roles in response to cancer. The host immune system protects against tumor formation via immunosurveillance; however, recognition of the tumor by immune cells also induces sculpting mechanisms leading to a Darwinian selection of tumor cell variants with reduced immunogenicity. Cancer immunoediting is the concept used to describe the complex interplay between tumor cells and the immune system. This concept, commonly referred to as the three E's, is encompassed by 3 distinct phases of elimination, equilibrium, and escape. Despite impressive results in the clinic, cancer immunotherapy still has room for improvement as many patients remain unresponsive to therapy. Moreover, many of the preclinical results obtained in the widely used mouse models of cancer are lost in translation to human patients. To improve the success rate of immuno-oncology research and preclinical testing of immune-based anticancer therapies, using alternative animal models more closely related to humans is a promising approach. Here, we describe 2 of the major alternative model systems: canine (spontaneous) and porcine (experimental) cancer models. Although dogs display a high rate of spontaneous tumor formation, an increased number of genetically modified porcine models exist. We suggest that the optimal immuno-oncology model may depend on the stage of cancer immunoediting in question. In particular, the spontaneous canine tumor models provide a unique platform for evaluating therapies aimed at the escape phase of cancer, while genetically engineered swine allow for elucidation of tumor-immune cell interactions especially during the phases of elimination and equilibrium.
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Affiliation(s)
- Nana H Overgaard
- Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs Lyngby, Denmark
| | - Timothy M Fan
- Department of Veterinary Clinical Medicine, University of Illinois, Urbana-Champaign, Illinois
| | | | - Daniel R Principe
- Medical Scientist Training Program, University of Illinois College of Medicine, Chicago, Illinois
| | - Lawrence B Schook
- Department of Radiology, University of Illinois, Chicago, Illinois.,Department of Animal Sciences, University of Illinois, Urbana-Champaign, Illinois
| | - Gregers Jungersen
- Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark
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16
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Hu Q, Myers M, Fang W, Yao M, Brummer G, Hawj J, Smart C, Berkland C, Cheng N. Role of ALDH1A1 and HTRA2 expression in CCL2/CCR2-mediated breast cancer cell growth and invasion. Biol Open 2019; 8:bio.040873. [PMID: 31208996 PMCID: PMC6679398 DOI: 10.1242/bio.040873] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Chemokines mediate immune cell trafficking during tissue development, wound healing and infection. The chemokine CCL2 is best known to regulate macrophage recruitment during wound healing, infection and inflammatory diseases. While the importance of CCL2/CCR2 signaling in macrophages during cancer progression is well documented, we recently showed that CCL2-mediated breast cancer progression depends on CCR2 expression in carcinoma cells. Using 3D Matrigel: Collagen cultures of SUM225 and DCIS.com breast cancer cells, this study characterized the mechanisms of CCL2/CCR2 signaling in cell growth and invasion. SUM225 cells, which expressed lower levels of CCR2 than DCIS.com cells, formed symmetrical spheroids in Matrigel: Collagen, and were not responsive to CCL2 treatment. DCIS.com cells formed asymmetric cell clusters in Matrigel: Collagen. CCL2 treatment increased growth, decreased expression of E-cadherin and increased TWIST1 expression. CCR2 overexpression in SUM225 cells increased responsiveness to CCL2 treatment, enhancing growth and invasion. These phenotypes corresponded to increased expression of Aldehyde Dehydrogenase 1A1 (ALDH1A1) and decreased expression of the mitochondrial serine protease HTRA2. CCR2 deficiency in DCIS.com cells inhibited CCL2-mediated growth and invasion, corresponding to decreased ALDH1A1 expression and increased HTRA2 expression. ALDH1A1 and HTRA2 expression were modulated in CCR2-deficient and CCR2-overexpressing cell lines. We found that ALDH1A1 and HTRA2 regulates CCR2-mediated breast cancer cell growth and cellular invasion in a CCL2/CCR2 context-dependent manner. These data provide novel insight on the mechanisms of chemokine signaling in breast cancer cell growth and invasion, with important implications on targeted therapeutics for anti-cancer treatment. This article has an associated First Person interview with the first author of the paper. Summary: Chemokines are known to regulate immune cell recruitment during inflammation. This report characterizes novel molecular mechanisms through which CCL2/CCR2 chemokine signaling in breast cancer cells regulates growth and invasion.
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Affiliation(s)
- Qingting Hu
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Megan Myers
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Wei Fang
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Min Yao
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Gage Brummer
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Justin Hawj
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Curtis Smart
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA
| | - Cory Berkland
- Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS 66047, USA
| | - Nikki Cheng
- Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS 66160, USA .,Department of Cancer Biology, University of Kansas Medical Center, Kansas City, KS 66160, USA
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17
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Garziera M, Roncato R, Montico M, De Mattia E, Gagno S, Poletto E, Scalone S, Canzonieri V, Giorda G, Sorio R, Cecchin E, Toffoli G. New Challenges in Tumor Mutation Heterogeneity in Advanced Ovarian Cancer by a Targeted Next-Generation Sequencing (NGS) Approach. Cells 2019; 8:cells8060584. [PMID: 31197119 PMCID: PMC6627128 DOI: 10.3390/cells8060584] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Revised: 06/06/2019] [Accepted: 06/13/2019] [Indexed: 12/12/2022] Open
Abstract
Next-generation sequencing (NGS) technology has advanced knowledge of the genomic landscape of ovarian cancer, leading to an innovative molecular classification of the disease. However, patient survival and response to platinum-based treatments are still not predictable based on the tumor genetic profile. This retrospective study characterized the repertoire of somatic mutations in advanced ovarian cancer to identify tumor genetic markers predictive of platinum chemo-resistance and prognosis. Using targeted NGS, 79 primary advanced (III-IV stage, tumor grade G2-3) ovarian cancer tumors, including 64 high-grade serous ovarian cancers (HGSOCs), were screened with a 26 cancer-genes panel. Patients, enrolled between 1995 and 2011, underwent primary debulking surgery (PDS) with optimal residual disease (RD < 1 cm) and platinum-based chemotherapy as first-line treatment. We found a heterogeneous mutational landscape in some uncommon ovarian histotypes and in HGSOC tumor samples with relevance in predicting platinum sensitivity. In particular, we identified a poor prognostic signature in patients with HGSOC harboring concurrent mutations in two driver actionable genes of the panel. The tumor heterogeneity described, sheds light on the translational potential of targeted NGS approach for the identification of subgroups of patients with distinct therapeutic vulnerabilities, that are modulated by the specific mutational profile expressed by the ovarian tumor.
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Affiliation(s)
- Marica Garziera
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Rossana Roncato
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Marcella Montico
- Scientific Directorate, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Elena De Mattia
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Sara Gagno
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Elena Poletto
- Medical Oncology, "Santa Maria della Misericordia" University Hospital, ASUIUD, 33100 Udine, Italy.
| | - Simona Scalone
- Medical Oncology Unit C, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Vincenzo Canzonieri
- Pathology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
- Department of Medical, Surgical and Health Sciences, University of Trieste, 34127 Trieste, Italy.
| | - Giorgio Giorda
- Gynecological Oncology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Roberto Sorio
- Medical Oncology Unit C, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Erika Cecchin
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
| | - Giuseppe Toffoli
- Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico (CRO), IRCCS, 33081 Aviano, Italy.
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18
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Liu Y, Feng W, Gu S, Wang H, Zhang Y, Chen W, Xu W, Lin C, Gong A, Xu M. The UCA1/KRAS axis promotes human pancreatic ductal adenocarcinoma stem cell properties and tumor growth. Am J Cancer Res 2019; 9:496-510. [PMID: 30949406 PMCID: PMC6448060] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Accepted: 01/28/2019] [Indexed: 06/09/2023] Open
Abstract
Emerging evidence indicates that the long noncoding RNA UCA1 is upregulated in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC), and plays a critical role in various complex biological processes. However, the functional roles of UCA1 in PDAC remain to be clarified. In the current study, we showed that UCA1 significantly promoted cell proliferation and tumor growth both in vitro and in vivo, and enhanced stemness maintenance of PDAC cell lines. Moreover, we found that UCA1 overexpression increased the activity and expression of oncogenic KRAS. Mechanistically, upregulated UCA1 increased phospho-KRAS protein levels by interacting with hnRNPA2B1, and KRAS facilitated high cytoplasmic accumulation of hnRNPA2B1. Additionally, we identified that UCA1 functioned as a competing endogenous RNA (ceRNA) to increase the expression of KRAS via sponging miR-590-3p, and in turn, KRAS promoted UCA1 expression. Collectively, these findings suggest that the UCA1-KRAS axis plays a crucial role in PDAC progression and that UCA1 may serve as a target for new PDAC therapies.
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Affiliation(s)
- Yawen Liu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Wen Feng
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
- Department of Gastroenterology, Songjiang Hospital Affiliated Shanghai First People’s Hospital, Shanghai Jiao Tong UniversityShanghai 201600, China
| | - Shumin Gu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Huizhi Wang
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Youli Zhang
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Wei Chen
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Wei Xu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Chen Lin
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
| | - Aihua Gong
- Department of Cell Biology, School of Medicine, Jiangsu UniversityZhenjiang 212003, Jiangsu Province, China
| | - Min Xu
- Department of Gastroenterology, Affiliated Hospital of Jiangsu University, Jiangsu UniversityZhenjiang 212001, Jiangsu Province, China
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19
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Sun H, Huang D, Liu G, Jian F, Zhu J, Zhang L. SIRT4 acts as a tumor suppressor in gastric cancer by inhibiting cell proliferation, migration, and invasion. Onco Targets Ther 2018; 11:3959-3968. [PMID: 30022839 PMCID: PMC6044351 DOI: 10.2147/ott.s156143] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Background Previous study has proven that SIRT4 is downregulated in gastric cancer (GC), but the role of SIRT4 has not been clearly understood. The aim of our work was to explore in detail the function and mechanism of SIRT4 in GC. Methods A total of 86 pairs of GC tumor tissues and adjacent normal tissues were collected, and quantitative real-time polymerase chain reaction and Western blotting analyses were used to determine the expression of SIRT4. Results Our study revealed that the expression of SIRT4 was downregulated in GC tissues and cells. In addition, the low expression of SIRT4 was negatively correlated with tumor size, pathological grade, and lymph node metastasis, which predicted a poor prognosis. Multiple functional experiments, including Cell Counting Kit-8 assay as well as colony formation assay, demonstrated SIRT4 suppressed cell proliferation. Moreover, we found epithelial-mesenchymal transition was regulated by SIRT4, thereby regulating cell migration and invasion. Conclusion Overall, our findings show that SIRT4 serves as a tumor suppressor in GC and might act as a novel biomarker and a therapeutic target of GC.
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Affiliation(s)
- Hongjie Sun
- Department of General Surgery, Changyi People's Hospital, Changyi, Shandong, People's Republic of China
| | - Dongli Huang
- Department of General Surgery, Changyi People's Hospital, Changyi, Shandong, People's Republic of China
| | - Guozheng Liu
- Department of General Surgery, Changyi People's Hospital, Changyi, Shandong, People's Republic of China
| | - Fengguo Jian
- Department of General Surgery, Changyi People's Hospital, Changyi, Shandong, People's Republic of China
| | - Jianfang Zhu
- Department of General Surgery, Changyi People's Hospital, Changyi, Shandong, People's Republic of China
| | - Lixia Zhang
- Department of Nuclear Medicine, Zhejiang Provincial Hospital of Traditional Chinese Medicine (The First Affiliated Hospital of Zhejiang Chinese Medical University), Hangzhou, Zhejiang, People's Republic of China,
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20
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Jahan R, Macha MA, Rachagani S, Das S, Smith LM, Kaur S, Batra SK. Axed MUC4 (MUC4/X) aggravates pancreatic malignant phenotype by activating integrin-β1/FAK/ERK pathway. Biochim Biophys Acta Mol Basis Dis 2018; 1864:2538-2549. [PMID: 29777904 DOI: 10.1016/j.bbadis.2018.05.008] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Revised: 05/09/2018] [Accepted: 05/14/2018] [Indexed: 12/13/2022]
Abstract
Alternative splicing is evolving as an eminent player of oncogenic signaling for tumor development and progression. Mucin 4 (MUC4), a type I membrane-bound mucin, is differentially expressed in pancreatic cancer (PC) and plays a critical role in its progression and metastasis. However, the molecular implications of MUC4 splice variants during disease pathogenesis remain obscure. The present study delineates the pathological and molecular significance of a unique splice variant of MUC4, MUC4/X, which lacks the largest exon 2, along with exon 3. Exon 2 encodes for the highly glycosylated tandem repeat (TR) domain of MUC4 and its absence creates MUC4/X, which is devoid of TR. Expression analysis from PC clinical samples revealed significant upregulation of MUC4/X in PC tissues with most differential expression in poorly differentiated tumors. In vitro studies suggest that overexpression of MUC4/X in wild-type-MUC4 (WT-MUC4) null PC cell lines markedly enhanced PC cell proliferation, invasion, and adhesion to extracellular matrix (ECM) proteins. Furthermore, MUC4/X overexpression leads to an increase in the tumorigenic potential of PC cells in orthotopic transplantation studies. In line with these findings, doxycycline-induced expression of MUC4/X in an endogenous WT-MUC4 expressing PC cell line (Capan-1) also displayed enhanced cell proliferation, invasion, and adhesion to ECM, compared to WT-MUC4 alone, emphasizing its direct involvement in the aggressive behavior of PC cells. Investigation into the molecular mechanism suggested that MUC4/X facilitated PC tumorigenesis via integrin-β1/FAK/ERK signaling pathway. Overall, these findings revealed the novel role of MUC4/X in promoting and sustaining the oncogenic features of PC.
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Affiliation(s)
- Rahat Jahan
- Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA
| | - Muzafar A Macha
- Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA; Department of Otolaryngology-Head and Neck Surgery, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA
| | - Satyanarayana Rachagani
- Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA
| | - Srustidhar Das
- Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA
| | - Lynette M Smith
- Department of Biostatistics, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA
| | - Sukhwinder Kaur
- Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA
| | - Surinder K Batra
- Department of Biochemistry and Molecular Biology, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA; Eppley Institute for Research in Cancer and Allied Diseases, Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, NE-68198, USA.
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21
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Mendt M, Kamerkar S, Sugimoto H, McAndrews KM, Wu CC, Gagea M, Yang S, Blanko EVR, Peng Q, Ma X, Marszalek JR, Maitra A, Yee C, Rezvani K, Shpall E, LeBleu VS, Kalluri R. Generation and testing of clinical-grade exosomes for pancreatic cancer. JCI Insight 2018; 3:99263. [PMID: 29669940 DOI: 10.1172/jci.insight.99263] [Citation(s) in RCA: 565] [Impact Index Per Article: 80.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2017] [Accepted: 03/14/2018] [Indexed: 12/13/2022] Open
Abstract
Exosomes are extracellular vesicles produced by all cells with a remarkable ability to efficiently transfer genetic material, including exogenously loaded siRNA, to cancer cells. Here, we report on a bioreactor-based, large-scale production of clinical-grade exosomes employing good manufacturing practice (GMP) standards. A standard operating procedure was established to generate engineered exosomes with the ability to target oncogenic Kras (iExosomes). The clinical-grade GMP iExosomes were tested in multiple in vitro and in vivo studies to confirm suppression of oncogenic Kras and an increase in the survival of several mouse models with pancreatic cancer. We perform studies to determine the shelf life, biodistribution, toxicology profile, and efficacy in combination with chemotherapy to inform future clinical testing of GMP iExosomes. Collectively, this report illustrates the process and feasibility of generating clinical-grade exosomes for various therapies of human diseases.
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Affiliation(s)
- Mayela Mendt
- Department of Cancer Biology, Metastasis Research Center.,Department of Stem Cell Transplantation and Cellular Therapy
| | | | | | | | | | | | - Sujuan Yang
- Department of Cancer Biology, Metastasis Research Center
| | | | - Qian Peng
- Department of Cancer Biology, Metastasis Research Center
| | - Xiaoyan Ma
- Center for Co-Clinical Trials and Institute for Applied Cancer Science
| | | | - Anirban Maitra
- Departments of Pathology and Translational Molecular Pathology, Ahmad Center for Pancreatic Cancer Research, and
| | - Cassian Yee
- Departments of Medical Melanoma and Stem Cell Transplantation and Cellular Therapy, University of Texas MD Anderson Cancer Center, Houston, Texas, USA
| | | | | | | | - Raghu Kalluri
- Department of Cancer Biology, Metastasis Research Center
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22
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Tsuchida N, Murugan AK, Grieco M. Kirsten Ras* oncogene: significance of its discovery in human cancer research. Oncotarget 2018; 7:46717-46733. [PMID: 27102293 PMCID: PMC5216832 DOI: 10.18632/oncotarget.8773] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2015] [Accepted: 04/10/2016] [Indexed: 12/24/2022] Open
Abstract
The KRAS/ K-RAS oncogene is crucially involved in human cancer. The term "oncogene" -- i.e., a gene able to transform a normal cell into a tumor cell - was introduced in 1969, but the word was not used in the human carcinogenesis literature until much later. Transforming Kras and Hras oncogenes from the Kirsten and Harvey sarcoma viruses were not identified until the early 1980s due to the complicated structures of the viral genomes. Orthologs of these viral oncogenes were then found in transforming DNA fragments in human cancers in the form of mutated versions of the HRAS and KRAS proto-oncogenes. Thus, RAS genes were the first human oncogenes to be identified. Subsequent studies showed that mutated KRAS acted as an in vivo oncogenic driver, as indicated by studies of anti-EGFR therapy for metastatic colorectal cancers. This review addresses the historical background and experimental studies that led to the discovery of Kirsten Ras as an oncogene, the role of mutated KRAS in human carcinogenesis, and recent therapeutic studies of cancer cells with KRAS mutations.
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Affiliation(s)
- Nobuo Tsuchida
- Graduate School of Medical and Dental Sciences, Tokyo Medical Dental University, Yushima, Bunkyo-ku, Tokyo, Japan
| | | | - Michele Grieco
- DiSTABiF, Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli, Caserta, Italy
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23
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Long-term vemurafenib treatment drives inhibitor resistance through a spontaneous KRAS G12D mutation in a BRAF V600E papillary thyroid carcinoma model. Oncotarget 2017; 7:30907-23. [PMID: 27127178 PMCID: PMC5058727 DOI: 10.18632/oncotarget.9023] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2015] [Accepted: 03/31/2016] [Indexed: 01/04/2023] Open
Abstract
The BRAF V600E mutation is commonly observed in papillary thyroid cancer (PTC) and predominantly activates the MAPK pathway. Presence of BRAF V600E predicts increasing risk of recurrence and higher mortality rate, and treatment options for such patients are limited. Vemurafenib, a BRAF V600E inhibitor, is initially effective, but cells inevitably develop alternative mechanisms of pathway activation. Mechanisms of primary resistance have been described in short-term cultures of PTC cells; however, mechanisms of acquired resistance have not. In the present study, we investigated possible adaptive mechanisms of BRAF V600E inhibitor resistance in KTC1 thyroid cancer cells following long-term vemurafenib exposure. We found that a subpopulation of KTC1 cells acquired resistance to vemurafenib following 5 months of treatment with the inhibitor. Resistance coincided with the spontaneous acquisition of a KRAS G12D activating mutation. Increases in activated AKT, ERK1/2, and EGFR were observed in these cells. In addition, the resistant cells were less sensitive to combinations of vemurafenib and MEK1 inhibitor or AKT inhibitor. These results support the KRAS G12D mutation as a genetic mechanism of spontaneously acquired secondary BRAF inhibitor resistance in BRAF V600E thyroid cancer cells.
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24
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Till JE, Yoon SS, Ryeom S. E-cadherin and K-ras: implications of a newly developed model of gastric cancer. Oncoscience 2017; 4:162-163. [PMID: 29344549 PMCID: PMC5769975 DOI: 10.18632/oncoscience.379] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2017] [Accepted: 11/12/2017] [Indexed: 12/17/2022] Open
Affiliation(s)
- Jacob E Till
- Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| | - Sam S Yoon
- Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
| | - Sandra Ryeom
- Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA
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25
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Hu J, Sheng Y, Kwak KJ, Shi J, Yu B, Lee LJ. A signal-amplifiable biochip quantifies extracellular vesicle-associated RNAs for early cancer detection. Nat Commun 2017; 8:1683. [PMID: 29162835 PMCID: PMC5698315 DOI: 10.1038/s41467-017-01942-1] [Citation(s) in RCA: 116] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2016] [Accepted: 10/26/2017] [Indexed: 11/09/2022] Open
Abstract
Detection of extracellular vesicle (EV)-associated RNAs with low expression levels in early-stage cancer remains a challenge and is highly valuable. Here, we report a nanoparticle-based biochip that could capture circulating EVs without isolation, brighten encapsulated RNAs, and amplify fluorescence signals in situ in a single step. We confine catalyzed hairpin DNA circuit (CHDC) in cationic lipid-polymer hybrid nanoparticles (LPHNs) that are tethered on a chip. LPHN features a core-shell-corona structure that facilitates the transfer and mixing of CHDC with EV-associated RNAs when forming the LPHN–EV nanocomplex. CHDC is triggered upon target RNA binding and quickly generate amplified signals. The signal amplification efficiency of LPHN–CHDC is demonstrated in artificial EVs, cancer cells, and cancer cell-derived EVs. We show that LPHN–CHDC biochip with signal amplification capability could selectively and sensitively identify low expression glypican-1 mRNA in serum EVs, distinguishing patients with early- and late-stage pancreatic cancer from healthy donors and patients with benign pancreatic disease. Extracellular vesicles (EV)-associated RNAs are serum biomarkers potentially exploitable for early cancer diagnosis. Here the authors develop a catalyzed hairpin DNA circuit within a cationic lipid-polymer hybrid nanoparticle that can detect low–level EV-associated RNAs in early stage cancer patients.
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Affiliation(s)
- Jiaming Hu
- Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA.
| | - Yan Sheng
- Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA. .,College of Chemistry and Chemical Engineering, Yantai University, Yantai, 264005, People's Republic of China.
| | - Kwang Joo Kwak
- Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA
| | - Junfeng Shi
- Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA
| | - Bohao Yu
- School of Chemistry and Molecular Engineering, East China University of Science & Technology, Shanghai, 200237, People's Republic of China
| | - L James Lee
- Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, 43210, USA.
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26
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Autocrine hGH stimulates oncogenicity, epithelial-mesenchymal transition and cancer stem cell-like behavior in human colorectal carcinoma. Oncotarget 2017; 8:103900-103918. [PMID: 29262609 PMCID: PMC5732775 DOI: 10.18632/oncotarget.21812] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2017] [Accepted: 09/08/2017] [Indexed: 11/25/2022] Open
Abstract
Tumor derived human growth hormone (hGH) has been implicated in cancer development and progression. However, the specific functional role of autocrine/paracrine hGH in colorectal cancer (CRC) remains largely to be determined. Herein, we demonstrated a crucial oncogenic role of autocrine hGH in CRC progression. Elevated hGH expression was detected in CRC compared to normal colorectal tissue, and hGH expression in CRC was positively associated with tumor size and lymph node metastasis. Forced expression of hGH stimulated cell proliferation, survival, oncogenicity and epithelial to mesenchymal transition (EMT) of CRC cells, and promoted xenograft growth and local invasion in vivo. Autocrine hGH expression in CRC cells stimulated the activation of the ERK1/2 pathway, which in turn resulted in increased transcription of the mesenchymal marker FIBRONECTIN 1 and transcriptional repression of the epithelial marker E-CADHERIN. The autocrine hGH-stimulated increase in CRC cell proliferation, cell survival and EMT was abrogated upon ERK1/2 inhibition. Furthermore, autocrine hGH-stimulated CRC cell migration and invasion was dependent on the ERK1/2-mediated increase in FIBRONECTIN 1 expression and decrease in E-CADHERIN expression. Forced expression of hGH also enhanced CSC-like behavior of CRC cells, as characterized by increased colonosphere formation, ALDH-positive population and CSC marker expression. Autocrine hGH-enhanced cancer stem cell (CSC)-like behavior in CRC cells was also observed to be E-CADHERIN-dependent. Thus, autocrine hGH plays a critical role in CRC progression, and inhibition of hGH could be a promising targeted therapeutic approach to limit disease progression in metastatic CRC patients.
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27
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Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 2017; 546:498-503. [PMID: 28607485 PMCID: PMC5538883 DOI: 10.1038/nature22341] [Citation(s) in RCA: 1877] [Impact Index Per Article: 234.6] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Accepted: 04/03/2017] [Indexed: 02/06/2023]
Abstract
The mutant form of the GTPase KRAS is a key driver of pancreatic cancer
but remains a challenging therapeutic target. Exosomes, extracellular vesicles
generated by all cells, are naturally present in the blood. Here we demonstrate
that enhanced retention of exosomes in circulation, compared to liposomes, is
due to CD47 mediated protection of exosomes from phagocytosis by monocytes and
macrophages. Exosomes derived from normal fibroblast-like mesenchymal cells were
engineered to carry siRNA or shRNA specific to oncogenic KRASG12D
(iExosomes), a common mutation in pancreatic cancer. Compared to liposomes,
iExosomes target oncogenic Kras with an enhanced efficacy that is dependent on
CD47, and is facilitated by macropinocytosis. iExosomes treatment suppressed
cancer in multiple mouse models of pancreatic cancer and significantly increased
their overall survival. Our results inform on a novel approach for direct and
specific targeting of oncogenic Kras in tumors using iExosomes.
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28
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Słotwiński R, Słotwińska SM. Diagnostic value of selected markers and apoptotic pathways for pancreatic cancer. Cent Eur J Immunol 2017; 41:392-403. [PMID: 28450803 PMCID: PMC5382885 DOI: 10.5114/ceji.2016.65139] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2016] [Accepted: 09/26/2016] [Indexed: 12/19/2022] Open
Abstract
Pancreatic cancer occupies the fourth place as a cause of death from cancer, and the mortality rate is similar to the number of newly detected cases. Due to the late diagnosis, only 5-6% of patients with pancreatic cancer survive for five years. Given that early diagnosis is critical for improving patients' survival rates, there is an urgent need for the discovery and validation of new biomarkers with sufficient sensitivity and specificity to help diagnose pancreatic cancer early. Detection of serum tumor markers (CA19-9, CEA, CA125 and CA242) is conducive to the early diagnosis of pancreatic cancer. The combination of miR-16, miR-196a and CA19-9 plasma level was more effective, especially in early tumor screening. Furthermore, recent studies reported that mainly miR-21, miR-155 and miR-196 were dysregulated in IPMN (intraductal papillary mucinous neoplasms) and PanIN (pancreatic intraepithelial neoplasia) lesions, suggesting their usefulness as early biomarkers of these diseases. The reduced rate of apoptosis plays a crucial role in carcinogenesis, and it is one of the most important characteristics acquired by pancreatic cancer cells, which protects them from attack by the immune system and reduces the effectiveness of pharmacological treatment. This review summarizes the data concerning the clinical utility of selected biomarkers in pancreatic cancer patients. The review mainly focuses on the genetic aspects of signaling pathway disorders associated with apoptosis in the pathogenesis and diagnosis of pancreatic cancer.
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Affiliation(s)
- Robert Słotwiński
- Department of Surgical Research and Transplantology, Mossakowski Medical Research Centre, Polish Academy of Sciences, Poland
- Department of Immunology, Biochemistry and Nutrition, Medical University of Warsaw, Poland
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29
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The cornerstone K-RAS mutation in pancreatic adenocarcinoma: From cell signaling network, target genes, biological processes to therapeutic targeting. Crit Rev Oncol Hematol 2017; 111:7-19. [PMID: 28259298 DOI: 10.1016/j.critrevonc.2017.01.002] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2016] [Revised: 11/15/2016] [Accepted: 01/05/2017] [Indexed: 01/17/2023] Open
Abstract
RAS belongs to the super family of small G proteins and plays crucial roles in signal transduction from membrane receptors in the cell. Mutations of K-RAS oncogene lead to an accumulation of GTP-bound proteins that maintains an active conformation. In the pancreatic ductal adenocarcinoma (PDAC), one of the most deadly cancers in occidental countries, mutations of the K-RAS oncogene are nearly systematic (>90%). Moreover, K-RAS mutation is the earliest genetic alteration occurring during pancreatic carcinogenetic sequence. In this review, we discuss the central role of K-RAS mutations and their tremendous diversity of biological properties by the interconnected regulation of signaling pathways (MAPKs, NF-κB, PI3K, Ral…). In pancreatic ductal adenocarcinoma, transcriptome analysis and preclinical animal models showed that K-RAS mutation alters biological behavior of PDAC cells (promoting proliferation, migration and invasion, evading growth suppressors, regulating mucin pattern, and miRNA expression). K-RAS also impacts tumor microenvironment and PDAC metabolism reprogramming. Finally we discuss therapeutic targeting strategies of K-RAS that have been developed without significant clinical success so far. As K-RAS is considered as the undruggable target, targeting its multiple effectors and target genes should be considered as potential alternatives.
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30
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Purohit A, Varney M, Rachagani S, Ouellette MM, Batra SK, Singh RK. CXCR2 signaling regulates KRAS(G¹²D)-induced autocrine growth of pancreatic cancer. Oncotarget 2016; 7:7280-96. [PMID: 26771140 PMCID: PMC4872785 DOI: 10.18632/oncotarget.6906] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2015] [Accepted: 11/25/2015] [Indexed: 12/27/2022] Open
Abstract
Pharmacological inhibition of RAS, the master regulator of pancreatic ductal adenocarcinoma (PDAC), continues to be a challenge. Mutations in various isoforms of RAS gene, including KRAS are known to upregulate CXC chemokines; however, their precise role in KRAS-driven pancreatic cancer remains unclear. In this report, we reveal a previously unidentified tumor cell-autonomous role of KRAS(G12D)-induced CXCR2 signaling in mediating growth of neoplastic PDAC cells. Progressively increasing expression of mCXCR2 and its ligands was detected in the malignant ductal cells of Pdx1-cre;LSL-Kras(G12D) mice. Knocking-down CXCR2 in KRAS(G12D)-bearing human pancreatic duct-derived cells demonstrated a significant decrease in the in vitro and in vivo tumor cell proliferation. Furthermore, CXCR2 antagonists showed selective growth inhibition of KRAS(G12D)-bearing cells in vitro. Intriguingly, both genetic and pharmacological inhibition of CXCR2 signaling in KRAS(G12D)-bearing pancreatic ductal cells reduced the levels of KRAS protein, strongly implying the presence of a KRAS-CXCR2 feed-forward loop. Together, these data demonstrate the role of CXCR2 signaling in KRAS(G12D)-induced growth transformation and progression in PDAC.
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Affiliation(s)
- Abhilasha Purohit
- Department of Pathology and Microbiology, 985900 Nebraska Medical Center, Omaha, NE, USA
| | - Michelle Varney
- Department of Pathology and Microbiology, 985900 Nebraska Medical Center, Omaha, NE, USA
| | - Satyanarayana Rachagani
- Department of Biochemistry and Molecular Biology, 985870 Nebraska Medical Center, Omaha, NE, USA
| | | | - Surinder K Batra
- Department of Pathology and Microbiology, 985900 Nebraska Medical Center, Omaha, NE, USA.,Department of Biochemistry and Molecular Biology, 985870 Nebraska Medical Center, Omaha, NE, USA.,Eppley Institute, 985950 Nebraska Medical Center, Omaha, NE, USA
| | - Rakesh K Singh
- Department of Pathology and Microbiology, 985900 Nebraska Medical Center, Omaha, NE, USA
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31
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Iyer SV, Parrales A, Begani P, Narkar A, Adhikari AS, Martinez LA, Iwakuma T. Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities. Oncotarget 2016; 7:5401-15. [PMID: 26700961 PMCID: PMC4868694 DOI: 10.18632/oncotarget.6634] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2015] [Accepted: 12/12/2015] [Indexed: 12/19/2022] Open
Abstract
Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.
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Affiliation(s)
- Swathi V Iyer
- Department of Cancer Biology, The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
| | - Alejandro Parrales
- Department of Cancer Biology, The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
| | - Priya Begani
- Department of Cancer Biology, The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
| | - Akshay Narkar
- Department of Cancer Biology, The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
| | - Amit S Adhikari
- Center for Advanced Preclinical Research, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA
| | - Luis A Martinez
- Department of Pathology, Stony Brook School of Medicine, Stony Brook, NY, USA
| | - Tomoo Iwakuma
- Department of Cancer Biology, The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA
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32
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Estrada-Bernal A, Chatterjee M, Haque SJ, Yang L, Morgan MA, Kotian S, Morrell D, Chakravarti A, Williams TM. MEK inhibitor GSK1120212-mediated radiosensitization of pancreatic cancer cells involves inhibition of DNA double-strand break repair pathways. Cell Cycle 2016; 14:3713-24. [PMID: 26505547 DOI: 10.1080/15384101.2015.1104437] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
PURPOSE Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK inhibitors have shown promising anti-tumor responses in recent preclinical and clinical studies, and are currently being tested in combination with radiation in clinical trials. Here, we have evaluated the radiosensitizing potential of a novel MEK1/2 inhibitor GSK1120212 (GSK212,or trametinib) and evaluated whether MEK1/2 inhibition alters DNA repair mechanisms in multiple PC cell lines. METHODS Radiosensitization and DNA double-strand break (DSB) repair were evaluated by clonogenic assays, comet assay, nuclear foci formation (γH2AX, DNA-PK, 53BP1, BRCA1, and RAD51), and by functional GFP-reporter assays for homologous recombination (HR) and non-homologous end-joining (NHEJ). Expression and activation of DNA repair proteins were measured by immunoblotting. RESULTS GSK212 blocked ERK1/2 activity and radiosensitized multiple KRAS mutant PC cell lines. Prolonged pre-treatment with GSK212 for 24-48 hours was required to observe significant radiosensitization. GSK212 treatment resulted in delayed resolution of DNA damage by comet assays and persistent γH2AX nuclear foci. GSK212 treatment also resulted in altered BRCA1, RAD51, DNA-PK, and 53BP1 nuclear foci appearance and resolution after radiation. Using functional reporters, GSK212 caused repression of both HR and NHEJ repair activity. Moreover, GSK212 suppressed the expression and activation of a number of DSB repair pathway intermediates including BRCA1, DNA-PK, RAD51, RRM2, and Chk-1. CONCLUSION GSK212 confers radiosensitization to KRAS-driven PC cells by suppressing major DNA-DSB repair pathways. These data provide support for the combination of MEK1/2 inhibition and radiation in the treatment of PC.
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Affiliation(s)
- Adriana Estrada-Bernal
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - Moumita Chatterjee
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - S Jaharul Haque
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - Linlin Yang
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - Meredith A Morgan
- b University of Michigan Comprehensive Cancer Center ; Ann Arbor , MI , USA
| | - Shweta Kotian
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - David Morrell
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - Arnab Chakravarti
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
| | - Terence M Williams
- a The Ohio State University Medical Center; Arthur G James Comprehensive Cancer Center and Richard J Solove Research Institute ; Columbus , OH USA
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Rabi T, Catapano CV. Aphanin, a triterpenoid from Amoora rohituka inhibits K-Ras mutant activity and STAT3 in pancreatic carcinoma cells. Tumour Biol 2016; 37:12455-12464. [PMID: 27333990 DOI: 10.1007/s13277-016-5102-2] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2015] [Accepted: 06/09/2016] [Indexed: 01/05/2023] Open
Abstract
Mutations of the K-Ras gene occur in over 90 % of pancreatic carcinomas, and to date, no targeted therapies exist for this genetically defined subset of cancers. STAT3 plays a critical role in KRAS-driven pancreatic tumorigenesis, suggesting its potential as a therapeutic target in this cancer. Therefore, finding novel and potential drugs to inhibit oncogenic K-Ras is a major challenge in cancer therapy. In an attempt to develop novel anti-KRAS mutant chemotherapeutics, we isolated three novel triterpenoids from Amoora rohituka stem and their chemical structures were characterized by extensive 1H-NMR, 13C-NMR, Mass, IR spectroscopic studies and chemical transformations. Aphanin (3 alpha-angeloyloxyolean-12-en-28-oic acid) is one of the isolated novel triterpenoid compounds. We found aphanin exhibited antiproliferative effects, caused G0-G1 cell cycle arrest, inhibits K-Ras G12D mutant activity by decreased STAT3, p-STAT3, Akt, p-Akt, cyclin D1 and c-Myc expressions, and induced apoptosis in pancreatic cancer HPAF-II (ΔKRAS G12D ) cells. The apoptosis proceeded through depletion of GSH with a concomitant increase in the reactive oxygen species production. The results of our study have important implications for the development of aphanin as potential novel agent for the treatment of K-Ras mutant pancreatic cancer, and STAT3-cMyc-cyclinD1 axis may serve as an important predictive biomarker for the therapeutic efficacy.
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Affiliation(s)
- Thangaiyan Rabi
- Siddha Clinic and Research Center SVA, Kanyakumari, Tamil Nadu, India.
- Tumor Biology and Experimental Therapeutics Program, Institute of Oncology Research (IOR), Bellinzona, Switzerland.
| | - Carlo V Catapano
- Tumor Biology and Experimental Therapeutics Program, Institute of Oncology Research (IOR), Bellinzona, Switzerland
- Department of Oncology, Faculty of Biology and Medicine, University of Lausanne, Lausanne, Switzerland
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KRAS, NRAS, BRAF mutations and high counts of poorly differentiated clusters of neoplastic cells in colorectal cancer: observational analysis of 175 cases. Pathology 2016; 47:551-6. [PMID: 26352110 DOI: 10.1097/pat.0000000000000300] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
A novel grading system based on the counting of poorly differentiated clusters (PDC) of neoplastic cells at the invasive margin and in the tumour stroma was recently introduced among the histological parameters predictive of adverse clinical outcome in colorectal cancer (CRC). The aim of this study was to correlate the histological grade based on PDC and the mutational status of KRAS, NRAS and BRAF genes in 175 consecutive CRCs. The highest PDC count under the objective lens of a ×20 microscopic field in each tumour was considered for grading assessment, so that PDC counts <5, 5-9 and ≥10 PDCs were defined grade 1, grade 2 and grade 3, respectively. Hotspots mutations were identified using the MassArray platform. Overall, there were 42 (24%) mutated tumours. Mutational status was significantly associated with high pT stage (p = 0.0021), advanced pTNM stage (p = 0.0018), nodal metastases (p = 0.006), tumour budding (p = 0.022) and high PDC grade (p = 0.0022). KRAS mutations were significantly associated with PDC grade (p = 0.0379), while BRAF mutations were associated with PDC-G3 although statistical significance was not reached. No significant associations were found between NRAS and PDC. The significant association between mutated KRAS and PDC grade suggests that KRAS mutations may be involved in the formation of PDC.
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Puniya BL, Allen L, Hochfelder C, Majumder M, Helikar T. Systems Perturbation Analysis of a Large-Scale Signal Transduction Model Reveals Potentially Influential Candidates for Cancer Therapeutics. Front Bioeng Biotechnol 2016; 4:10. [PMID: 26904540 PMCID: PMC4750464 DOI: 10.3389/fbioe.2016.00010] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2015] [Accepted: 01/25/2016] [Indexed: 12/20/2022] Open
Abstract
Dysregulation in signal transduction pathways can lead to a variety of complex disorders, including cancer. Computational approaches such as network analysis are important tools to understand system dynamics as well as to identify critical components that could be further explored as therapeutic targets. Here, we performed perturbation analysis of a large-scale signal transduction model in extracellular environments that stimulate cell death, growth, motility, and quiescence. Each of the model’s components was perturbed under both loss-of-function and gain-of-function mutations. Using 1,300 simulations under both types of perturbations across various extracellular conditions, we identified the most and least influential components based on the magnitude of their influence on the rest of the system. Based on the premise that the most influential components might serve as better drug targets, we characterized them for biological functions, housekeeping genes, essential genes, and druggable proteins. The most influential components under all environmental conditions were enriched with several biological processes. The inositol pathway was found as most influential under inactivating perturbations, whereas the kinase and small lung cancer pathways were identified as the most influential under activating perturbations. The most influential components were enriched with essential genes and druggable proteins. Moreover, known cancer drug targets were also classified in influential components based on the affected components in the network. Additionally, the systemic perturbation analysis of the model revealed a network motif of most influential components which affect each other. Furthermore, our analysis predicted novel combinations of cancer drug targets with various effects on other most influential components. We found that the combinatorial perturbation consisting of PI3K inactivation and overactivation of IP3R1 can lead to increased activity levels of apoptosis-related components and tumor-suppressor genes, suggesting that this combinatorial perturbation may lead to a better target for decreasing cell proliferation and inducing apoptosis. Finally, our approach shows a potential to identify and prioritize therapeutic targets through systemic perturbation analysis of large-scale computational models of signal transduction. Although some components of the presented computational results have been validated against independent gene expression data sets, more laboratory experiments are warranted to more comprehensively validate the presented results.
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Affiliation(s)
- Bhanwar Lal Puniya
- Department of Biochemistry, University of Nebraska-Lincoln , Lincoln, NE , USA
| | - Laura Allen
- Department of Mathematics, University of Nebraska at Omaha , Omaha, NE , USA
| | | | - Mahbubul Majumder
- Department of Mathematics, University of Nebraska at Omaha , Omaha, NE , USA
| | - Tomáš Helikar
- Department of Biochemistry, University of Nebraska-Lincoln , Lincoln, NE , USA
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Melo SA, Luecke LB, Kahlert C, Fernandez AF, Gammon ST, Kaye J, LeBleu VS, Mittendorf EA, Weitz J, Rahbari N, Reissfelder C, Pilarsky C, Fraga MF, Piwnica-Worms D, Kalluri R. Glypican-1 identifies cancer exosomes and detects early pancreatic cancer. Nature 2015; 523:177-82. [PMID: 26106858 PMCID: PMC4825698 DOI: 10.1038/nature14581] [Citation(s) in RCA: 2166] [Impact Index Per Article: 216.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2014] [Accepted: 05/22/2015] [Indexed: 12/12/2022]
Abstract
Exosomes are lipid-bilayer-enclosed extracellular vesicles that contain proteins and nucleic acids. They are secreted by all cells and circulate in the blood. Specific detection and isolation of cancer-cell-derived exosomes in the circulation is currently lacking. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC1(+) circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1(+) crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1(+) crExos correlate with tumour burden and the survival of pre- and post-surgical patients. GPC1(+) crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations, and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by magnetic resonance imaging. GPC1(+) crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.
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Affiliation(s)
- Sonia A. Melo
- Department of Cancer Biology, Metastasis Research Center, University
of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
| | - Linda B. Luecke
- Department of Cancer Biology, Metastasis Research Center, University
of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
| | - Christoph Kahlert
- Department of Cancer Biology, Metastasis Research Center, University
of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
| | - Agustin F. Fernandez
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias
(IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain
| | - Seth T. Gammon
- Department of Cancer Systems Imaging, The University of Texas M.D.
Anderson Cancer Center, Houston, TX 77054, USA
| | - Judith Kaye
- Department of Cancer Biology, Metastasis Research Center, University
of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
| | - Valerie S. LeBleu
- Department of Cancer Biology, Metastasis Research Center, University
of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
| | - Elizabeth A. Mittendorf
- Department of Surgical Oncology, The University of Texas MD Anderson
Cancer Center, Houston, TX 77030, USA
| | - Juergen Weitz
- Department of Gastrointestinal, Thoracic and Vascular Surgery,
Medizinische Fakultät Carl Gustav Carus, Technische Universität
Dresden, Fetscherstr. 74, 01307 Dresden, Germany
| | - Nuh Rahbari
- Department of Gastrointestinal, Thoracic and Vascular Surgery,
Medizinische Fakultät Carl Gustav Carus, Technische Universität
Dresden, Fetscherstr. 74, 01307 Dresden, Germany
| | - Christoph Reissfelder
- Department of Gastrointestinal, Thoracic and Vascular Surgery,
Medizinische Fakultät Carl Gustav Carus, Technische Universität
Dresden, Fetscherstr. 74, 01307 Dresden, Germany
| | - Christian Pilarsky
- Department of Gastrointestinal, Thoracic and Vascular Surgery,
Medizinische Fakultät Carl Gustav Carus, Technische Universität
Dresden, Fetscherstr. 74, 01307 Dresden, Germany
| | - Mario F. Fraga
- Cancer Epigenetics Laboratory, Institute of Oncology of Asturias
(IUOPA), HUCA, Universidad de Oviedo, Oviedo, Spain
- Department of Immunology and Oncology, National Center for
Biotechnology, CNB-CSIC, Cantoblanco, 28049 Madrid, Spain
| | - David Piwnica-Worms
- Department of Cancer Systems Imaging, The University of Texas M.D.
Anderson Cancer Center, Houston, TX 77054, USA
| | - Raghu Kalluri
- Department of Cancer Biology, Metastasis Research Center, University
of Texas MD Anderson Cancer Center, Houston, TX 77054, USA
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Tan C, Zhang L, Cheng X, Lin XF, Lu RR, Bao JD, Yu HX. Curcumin inhibits hypoxia-induced migration in K1 papillary thyroid cancer cells. Exp Biol Med (Maywood) 2015; 240:925-35. [PMID: 25349216 PMCID: PMC4935405 DOI: 10.1177/1535370214555665] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/10/2014] [Accepted: 09/07/2014] [Indexed: 12/20/2022] Open
Abstract
Curcumin, traditionally used as food and medicinal purposes, has recently been reported to have protective efficacy against hypoxia. Hypoxia is one of the important reactive factors in tumor metastasis, which is a key problem in clinical thyroid cancer therapy. In present study, we investigate the anti-metastatic effect of curcumin on the K1 papillary thyroid cancer cells as well as its potential mechanisms. The results show that curcumin effectively inhibits hypoxia-induced reactive oxygen species (ROS) upregulation and significantly decreases the mRNA and protein expression levels of hypoxia-inducible factor-1α (HIF-1α) in K1 cells. Curcumin also decreases the DNA binding ability of HIF-1α to hypoxia response element (HRE). Furthermore, curcumin enhances E-cadherin expression, inhibits metalloproteinase-9 (MMP-9) enzyme activity, and weakens K1 cells migration under hypoxic conditions. In summary, these results indicate that curcumin possesses a potent anti-metastatic effect and might be an effective tumoristatic agent for the treatment of aggressive papillary thyroid cancers.
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Affiliation(s)
- Cheng Tan
- Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
| | - Li Zhang
- Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
| | - Xian Cheng
- Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
| | - Xiu-Feng Lin
- Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
| | - Rong-Rong Lu
- State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China
| | - Jian-Dong Bao
- Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
| | - Hui-Xin Yu
- Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu 214063, China
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Tumour-suppressive function of SIRT4 in human colorectal cancer. Br J Cancer 2015; 113:492-9. [PMID: 26086877 PMCID: PMC4522635 DOI: 10.1038/bjc.2015.226] [Citation(s) in RCA: 122] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2015] [Revised: 05/01/2015] [Accepted: 05/27/2015] [Indexed: 12/16/2022] Open
Abstract
Background: SIRT4, which is localised in the mitochondria, is one of the least characterised members of the sirtuin family of nicotinamide adenine dinucleotide-dependent enzymes that play key roles in multiple cellular processes such as metabolism, stress response and longevity. There are only a few studies that have characterised its function and assessed its clinical significance in human cancers. Methods: We established colorectal cancer cell lines (SW480, HCT116, and HT29) overexpressing SIRT4 and investigated their effects on proliferation, migration and invasion, as well as E-cadherin expression, that negatively regulates tumour invasion and metastases. The associations between SIRT4 expression in colorectal cancer specimens and clinicopathological features including prognosis were assessed by immunohistochemistry. Results: SIRT4 upregulated E-cadherin expression and suppressed proliferation, migration and invasion through inhibition of glutamine metabolism in colorectal cancer cells. Moreover, SIRT4 expression in colorectal cancer decreased with the progression of invasion and metastasis, and a low expression level of SIRT4 was correlated with a worse prognosis. Conclusions: SIRT4 has a tumour-suppressive function and may serve as a novel therapeutic target in colorectal cancer.
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Beuran M, Negoi I, Paun S, Ion AD, Bleotu C, Negoi RI, Hostiuc S. The epithelial to mesenchymal transition in pancreatic cancer: A systematic review. Pancreatology 2015; 15:217-225. [PMID: 25794655 DOI: 10.1016/j.pan.2015.02.011] [Citation(s) in RCA: 102] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/01/2014] [Revised: 02/03/2015] [Accepted: 02/23/2015] [Indexed: 12/11/2022]
Abstract
BACKGROUND/OBJECTIVES The present article summarizes and analyzes the current knowledge about the role of the epithelial to mesenchymal transition (EMT) in the systemic invasiveness of pancreatic cancer. METHOD An electronic search of PubMed/MEDLINE, EMBASE, and the Web of Science was used to identify relevant original articles and reviews. RESULTS The EMT represents a key step during normal embryogenesis. However, increasing evidence reveals its essential role in the local progression and metastasis of pancreatic cancer. Areas of interest are the cross-linking between cells undergoing the EMT and pancreatic cancer stem cells, and the correlation between the EMT and chemoresistance to standard therapies. During carcinogenesis, malignant pancreatic cells at the primary site acquire the ability to undergo the EMT, a transformation associated with increased mobility. The reverse process at secondary sites, the mesenchymal to epithelial transition (MET), has devastating consequences, allowing neoplastic epithelial cells to invade surrounding tissues and spread to distant sites. Consequences of the EMT are the loss of E-cadherin expression and the acquisition of mesenchymal markers including fibronectin or vimentin. Detailed knowledge of the molecular processes underlying the EMT has opened possibilities for new therapeutic agents. These include an EMT approach for patients with early cancers, to prevent invasion and dissemination, and anti-MET therapy for patients with established metastasis. CONCLUSIONS The current literature shows a strong correlation between the EMT and the systemic aggressiveness of pancreatic tumors. Individualized therapy, targeting the process of EMT and its cross-linking with cancer stem cells, may increase survival of patients with pancreatic cancer.
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Affiliation(s)
- Mircea Beuran
- Emergency Hospital of Bucharest, Romania; Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
| | - Ionut Negoi
- Emergency Hospital of Bucharest, Romania; Carol Davila University of Medicine and Pharmacy, Bucharest, Romania.
| | - Sorin Paun
- Emergency Hospital of Bucharest, Romania; Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
| | - Adriana Daniela Ion
- Physiopathology Department, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
| | - Coralia Bleotu
- Stefan S. Nicolau Institute of Virology, Romanian Academy, Bucharest, Romania
| | - Ruxandra Irina Negoi
- Embriology Department, Carol Davila University of Medicine and Pharmacy, Bucharest, Romania
| | - Sorin Hostiuc
- Carol Davila University of Medicine and Pharmacy, Bucharest, Romania; National Institute of Legal Medicine Mina Minovici, Bucharest, Romania
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Zhang W, Li H, Yang Y, Liao J, Yang GY. Knockdown or inhibition of aldo-keto reductase 1B10 inhibits pancreatic carcinoma growth via modulating Kras-E-cadherin pathway. Cancer Lett 2014; 355:273-80. [PMID: 25304374 PMCID: PMC4462172 DOI: 10.1016/j.canlet.2014.09.031] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2014] [Revised: 09/12/2014] [Accepted: 09/24/2014] [Indexed: 01/13/2023]
Abstract
Aldo-keto reductase 1B10 (AKR1B10) has relatively specific lipid substrates including carbonyls, retinal and farnesal/geranylgeranial. Metabolizing these lipid substrates appears crucial to carcinogenesis, particularly for farnesal/geranylgeranial that involves protein prenylation. Mutant Kras is a most common active oncogene in pancreatic cancer, and its activation requires protein prenylation. To directly determine the role of AKR1B10 in pancreatic carcinogenesis, we knocked down AKR1B10 in CD18 human pancreatic carcinoma cells using shRNA approach. Silencing AKR1B10 resulted in a significant inhibition of anchor-dependent growth (knockdown cells vs. vector-control cells: 67 ± 9.5 colonies/HPF vs. 170 ± 3.7 colonies/HPF, p < 0.01), invasion index (0.27 vs. 1.00, p < 0.05), and cell migration (at 16 hours 9.2 ± 1.2% vs. 14.0 ± 1.8%, at 24 hours 21.0 ± 1.1% vs. 30.5 ± 3.5%, and at 48 hours 51.9 ± 5.7% vs. 88.9 ± 3.0%, p < 0.01). Inhibition of AKR1B10 by oleanolic acid (OA) showed a dose-dependent inhibition of cell growth with IC50 at 30 µM. Kras pull-down and Western blot analysis revealed a significant down-regulation of active form Kras and phosphorylated C-Raf, and Erk, as well as an up-regulation of E-cadherin. A significant reduction of in vivo tumor growth was observed in nude mice implanted with the CD18 pancreatic carcinoma cells with AKR1B10 knockdown (tumor weight: 0.25 ± 0.06 g vs. 0.52 ± 0.07 g, p = 0.01), and with OA treatment (tumor weight: 0.35 ± 0.05 g vs. 0.52 ± 0.07 g, p = 0.05). Our findings indicate AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis via modulation of the Kras-E-cadherin pathway.
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Affiliation(s)
- Wanying Zhang
- Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Haonan Li
- Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Yihe Yang
- Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Jie Liao
- Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA
| | - Guang-Yu Yang
- Department of Pathology, Northwestern University, Feinberg School of Medicine, Chicago, IL 60611, USA.
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Phospholipase Cδ1 induces E-cadherin expression and suppresses malignancy in colorectal cancer cells. Proc Natl Acad Sci U S A 2014; 111:13505-10. [PMID: 25197077 DOI: 10.1073/pnas.1405374111] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Colorectal cancer (CRC) is one of the most common causes of cancer-related deaths worldwide, and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in CRC predict the ineffectiveness of EGF receptor-targeted therapy. Previous transcriptional microarray analysis suggests the association between phospholipase Cδ1 (PLCδ1) expression and KRAS mutation status in CRC. However, both the roles and the regulatory mechanisms of PLCδ1 in CRC are not known. Here, we found that the expression of PLCδ1, one of the most basal PLCs, is down-regulated in CRC specimens compared with normal colon epithelium by immunohistochemistry. Furthermore, we examined the roles of PLCδ1 in CRC cell lines that harbor an activating KRAS mutation. Ectopic expression of PLCδ1 in CRC cells induced the expression of E-cadherin, whereas knockdown of PLCδ1 repressed the expression of E-cadherin. Moreover, the overexpression of PLCδ1 suppressed the expression of several mesenchymal genes and reduced cell motility, invasiveness, and in vivo tumorigenicity of SW620 CRC cells. We also showed that PLCδ1 expression is repressed by the KRAS/mitogen-activated protein kinase kinase (MEK) pathway. Furthermore, PLCδ1 suppressed the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 through E-cadherin induction in CRC cells, suggesting the presence of a negative regulatory loop between KRAS/MEK/ERK signaling and PLCδ1. These data indicate that PLCδ1 has tumor-suppressive functions in CRC through E-cadherin induction and KRAS/MEK/ERK signal attenuation.
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Ferro R, Falasca M. Emerging role of the KRAS-PDK1 axis in pancreatic cancer. World J Gastroenterol 2014; 20:10752-10757. [PMID: 25152578 PMCID: PMC4138455 DOI: 10.3748/wjg.v20.i31.10752] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/29/2013] [Accepted: 03/19/2014] [Indexed: 02/06/2023] Open
Abstract
Pancreatic cancer is a highly aggressive tumour that is very resistant to treatments and it is rarely diagnosed early because of absence of specific symptoms. Therefore, the prognosis for this disease is very poor and it has the grim supremacy in terms of unfavourable survival rates. There have been great advances in survival rates for many types of cancers over the past few decades but hardly any change for pancreatic cancer. Mutations of the Ras oncogene are the most frequent oncogenic alterations in human cancers. The frequency of KRAS mutations in pancreatic cancer is around 90%. Given the well-established role of KRAS in cancer it is not surprising that it is one of the most attractive targets for cancer therapy. Nevertheless, during the last thirty years all attempts to target directly KRAS protein have failed. Therefore, it is crucial to identify downstream KRAS effectors in order to develop specific drugs able to counteract activation of this pathway. Among the different signalling pathways activated by oncogenic KRAS, the phosphoinositide 3-Kinase (PI3K) pathway is emerging as one of the most critical KRAS effector. In turn, PI3K activates several parallel pathways making the identification of the precise effectors activated by KRAS/PI3K more difficult. Recent data identify 3-phosphoinositide-dependent protein kinase 1 as a key tumour-initiating event downstream KRAS interaction with PI3K in pancreatic cancer.
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Schiavone M, Rampazzo E, Casari A, Battilana G, Persano L, Moro E, Liu S, Leach SD, Tiso N, Argenton F. Zebrafish reporter lines reveal in vivo signaling pathway activities involved in pancreatic cancer. Dis Model Mech 2014; 7:883-94. [PMID: 24878567 PMCID: PMC4073277 DOI: 10.1242/dmm.014969] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Pancreatic adenocarcinoma, one of the worst malignancies of the exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. This condition is usually driven by oncogenic KRAS point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and unbalanced expression of genes involved in the specific signaling pathways. To examine in vivo the effects of KRASG12D during pancreatic cancer progression and time correlation with cancer signaling pathway activities, we have generated a zebrafish model of pancreatic adenocarcinoma in which eGFP-KRASG12D expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic KRASG12D line with transgenic zebrafish reporters, harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-KRASG12D showed normal exocrine pancreas development until 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degrees of acinar lesions, characterized by an increase in mesenchymal cells and mixed acinar/ductal features, followed by progressive bowel and liver infiltrations and, finally, highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of KRAS-positive cells and progressive activation of TGFβ and Notch pathways. Increase in TGFβ, following KRASG12D activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma, indicating a tissue-specific regulation of cell signaling pathways. Moreover, our results show that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool for describing in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs.
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Affiliation(s)
- Marco Schiavone
- Department of Biology, University of Padua, 35131 Padua, Italy
| | - Elena Rampazzo
- Department of Molecular Medicine, University of Padua, 35131 Padua, Italy
| | | | - Giusy Battilana
- Department of Molecular Medicine, University of Padua, 35131 Padua, Italy
| | - Luca Persano
- Department of Woman and Child Health, University of Padua, 35131 Padua, Italy
| | - Enrico Moro
- Department of Molecular Medicine, University of Padua, 35131 Padua, Italy
| | - Shu Liu
- Department of Surgery and The McKusick-Nathans Institute of Genetic Medicine Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Steve D Leach
- Department of Surgery and The McKusick-Nathans Institute of Genetic Medicine Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
| | - Natascia Tiso
- Department of Biology, University of Padua, 35131 Padua, Italy
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Rabi T, Venkatanarashiman M. Novel synthetic oleanane triterpenoid AMR-MeOAc inhibits K-Ras through ERK, Akt and survivin in pancreatic cancer cells. PHYTOMEDICINE : INTERNATIONAL JOURNAL OF PHYTOTHERAPY AND PHYTOPHARMACOLOGY 2014; 21:491-496. [PMID: 24215674 DOI: 10.1016/j.phymed.2013.09.017] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/30/2013] [Revised: 08/28/2013] [Accepted: 09/29/2013] [Indexed: 06/02/2023]
Abstract
K-Ras activating mutations are a major problem that drives aggressive tumor growth and metastasis in pancreatic cancer. Currently, there are no effective targeted therapies for this genetically defined subset of cancers harboring oncogenic K-Ras mutations that confer drug resistance, aggressive tumor growth, metastasis and poor clinical outcome. We identified a novel synthetic oleanane triterpenoid compound designated AMR-MeOAc that effectively kills K-Ras mutant pancreatic cancer HPAF-II cells. The cytotoxic effects correlated with apoptosis induction, as was evidenced by increase of apoptosis cells upon the treatment of AMR-MeOAc in HPAF-II cells. Our studies revealed that AMR-MeOAc treatment inhibits cancer associated survival gene survivin. Moreover, AMR-MeOAc also led to down regulation of Akt, ERK1/2 and survivin protein levels. Our results indicate that AMR-MeOAc or its active analogs could be a novel class of anticancer agents against K-Ras driven human pancreatic cancer.
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Bosutinib inhibits migration and invasion via ACK1 in KRAS mutant non-small cell lung cancer. Mol Cancer 2014; 13:13. [PMID: 24461128 PMCID: PMC3930897 DOI: 10.1186/1476-4598-13-13] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2013] [Accepted: 01/21/2014] [Indexed: 01/13/2023] Open
Abstract
The advent of effective targeted therapeutics has led to increasing emphasis on precise biomarkers for accurate patient stratification. Here, we describe the role of ACK1, a non-receptor tyrosine kinase in abrogating migration and invasion in KRAS mutant lung adenocarcinoma. Bosutinib, which inhibits ACK1 at 2.7 nM IC50, was found to inhibit cell migration and invasion but not viability in a panel of non-small cell lung cancer (NSCLC) cell lines. Knockdown of ACK1 abrogated bosutinib-induced inhibition of cell migration and invasion specifically in KRAS mutant cells. This finding was further confirmed in an in vivo zebrafish metastatic model. Tissue microarray data on 210 Singaporean lung adenocarcinomas indicate that cytoplasmic ACK1 was significantly over-expressed relative to paired adjacent non-tumor tissue. Interestingly, ACK1 expression in “normal” tissue adjacent to tumour, but not tumour, was independently associated with poor overall and relapse-free survival. In conclusion, inhibition of ACK1 with bosutinib attenuates migration and invasion in the context of KRAS mutant NSCLC and may fulfil a therapeutic niche through combinatorial treatment approaches.
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Gao L, Antic T, Hyjek E, Gong C, Mueller J, Waxman I, DeMay RM, Reeves W. Immunohistochemical analysis of E-cadherin and zeste homolog 2 expression in endoscopic ultrasound-guided fine-needle aspiration of pancreatic adenocarcinoma. Cancer Cytopathol 2013; 121:644-52. [PMID: 23674382 DOI: 10.1002/cncy.21310] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2013] [Revised: 04/10/2013] [Accepted: 04/11/2013] [Indexed: 12/16/2023]
Abstract
BACKGROUND Recent studies have demonstrated that partial or complete loss of E-cadherin (EC) and nuclear accumulation of zeste homolog 2 (EZH2) are hallmarks of poorly differentiated pancreatic adenocarcinoma (PAC). Depletion of EZH2 sensitizes cancer cells to chemotherapy in vitro. The objective of this study was to determine EC and EZH2 expression by immunohistochemistry (IHC) in samples obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) as potential biomarkers for treatment and disease prognosis. METHODS Thirty-eight EUS-FNA samples from patients with a PAC diagnosis were analyzed by IHC for EC and EZH2 expression. Seven corresponding surgical resection specimens were included in the study. The intensity of EZH2 and EC expression in PAC and in normal gastrointestinal pick-ups (internal positive control) was scored by using a 4-tier grading system. RESULTS EC demonstrated a focal, weak-to-moderate decrease in 24 PAC samples. Complete loss of EC expression was observed in the poorly differentiated areas represented by single tumor cells. The average staining intensity of EC in samples of poorly differentiated PAC was significantly lower than that of moderately differentiated PAC samples (P = .0005). EZH2 was variably positive in 31 of 38 PAC samples. The average staining intensity of EZH2 in moderately and poorly differentiated PACs did not differ significantly (P = .81). CONCLUSIONS EC and EZH2 expression was determined reliably by IHC on paraffin sections of EUS-FNA cell block specimens. The current results were consistent with prior reports indicating a decrease or loss of EC expression in poorly differentiated PAC. However, EZH2 expression did not always correlate inversely with EC expression and was more heterogeneous.
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Affiliation(s)
- Ling Gao
- Department of Pathology, The University of Chicago, Chicago, Illinois
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Hirano T, Satow R, Kato A, Tamura M, Murayama Y, Saya H, Kojima H, Nagano T, Okabe T, Fukami K. Identification of novel small compounds that restore E-cadherin expression and inhibit tumor cell motility and invasiveness. Biochem Pharmacol 2013; 86:1419-29. [PMID: 24035834 DOI: 10.1016/j.bcp.2013.09.001] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2013] [Revised: 09/05/2013] [Accepted: 09/05/2013] [Indexed: 01/27/2023]
Abstract
Tumor dissemination and invasive behavior are associated with a majority of cancer-related mortality cases. Loss of E-cadherin, which is caused by several tumor-promoting factors, is associated with metastasis and poor prognosis in many neoplasms. In this study, we aimed to identify small molecule compounds that restore the expression of E-cadherin, because these molecules are most likely to suppress tumor malignancy by restoring E-cadherin function and/or by inhibiting signals that suppress E-cadherin expression. Here, we developed a fluorescence screen system based on E-cadherin expression. A pilot drug library screen revealed that methotrexate (MTX) strongly induces E-cadherin expression in a colorectal cancer cell line, SW620. From the screen for 9600 compounds, we identified 9 hit compounds, which restored the expression of E-cadherin in SW620 and/or a melanoma cell line, SK-MEL-28. We confirmed that MTX and the other identified compounds transcriptionally promote E-cadherin expression. Among these, 2 compounds suppressed migration/invasion capacity in colorectal cancer cells and 3 in melanoma cells. A compound reduced SW620 migration and invasion with subtle effects on cell viability in SW620, SK-MEL-28, and a non-tumor cell line, HaCaT, with decrease in AKT and ERK1/2 protein levels. One of the other compounds reduced SK-MEL-28 cell migration and invasion and affected the viability only of SW620 and SK-MEL-28 cells but not HaCaT cells. These results suggest that these compounds would be attractive lead molecules as anti-metastasis agents.
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Affiliation(s)
- Tamaki Hirano
- Laboratory of Genome and Biosignal, Tokyo University of Pharmacy and Life Sciences, Hachioji-shi, Tokyo 192-0392, Japan
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48
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ETV4 promotes metastasis in response to activation of PI3-kinase and Ras signaling in a mouse model of advanced prostate cancer. Proc Natl Acad Sci U S A 2013; 110:E3506-15. [PMID: 23918374 DOI: 10.1073/pnas.1303558110] [Citation(s) in RCA: 111] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Combinatorial activation of PI3-kinase and RAS signaling occurs frequently in advanced prostate cancer and is associated with adverse patient outcome. We now report that the oncogenic Ets variant 4 (Etv4) promotes prostate cancer metastasis in response to coactivation of PI3-kinase and Ras signaling pathways in a genetically engineered mouse model of highly penetrant, metastatic prostate cancer. Using an inducible Cre driver to simultaneously inactivate Pten while activating oncogenic Kras and a fluorescent reporter allele in the prostate epithelium, we performed lineage tracing in vivo to define the temporal and spatial occurrence of prostate tumors, disseminated tumor cells, and metastases. These analyses revealed that though disseminated tumors cells arise early following the initial occurrence of prostate tumors, there is a significant temporal lag in metastasis, which is temporally coincident with the up-regulation of Etv4 expression in primary tumors. Functional studies showed that knockdown of Etv4 in a metastatic cell line derived from the mouse model abrogates the metastatic phenotype but does not affect tumor growth. Notably, expression and activation of ETV4, but not other oncogenic ETS genes, is correlated with activation of both PI3-kinase and Ras signaling in human prostate tumors and metastases. Our findings indicate that ETV4 promotes metastasis in prostate tumors that have activation of PI3-kinase and Ras signaling, and therefore, ETV4 represents a potential target of therapeutic intervention for metastatic prostate cancer.
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49
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Impaired expression of protein phosphatase 2A subunits enhances metastatic potential of human prostate cancer cells through activation of AKT pathway. Br J Cancer 2013; 108:2590-600. [PMID: 23598299 PMCID: PMC3694226 DOI: 10.1038/bjc.2013.160] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Background: Protein phosphatase 2A (PP2A) is a dephosphorylating enzyme, loss of which can contribute to prostate cancer (PCa) pathogenesis. The aim of this study was to analyse the transcriptional and translational expression patterns of individual subunits of the PP2A holoenzyme during PCa progression. Methods: Immunohistochemistry (IHC), western blot, and real-time PCR was performed on androgen-dependent (AD) and androgen-independent (AI) PCa cells, and benign and malignant prostate tissues for all the three PP2A (scaffold, regulatory, and catalytic) subunits. Mechanistic and functional studies were performed using various biochemical and cellular techniques. Results: Through immunohistochemical analysis we observed significantly reduced levels of PP2A-A and -B′γ subunits (P<0.001 and P=0.0002) in PCa specimens compared with benign prostate. Contemporarily, there was no significant difference in PP2A-C subunit expression between benign and malignant tissues. Similar to the expression pattern observed in tissues, the endogenous levels of PP2A-A and B′γ subunits were abrogated from the low metastatic to high metastatic and AD to AI cell line models, without any change in the catalytic subunit expression. Furthermore, using in vitro studies we demonstrated that PP2A-Aα scaffold subunit has a role in dampening AKT, β-catenin, and FAK (focal adhesion kinase) signalling. Conclusion: We conclude that loss of expression of scaffold and regulatory subunits of PP2A is responsible for its altered function during PCa pathogenesis.
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50
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Guo S, Li Y, Tong Q, Gu F, Zhu T, Fu L, Yang S. δEF1 down-regulates ER-α expression and confers tamoxifen resistance in breast cancer. PLoS One 2012; 7:e52380. [PMID: 23285017 PMCID: PMC3528679 DOI: 10.1371/journal.pone.0052380] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2012] [Accepted: 11/12/2012] [Indexed: 12/26/2022] Open
Abstract
Resistance to tamoxifen therapy represents a major barrier to the successful treatment of breast cancer, where a loss of or reduced ER-α level is considered a primary mechanism. Understanding how ER-α expression is regulated would provide insights into new intervention points to overcome tamoxifen resistance. In this study, we report that the expression of δEF1 is up-regulated by 17β-estradiol (E2) in MCF-7 cells in an ER-α-dependent manner, through either PI3K or NF-κB pathway. Ectopic expression of δEF1 in turn repressed ER-α transcription by binding to the E(2)-box on the ER-α promoter. At the tissue level of breast cancer, there is a strong and inverse correlation between the expression levels of δEF1 and ER-α. In MCF-7 cells, an elevated expression of δEF1 made the cells less sensitive to tamoxifen treatment, whereas overexpression of ER-α compromised the effects of δEF1 and restored the sensitivity. Also, depletion of δEF1 by RNA interference in MDA-MB-231 cells restored the expression of ER-α and tamoxifen sensitivity. In conclusion, we have identified an important role of δEF1 in the development of tamoxifen resistance in breast cancer. Inhibiting δEF1 to restore ER-α expression might represent a potential therapeutic strategy for overcoming endocrine resistance in breast cancer.
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Affiliation(s)
- Shaocong Guo
- Medical College of Nankai University, Tianjin, China
| | - Yaqing Li
- Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
| | - Qi Tong
- Medical College of Nankai University, Tianjin, China
| | - Feng Gu
- Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
| | - Tianhui Zhu
- Medical College of Nankai University, Tianjin, China
| | - Li Fu
- Tianjin Medical University Cancer Institute and Hospital, Tianjin, China
| | - Shuang Yang
- Medical College of Nankai University, Tianjin, China
- * E-mail:
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