|
1
|
Tang M, Rong D, Gao X, Lu G, Tang H, Wang P, Shao NY, Xia D, Feng XH, He WF, Chen W, Lu JH, Liu W, Shen HM. A positive feedback loop between SMAD3 and PINK1 in regulation of mitophagy. Cell Discov 2025; 11:22. [PMID: 40064862 PMCID: PMC11894195 DOI: 10.1038/s41421-025-00774-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2024] [Accepted: 01/14/2025] [Indexed: 03/14/2025] Open
Abstract
PTEN-induced kinase-1 (PINK1) is a crucial player in selective clearance of damaged mitochondria via the autophagy-lysosome pathway, a process termed mitophagy. Previous studies on PINK1 mainly focused on its post-translational modifications, while the transcriptional regulation of PINK1 is much less understood. Herein, we reported a novel mechanism in control of PINK1 transcription by SMAD Family Member 3 (SMAD3), an essential component of the transforming growth factor beta (TGFβ)-SMAD signaling pathway. First, we observed that mitochondrial depolarization promotes PINK1 transcription, and SMAD3 is likely to be the nuclear transcription factor mediating PINK1 transcription. Intriguingly, SMAD3 positively transactivates PINK1 transcription independent of the canonical TGFβ signaling components, such as TGFβ-R1, SMAD2 or SMAD4. Second, we found that mitochondrial depolarization activates SMAD3 via PINK1-mediated phosphorylation of SMAD3 at serine 423/425. Therefore, PINK1 and SMAD3 constitute a positive feedforward loop in control of mitophagy. Finally, activation of PINK1 transcription by SMAD3 provides an important pro-survival signal, as depletion of SMAD3 sensitizes cells to cell death caused by mitochondrial stress. In summary, our findings identify a non-canonical function of SMAD3 as a nuclear transcriptional factor in regulation of PINK1 transcription and mitophagy and a positive feedback loop via PINK1-mediated SMAD3 phosphorylation and activation. Understanding this novel regulatory mechanism provides a deeper insight into the pathological function of PINK1 in the pathogenesis of neurodegenerative diseases such as Parkinson's disease.
Collapse
Affiliation(s)
- Mingzhu Tang
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China
| | - Dade Rong
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China
| | - Xiangzheng Gao
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China
| | - Guang Lu
- Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, China
| | - Haimei Tang
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China
- Department of Immunology, Shenzhen University School of Medicine, Shenzhen, Guangdong, China
| | - Peng Wang
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China
| | - Ning-Yi Shao
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China
| | - Dajing Xia
- Department of Toxicology of School of Public Health and Department of Gynecologic Oncology of Women's Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, China
| | - Xin-Hua Feng
- Life Science Institute, Zhejiang University, Hangzhou, Zhejiang, China
| | - Wei-Feng He
- State Key Laboratory of Trauma, Burn and Combined Injury, Institute of Burn Research, Southwest Hospital, Army Medical University, Chongqing, China
| | - Weilin Chen
- Department of Immunology, Shenzhen University School of Medicine, Shenzhen, Guangdong, China
| | - Jia-Hong Lu
- State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China
| | - Wei Liu
- Center for Metabolism Research, the Fourth Affiliated Hospital of Zhejiang University School of Medicine, and International School of Medicine, International Institutes of Medicine, Zhejiang University, Yiwu, Zhejiang, China
| | - Han-Ming Shen
- Faculty of Healthy Sciences, Ministry of Education Frontiers Science Center for Precision Oncology, University of Macau, Macau, China.
| |
Collapse
|
|
2
|
Hlophe YN, Joubert AM. Vascular endothelial growth
factor‐C
in activating vascular endothelial growth factor receptor‐3 and chemokine receptor‐4 in melanoma adhesion. J Cell Mol Med 2022; 26:5743-5754. [DOI: 10.1111/jcmm.17571] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2022] [Revised: 08/27/2022] [Accepted: 09/12/2022] [Indexed: 11/19/2022] Open
Affiliation(s)
- Yvette N. Hlophe
- Department of Physiology University of Pretoria Pretoria South Africa
| | - Anna M. Joubert
- Department of Physiology University of Pretoria Pretoria South Africa
| |
Collapse
|
|
3
|
Importance of Fibrosis in the Pathogenesis of Uterine Leiomyoma and the Promising Anti-fibrotic Effects of Dipeptidyl Peptidase-4 and Fibroblast Activation Protein Inhibitors in the Treatment of Uterine Leiomyoma. Reprod Sci 2022; 30:1383-1398. [PMID: 35969363 DOI: 10.1007/s43032-022-01064-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2022] [Accepted: 08/10/2022] [Indexed: 10/15/2022]
Abstract
Uterine fibroid or leiomyoma is the most common benign uterus tumor. The tumor is primarily composed of smooth muscle (fibroid) cells, myofibroblast, and a significant amount of extracellular matrix components. It mainly affects women of reproductive age. They are uncommon before menarche and usually disappear after menopause. The fibroids have excessive extracellular matrix components secreted by activated fibroblast cells (myofibroblast). Myofibroblast has the characteristics of fibroblast and smooth muscle cells. These cells possess contractile capability due to the expression of contractile proteins which are normally found only in muscle tissues. The rigid nature of the tumor is responsible for many side effects associated with uterine fibroids. The current drug treatment strategies are primarily hormone-driven and not anti-fibrotic. This paper emphasizes the fibrotic background of uterine fibroids and the mechanisms behind the deposition of excessive extracellular matrix components. The transforming growth factor-β, hippo, and focal adhesion kinase-mediated signaling pathways activate the fibroblast cells and deposit excessive extracellular matrix materials. We also exemplify how dipeptidyl peptidase-4 and fibroblast activation protein inhibitors could be beneficial in reducing the fibrotic process in leiomyoma. Dipeptidyl peptidase-4 and fibroblast activation protein inhibitors prevent the fibrotic process in organs such as the kidneys, lungs, liver, and heart. These inhibitors are proven to inhibit the signaling pathways mentioned above at various stages of their activation. Based on literature evidence, we constructed a narrative review on the mechanisms that support the beneficial effects of dipeptidyl peptidase-4 and fibroblast activation protein inhibitors for treating uterine fibroids.
Collapse
|
|
4
|
Golan T, Parikh R, Jacob E, Vaknine H, Zemser-Werner V, Hershkovitz D, Malcov H, Leibou S, Reichman H, Sheinboim D, Percik R, Amar S, Brenner R, Greenberger S, Kung A, Khaled M, Levy C. Adipocytes sensitize melanoma cells to environmental TGF-β cues by repressing the expression of miR-211. Sci Signal 2019; 12:12/591/eaav6847. [PMID: 31337739 DOI: 10.1126/scisignal.aav6847] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Transforming growth factor-β (TGF-β) superfamily members are critical signals in tissue homeostasis and pathogenesis. Melanoma grows in the epidermis and invades the dermis before metastasizing. This disease progression is accompanied by increased sensitivity to microenvironmental TGF-β. Here, we found that skin fat cells (adipocytes) promoted metastatic initiation by sensitizing melanoma cells to TGF-β. Analysis of melanoma clinical samples revealed that adipocytes, usually located in the deeper hypodermis layer, were present in the upper dermis layer within proximity to in situ melanoma cells, an observation that correlated with disease aggressiveness. In a coculture system, adipocytes secreted the cytokines IL-6 and TNF-α, which induced a proliferative-to-invasive phenotypic switch in melanoma cells by repressing the expression of the microRNA miR-211. In a xenograft model, miR-211 exhibited a dual role in melanoma progression, promoting cell proliferation while inhibiting metastatic spread. Bioinformatics and molecular analyses indicated that miR-211 directly targeted and repressed the translation of TGFBR1 mRNA, which encodes the type I TGF-β receptor. Hence, through this axis of cytokine-mediated repression of miR-211, adipocytes increased the abundance of the TGF-β receptor in melanoma cells, thereby enhancing cellular responsiveness to TGF-β ligands. The induction of TGF-β signaling, in turn, resulted in a proliferative-to-invasive phenotypic switch in cultured melanoma cells. Pharmacological inhibition of TGF-β prevented these effects. Our findings further reveal a molecular link between fat cells and metastatic progression in melanoma that might be therapeutically targeted in patients.
Collapse
Affiliation(s)
- Tamar Golan
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Roma Parikh
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Etai Jacob
- The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 5290002, Israel.,Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.,Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.,Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Hananya Vaknine
- Institute of Pathology, E. Wolfson Medical Center, Holon 58100, Israel
| | | | - Dov Hershkovitz
- Institute of Pathology, Tel Aviv Sourasky Medical Center, Tel Aviv 6423906, Israel.,Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Hagar Malcov
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Stav Leibou
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Hadar Reichman
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Danna Sheinboim
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
| | - Ruth Percik
- Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.,Institute of Endocrinology, Chaim Sheba Medical Center, Tel Hashomer, Israel
| | - Sarah Amar
- Institute of Pathology, E. Wolfson Medical Center, Holon 58100, Israel
| | - Ronen Brenner
- Institute of Pathology, E. Wolfson Medical Center, Holon 58100, Israel
| | | | - Andrew Kung
- Department of Pediatrics, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA
| | - Mehdi Khaled
- INSERM 1186, Gustave Roussy, Université Paris-Saclay, Villejuif 94805, France
| | - Carmit Levy
- Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
| |
Collapse
|
|
5
|
Valenti MT, Dalle Carbonare L, Mottes M. Ectopic expression of the osteogenic master gene RUNX2 in melanoma Maria Teresa Valenti, Luca Dalle Carbonare, Monica Mottes. World J Stem Cells 2018; 10:78-81. [PMID: 30079129 PMCID: PMC6068731 DOI: 10.4252/wjsc.v10.i7.78] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/17/2018] [Revised: 06/11/2018] [Accepted: 06/28/2018] [Indexed: 02/06/2023] Open
Abstract
The transcription factor RUNX2 is the osteogenic master gene expressed in mesenchymal stem cells during osteogenic commitment as well as in pre-osteoblasts and early osteoblasts. However, RUNX2 is also ectopically expressed in melanoma and other cancers. Malignant melanoma (MM) is a highly metastatic skin cancer. The incidence of MM has increased considerably in the past half-century. The expression levels and mutation rates of genes such as BRAF, KIT, NRAS, PTEN, P53, TERT and MITF are higher in melanoma than in other solid malignancies. Additionally, transcription factors can affect cellular processes and induce cellular transformation since they control gene expression. Recently, several studies have identified alterations in RUNX2 expression. In particular, the regulation of KIT by RUNX2 and the increased expression of RUNX2 in melanoma specimens have been shown. Melanocytes, whose transformation results in melanoma, arise from the neural crest and therefore show “stemness” features. RUNX2 plays an important role in the re-activation of the MAPK and PI3K/AKT pathways, thus endowing melanoma cells with a high metastatic potential. In melanoma, the most frequent metastatic sites are the lung, liver, brain and lymph nodes. In addition, bone metastatic melanoma has been described. Notably, studies focusing on RUNX2 may contribute to the identification of an appropriate oncotarget in melanoma.
Collapse
Affiliation(s)
| | | | - Monica Mottes
- Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Verona 37100, Italy
| |
Collapse
|
|
6
|
Hou L, Liu Q, Shen L, Liu Y, Zhang X, Chen F, Huang L. Nano-delivery of fraxinellone remodels tumor microenvironment and facilitates therapeutic vaccination in desmoplastic melanoma. Theranostics 2018; 8:3781-3796. [PMID: 30083259 PMCID: PMC6071534 DOI: 10.7150/thno.24821] [Citation(s) in RCA: 67] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2018] [Accepted: 03/16/2018] [Indexed: 01/05/2023] Open
Abstract
Rationale: Tumor-associated fibroblasts (TAFs) play a critical role in the suppressive immune tumor microenvironment (TME), compromising the efficacy of immunotherapy. To overcome this therapeutic hurdle, we developed a nanoemulsion (NE) formulation to deliver fraxinellone (Frax), an anti-fibrotic medicine, to TAFs, as an approach to reverse immunosuppressive TME of desmoplastic melanoma. Methods: Frax NE was prepared by an ultrasonic emulsification method. The tumor inhibition effect was evaluated by immunofluorescence staining, masson trichrome staining and western blot analysis. Immune cell populations in tumor and LNs were detected by flow cytometry. Results: This Frax NE, with a particle size of around 145 nm, can efficiently accumulate in the tumor site after systemic administration and was taken up by TAFs and tumor cells. A significant decrease in TAFs and stroma deposition was observed after intravenous administration of Frax NE, and Frax NE treatment also remolded the tumor immune microenvironment, as was reflected by an increase of natural-killer cells, cytotoxic T cells (CTLs) as well as a decrease of regulatory B cells, and myeloid-derived suppressor cells in the TME. In addition, after treatment by Frax NEs, T helper 1 (Th1) cytokines of interferon gamma (IFN-γ), which effectively elicit anti-tumor immunity, were enhanced. Transforming growth factor-β (TGF-β), chemokine (C-C motif) ligand 2 (CCL2) and interleukin 6 (IL6), which inhibit the development of anti-tumor immunity, were reduced. Although Frax NE demonstrated an inhibitory effect on tumor growth, this mono-therapy could only achieve partial antitumor efficacy, and the tumor growth effect was not maintained long-term after dosing stopped. Therefore, a tumor-specific peptide vaccine was combined with Frax NEs. The combination led to enhanced tumor-specific T-cell infiltration, activated death receptors on the tumor cell surface, and induced increased apoptotic tumor cell death. Conclusion: Collectively, Frax NE combined with tumor-specific peptide vaccine might be an effective and safe strategy to remodel fibrotic TME, thereby enhancing immune response activation, resulting in a prolonged efficiency for advanced desmoplastic melanoma.
Collapse
Affiliation(s)
- Lin Hou
- Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
- School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, China
- Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, China
- Key Laboratory of Targeting and Diagnosis for Critical Diseases, Henan Province, China
| | - Qi Liu
- Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Limei Shen
- Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Yun Liu
- Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Xueqiong Zhang
- Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| | - Fengqian Chen
- Department of Environmental Toxicology, The Institute of Environmental and Human Health (TIEHH) and the Center for Biotechnology & Genomics, Texas Tech University, Lubbock, TX 79416, USA
| | - Leaf Huang
- Division of Pharmacoengineering and Molecular Pharmaceutics and Center for Nanotechnology in Drug Delivery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
| |
Collapse
|
|
7
|
Grainyhead-like 2 (GRHL2) knockout abolishes oral cancer development through reciprocal regulation of the MAP kinase and TGF-β signaling pathways. Oncogenesis 2018; 7:38. [PMID: 29735981 PMCID: PMC5938237 DOI: 10.1038/s41389-018-0047-5] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2017] [Revised: 02/25/2018] [Accepted: 04/05/2018] [Indexed: 01/01/2023] Open
Abstract
Grainyhead-Like 2 (GRHL2) is an epithelial-specific transcription factor that regulates epithelial morphogenesis and differentiation. Prior studies suggested inverse regulation between GRHL2 and TGF-β in epithelial plasticity and potential carcinogenesis. Here, we report the role of GRHL2 in oral carcinogenesis in vivo using a novel Grhl2 knockout (KO) mouse model and the underlying mechanism involving its functional interaction with TGF-β signaling. We developed epithelial-specific Grhl2 conditional KO mice by crossing Grhl2 floxed mice with those expressing CreER driven by the K14 promoter. After induction of Grhl2 KO, we confirmed the loss of GRHL2 and its target proteins, while Grhl2 KO strongly induced TGF-β signaling molecules. When exposed to 4-nitroquinoline 1-oxide (4-NQO), a strong chemical carcinogen, Grhl2 wild-type (WT) mice developed rampant oral tongue tumors, while Grhl2 KO mice completely abolished tumor development. In cultured oral squamous cell carcinoma (OSCC) cell lines, TGF-β signaling was notably induced by GRHL2 knockdown while being suppressed by GRHL2 overexpression. GRHL2 knockdown or KO in vitro and in vivo, respectively, led to loss of active p-Erk1/2 and p-JNK MAP kinase levels; moreover, ectopic overexpression of GRHL2 strongly induced the MAP kinase activation. Furthermore, the suppressive effect of GRHL2 on TGF-β signaling was diminished in cells exposed to Erk and JNK inhibitors. These data indicate that GRHL2 activates the Erk and JNK MAP kinases, which in turn suppresses the TGF -β signaling. This novel signaling represents an alternative pathway by which GRHL2 regulates carcinogenesis, and is distinct from the direct transcriptional regulation by GRHL2 binding at its target gene promoters, e.g., E-cadherin, hTERT, p63, and miR-200 family genes. Taken together, the current study provides the first genetic evidence to support the role of GRHL2 in carcinogenesis and the underlying novel mechanism that involves the functional interaction between GRHL2 and TGF-β signaling through the MAPK pathways.
Collapse
|
|
8
|
Zecena H, Tveit D, Wang Z, Farhat A, Panchal P, Liu J, Singh SJ, Sanghera A, Bainiwal A, Teo SY, Meyskens FL, Liu-Smith F, Filipp FV. Systems biology analysis of mitogen activated protein kinase inhibitor resistance in malignant melanoma. BMC SYSTEMS BIOLOGY 2018; 12:33. [PMID: 29615030 PMCID: PMC5883534 DOI: 10.1186/s12918-018-0554-1] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 10/29/2017] [Accepted: 02/21/2018] [Indexed: 11/12/2022]
Abstract
BACKGROUND Kinase inhibition in the mitogen activated protein kinase (MAPK) pathway is a standard therapy for cancer patients with activating BRAF mutations. However, the anti-tumorigenic effect and clinical benefit are only transient, and tumors are prone to treatment resistance and relapse. To elucidate mechanistic insights into drug resistance, we have established an in vitro cellular model of MAPK inhibitor resistance in malignant melanoma. METHODS The cellular model evolved in response to clinical dosage of the BRAF inhibitor, vemurafenib, PLX4032. We conducted transcriptomic expression profiling using RNA-Seq and RT-qPCR arrays. Pathways of melanogenesis, MAPK signaling, cell cycle, and metabolism were significantly enriched among the set of differentially expressed genes of vemurafenib-resistant cells vs control. The underlying mechanism of treatment resistance and pathway rewiring was uncovered to be based on non-genomic adaptation and validated in two distinct melanoma models, SK-MEL-28 and A375. Both cell lines have activating BRAF mutations and display metastatic potential. RESULTS Downregulation of dual specific phosphatases, tumor suppressors, and negative MAPK regulators reengages mitogenic signaling. Upregulation of growth factors, cytokines, and cognate receptors triggers signaling pathways circumventing BRAF blockage. Further, changes in amino acid and one-carbon metabolism support cellular proliferation despite MAPK inhibitor treatment. In addition, treatment-resistant cells upregulate pigmentation and melanogenesis, pathways which partially overlap with MAPK signaling. Upstream regulator analysis discovered significant perturbation in oncogenic forkhead box and hypoxia inducible factor family transcription factors. CONCLUSIONS The established cellular models offer mechanistic insight into cellular changes and therapeutic targets under inhibitor resistance in malignant melanoma. At a systems biology level, the MAPK pathway undergoes major rewiring while acquiring inhibitor resistance. The outcome of this transcriptional plasticity is selection for a set of transcriptional master regulators, which circumvent upstream targeted kinases and provide alternative routes of mitogenic activation. A fine-woven network of redundant signals maintains similar effector genes allowing for tumor cell survival and malignant progression in therapy-resistant cancer.
Collapse
Affiliation(s)
- Helma Zecena
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| | - Daniel Tveit
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| | - Zi Wang
- Department of Medicine,
School of Medicine, Chao Family Comprehensive Cancer Center,
University of California Irvine,
Irvine, CA 92697 USA
- The State Key Laboratory of
Medical Genetics and School of Life Sciences, Department of Molecular
Biology, Central South University,
Changsha, 410078 China
| | - Ahmed Farhat
- Department of Medicine,
School of Medicine, Chao Family Comprehensive Cancer Center,
University of California Irvine,
Irvine, CA 92697 USA
| | - Parvita Panchal
- Department of Medicine,
School of Medicine, Chao Family Comprehensive Cancer Center,
University of California Irvine,
Irvine, CA 92697 USA
| | - Jing Liu
- Department of Medicine,
School of Medicine, Chao Family Comprehensive Cancer Center,
University of California Irvine,
Irvine, CA 92697 USA
- The State Key Laboratory of
Medical Genetics and School of Life Sciences, Department of Molecular
Biology, Central South University,
Changsha, 410078 China
| | - Simar J. Singh
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| | - Amandeep Sanghera
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| | - Ajay Bainiwal
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| | - Shuan Y. Teo
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| | - Frank L. Meyskens
- Department of Medicine,
School of Medicine, Chao Family Comprehensive Cancer Center,
University of California Irvine,
Irvine, CA 92697 USA
| | - Feng Liu-Smith
- Department of Medicine,
School of Medicine, Chao Family Comprehensive Cancer Center,
University of California Irvine,
Irvine, CA 92697 USA
- Department of Epidemiology,
School of Medicine, University of California,
Irvine, CA 92697 USA
| | - Fabian V. Filipp
- Systems Biology and Cancer
Metabolism, Program for Quantitative Systems Biology,
University of California Merced,
2500 North Lake Road, Merced, CA 95343 USA
| |
Collapse
|
|
9
|
D'Arcangelo D, Facchiano F, Nassa G, Stancato A, Antonini A, Rossi S, Senatore C, Cordella M, Tabolacci C, Salvati A, Tarallo R, Weisz A, Facchiano AM, Facchiano A. PDGFR-alpha inhibits melanoma growth via CXCL10/IP-10: a multi-omics approach. Oncotarget 2018; 7:77257-77275. [PMID: 27764787 PMCID: PMC5363585 DOI: 10.18632/oncotarget.12629] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Accepted: 09/25/2016] [Indexed: 12/21/2022] Open
Abstract
Melanoma is the most aggressive skin-cancer, showing high mortality at advanced stages. Platelet Derived Growth Factor Receptor-alpha (PDGFR-alpha) potently inhibits melanoma- and endothelium-proliferation and its expression is significantly reduced in melanoma-biopsies, suggesting that melanoma progression eliminates cells expressing PDGFR-alpha. In the present study transient overexpression of PDGFR-alpha in endothelial (HUVEC) and melanoma (SKMel-28, A375, Preyer) human-cells shows strong anti-proliferative effects, with profound transcriptome and miRNome deregulation. PDGFR-alpha overexpression strongly affects expression of 82 genes in HUVEC (41 up-, 41 down-regulated), and 52 genes in SKMel-28 (43 up-, 9 down-regulated). CXCL10/IP-10 transcript showed up to 20 fold-increase, with similar changes detectable at the protein level. miRNA expression profiling in cells overexpressing PDGFR-alpha identified 14 miRNAs up- and 40 down-regulated, with miR-503 being the most down-regulated (6.4 fold-reduction). miR-503, miR-630 and miR-424 deregulation was confirmed by qRT-PCR. Interestingly, the most upregulated transcript (i.e., CXCL10/IP-10) was a validated miR-503 target and CXCL10/IP-10 neutralization significantly reverted the anti-proliferative action of PDGFR-alpha, and PDGFR-alpha inhibition by Dasatinb totally reverted the CXCL10/IP10 induction, further supporting a functional interplay of these factors. Finally, integration of transcriptomics and miRNomics data highlighted several pathways affected by PDGFR-alpha. This study demonstrates for the first time that PDGFR-alpha strongly inhibits endothelial and melanoma cells proliferation in a CXCL10/IP-10 dependent way, via miR-503 down-regulation.
Collapse
Affiliation(s)
- Daniela D'Arcangelo
- Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Fondazione Luigi Maria Monti, Rome, Italy
| | - Francesco Facchiano
- Dipartimento Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy
| | - Giovanni Nassa
- Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery,University of Salerno, Baronissi (SA), Italy.,Genomix4Life srl, Department of Medicine and Surgery, University of Salerno, Baronissi (SA), Italy
| | - Andrea Stancato
- Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Fondazione Luigi Maria Monti, Rome, Italy
| | - Annalisa Antonini
- Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Fondazione Luigi Maria Monti, Rome, Italy
| | - Stefania Rossi
- Dipartimento Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy
| | - Cinzia Senatore
- Dipartimento Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy
| | - Martina Cordella
- Dipartimento Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy
| | - Claudio Tabolacci
- Dipartimento Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy
| | - Annamaria Salvati
- Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery,University of Salerno, Baronissi (SA), Italy
| | - Roberta Tarallo
- Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery,University of Salerno, Baronissi (SA), Italy
| | - Alessandro Weisz
- Laboratory of Molecular Medicine and Genomics, Department of Medicine and Surgery,University of Salerno, Baronissi (SA), Italy
| | | | - Antonio Facchiano
- Istituto Dermopatico dell'Immacolata, IDI-IRCCS, Fondazione Luigi Maria Monti, Rome, Italy
| |
Collapse
|
|
10
|
Spender LC, Ferguson GJ, Liu S, Cui C, Girotti MR, Sibbet G, Higgs EB, Shuttleworth MK, Hamilton T, Lorigan P, Weller M, Vincent DF, Sansom OJ, Frame M, Dijke PT, Marais R, Inman GJ. Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells. Oncotarget 2016; 7:81995-82012. [PMID: 27835901 PMCID: PMC5347669 DOI: 10.18632/oncotarget.13226] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2016] [Accepted: 07/18/2016] [Indexed: 12/25/2022] Open
Abstract
Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance.
Collapse
MESH Headings
- Animals
- Animals, Genetically Modified
- Antineoplastic Agents/pharmacology
- Benzamides/pharmacology
- Cell Line, Tumor
- Cell Proliferation/drug effects
- Dioxoles/pharmacology
- Dose-Response Relationship, Drug
- Drug Resistance, Neoplasm/drug effects
- Drug Resistance, Neoplasm/genetics
- Humans
- Indoles/pharmacology
- Melanocytes/drug effects
- Melanocytes/enzymology
- Melanocytes/pathology
- Melanoma/drug therapy
- Melanoma/enzymology
- Melanoma/genetics
- Melanoma/pathology
- Mice, Nude
- Mitosis/drug effects
- Mutation
- Protein Kinase Inhibitors/pharmacology
- Protein Serine-Threonine Kinases/antagonists & inhibitors
- Protein Serine-Threonine Kinases/genetics
- Protein Serine-Threonine Kinases/metabolism
- Proto-Oncogene Proteins B-raf/antagonists & inhibitors
- Proto-Oncogene Proteins B-raf/genetics
- Proto-Oncogene Proteins B-raf/metabolism
- RNA Interference
- Receptor, Transforming Growth Factor-beta Type I
- Receptors, Transforming Growth Factor beta/antagonists & inhibitors
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/metabolism
- Signal Transduction/drug effects
- Skin Neoplasms/drug therapy
- Skin Neoplasms/enzymology
- Skin Neoplasms/genetics
- Skin Neoplasms/pathology
- Smad4 Protein/genetics
- Smad4 Protein/metabolism
- Sulfonamides/pharmacology
- Time Factors
- Transfection
- Transforming Growth Factor beta1/pharmacology
- Vemurafenib
- Xenograft Model Antitumor Assays
- Zebrafish
Collapse
Affiliation(s)
- Lindsay C. Spender
- Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
- Division of Cancer Research, School of Medicine, University of Dundee, Dundee, United Kingdom
| | - G. John Ferguson
- Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
- Department of Respiratory, Inflammation and Autoimmunity Research, MedImmune Limited, Cambridge, United Kingdom
| | - Sijia Liu
- Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Einthovenweg, Leiden, Netherlands
| | - Chao Cui
- Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Einthovenweg, Leiden, Netherlands
| | - Maria Romina Girotti
- Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Withington, Manchester, United Kingdom
| | - Gary Sibbet
- Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
| | - Ellen B. Higgs
- Division of Cancer Research, School of Medicine, University of Dundee, Dundee, United Kingdom
| | - Morven K. Shuttleworth
- Division of Cancer Research, School of Medicine, University of Dundee, Dundee, United Kingdom
| | - Tom Hamilton
- Biological Services, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
| | - Paul Lorigan
- The University of Manchester, The Christie NHS Foundation Trust, Manchester, United Kingdom
| | - Michael Weller
- Department of Neurology, University Hospital Zurich, Frauenklinikstrasse, Zurich, Switzerland
| | - David F. Vincent
- Colorectal Cancer and Wnt Signalling, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
| | - Owen J. Sansom
- Colorectal Cancer and Wnt Signalling, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
| | - Margaret Frame
- The Institute of Genetics and Molecular Medicine, Edinburgh Cancer Research Centre, University of Edinburgh, Western General Hospital, Edinburgh, United Kingdom
| | - Peter ten Dijke
- Department of Molecular Cell Biology, Cancer Genomics Centre Netherlands, Leiden University Medical Center, Einthovenweg, Leiden, Netherlands
| | - Richard Marais
- Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Withington, Manchester, United Kingdom
| | - Gareth J. Inman
- Growth Factor Signalling Laboratory, The Beatson Institute for Cancer Research, Bearsden, Glasgow, United Kingdom
- Division of Cancer Research, School of Medicine, University of Dundee, Dundee, United Kingdom
| |
Collapse
|
|
11
|
Faião-Flores F, Alves-Fernandes DK, Pennacchi PC, Sandri S, Vicente ALSA, Scapulatempo-Neto C, Vazquez VL, Reis RM, Chauhan J, Goding CR, Smalley KS, Maria-Engler SS. Targeting the hedgehog transcription factors GLI1 and GLI2 restores sensitivity to vemurafenib-resistant human melanoma cells. Oncogene 2016; 36:1849-1861. [PMID: 27748762 PMCID: PMC5378933 DOI: 10.1038/onc.2016.348] [Citation(s) in RCA: 78] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2015] [Revised: 07/25/2016] [Accepted: 08/15/2016] [Indexed: 12/16/2022]
Abstract
BRAF inhibitor (BRAFi) therapy for melanoma patients harboring the V600E mutation is initially highly effective, but almost all patients relapse within a few months. Understanding the molecular mechanisms underpinning BRAFi-based therapy is therefore an important issue. Here we identified a previously unsuspected mechanism of BRAFi resistance driven by elevated Hedgehog (Hh) pathway activation that is observed in a cohort of melanoma patients after vemurafenib treatment. Specifically, we demonstrate that melanoma cell lines, with acquired in vitro-induced vemurafenib resistance, show increased levels of glioma-associated oncogene homolog 1 and 2 (GLI1/GLI2) compared with naïve cells. We also observed these findings in clinical melanoma specimens. Moreover, the increased expression of the transcription factors GLI1/GLI2 was independent of canonical Hh signaling and was instead correlated with the noncanonical Hh pathway, involving TGFβ/SMAD (transforming growth factor-β/Sma- and Mad-related family) signaling. Knockdown of GLI1 and GLI2 restored sensitivity to vemurafenib-resistant cells, an effect associated with both growth arrest and senescence. Treatment of vemurafenib-resistant cells with the GLI1/GLI2 inhibitor Gant61 led to decreased invasion of the melanoma cells in a three-dimensional skin reconstruct model and was associated with a decrease in metalloproteinase (MMP2/MMP9) expression and microphthalmia transcription factor upregulation. Gant61 monotherapy did not alter the drug sensitivity of naïve cells, but could reverse the resistance of melanoma cells chronically treated with vemurafenib. We further noted that alternating dosing schedules of Gant61 and vemurafenib prevented the onset of BRAFi resistance, suggesting that this could be a potential therapeutic strategy for the prevention of therapeutic escape. Our results suggest that targeting the Hh pathway in BRAFi-resistant melanoma may represent a viable therapeutic strategy to restore vemurafenib sensitivity, reducing or even inhibiting the acquired chemoresistance in melanoma patients.
Collapse
Affiliation(s)
- F Faião-Flores
- Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| | - D K Alves-Fernandes
- Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| | - P C Pennacchi
- Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| | - S Sandri
- Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| | - A L S A Vicente
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil
| | - C Scapulatempo-Neto
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil.,Department of Pathology, Barretos Cancer Hospital, Barretos, Brazil
| | - V L Vazquez
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil.,Department of Surgery Melanoma/Sarcoma, Barretos Cancer Hospital, Barretos, Brazil
| | - R M Reis
- Molecular Oncology Research Center, Barretos Cancer Hospital, Barretos, Brazil.,Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.,3B's - PT Government Associate Laboratory, Braga/Guimarães, Guimarães, Portugal
| | - J Chauhan
- Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Headington, Oxford, UK
| | - C R Goding
- Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Headington, Oxford, UK
| | - K S Smalley
- The Department of Tumor Biology, The Moffitt Cancer Center and Research Institute, Tampa, FL, USA
| | - S S Maria-Engler
- Department of Clinical Chemistry and Toxicological Analysis, School of Pharmaceutical Sciences, University of São Paulo, São Paulo, Brazil
| |
Collapse
|
|
12
|
Nahta R, Al-Mulla F, Al-Temaimi R, Amedei A, Andrade-Vieira R, Bay S, G. Brown D, Calaf GM, Castellino RC, Cohen-Solal KA, Colacci A, Cruickshanks N, Dent P, Di Fiore R, Forte S, Goldberg GS, Hamid RA, Krishnan H, Laird DW, Lasfar A, Marignani PA, Memeo L, Mondello C, Naus CC, Ponce-Cusi R, Raju J, Roy D, Roy R, P. Ryan E, Salem HK, Scovassi AI, Singh N, Vaccari M, Vento R, Vondráček J, Wade M, Woodrick J, Bisson WH. Mechanisms of environmental chemicals that enable the cancer hallmark of evasion of growth suppression. Carcinogenesis 2015; 36 Suppl 1:S2-S18. [PMID: 26106139 PMCID: PMC4565608 DOI: 10.1093/carcin/bgv028] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2014] [Revised: 09/01/2014] [Accepted: 09/19/2014] [Indexed: 12/18/2022] Open
Abstract
As part of the Halifax Project, this review brings attention to the potential effects of environmental chemicals on important molecular and cellular regulators of the cancer hallmark of evading growth suppression. Specifically, we review the mechanisms by which cancer cells escape the growth-inhibitory signals of p53, retinoblastoma protein, transforming growth factor-beta, gap junctions and contact inhibition. We discuss the effects of selected environmental chemicals on these mechanisms of growth inhibition and cross-reference the effects of these chemicals in other classical cancer hallmarks.
Collapse
Affiliation(s)
- Rita Nahta
- *To whom correspondence should be addressed.
| | - Fahd Al-Mulla
- Department of Pathology, Kuwait University, Safat 13110, Kuwait
| | | | - Amedeo Amedei
- Department of Experimental and Clinical Medicine, University of Firenze, 50134 Florence, Italy
| | - Rafaela Andrade-Vieira
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Sarah Bay
- Program in Genetics and Molecular Biology, Graduate Division of Biological and Biomedical Sciences, Emory University, Atlanta, GA 30322, USA
| | - Dustin G. Brown
- Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Gloria M. Calaf
- Center for Radiological Research, Columbia University Medical Center, New York, NY 10032, USA
- Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile
| | - Robert C. Castellino
- Division of Hematology and Oncology, Department of Pediatrics, Children’s Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA
| | - Karine A. Cohen-Solal
- Department of Medicine/Medical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ 08901-1914, USA
| | - Annamaria Colacci
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Nichola Cruickshanks
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA
| | - Paul Dent
- Departments of Neurosurgery and Biochemistry and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 980033, USA
| | - Riccardo Di Fiore
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy
| | - Stefano Forte
- Mediterranean Institute of Oncology, 95029 Viagrande, Italy
| | - Gary S. Goldberg
- Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA
| | - Roslida A. Hamid
- Department of Biomedical Science, Faculty of Medicine and Health Sciences, University Putra, Serdang, Selangor 43400, Malaysia
| | - Harini Krishnan
- Graduate School of Biomedical Sciences and Department of Molecular Biology, School of Osteopathic Medicine, Rowan University, Stratford, NJ 08084-1501, USA
| | - Dale W. Laird
- Department of Anatomy and Cell Biology, University of Western Ontario, London, Ontario N6A 5C1, Canada
| | - Ahmed Lasfar
- Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 60503, USA
| | - Paola A. Marignani
- Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada
| | - Lorenzo Memeo
- Mediterranean Institute of Oncology, 95029 Viagrande, Italy
| | - Chiara Mondello
- Institute of Molecular Genetics, National Research Council, 27100 Pavia, Italy
| | - Christian C. Naus
- Department of Cellular & Physiological Sciences, Life Sciences Institute, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
| | - Richard Ponce-Cusi
- Instituto de Alta Investigacion, Universidad de Tarapaca, Arica 8097877, Chile
| | - Jayadev Raju
- Toxicology Research Division, Bureau of Chemical Safety Food Directorate, Health Products and Food Branch Health Canada, Ottawa, Ontario K1A0K9, Canada
| | - Debasish Roy
- Department of Natural Science, The City University of New York at Hostos Campus, Bronx, NY 10451, USA
| | - Rabindra Roy
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - Elizabeth P. Ryan
- Department of Environmental and Radiological Health Sciences/Colorado State University/Colorado School of Public Health, Fort Collins, CO 80523-1680, USA
| | - Hosni K. Salem
- Urology Dept., kasr Al-Ainy School of Medicine, Cairo University, El Manial, Cairo 12515, Egypt
| | - A. Ivana Scovassi
- Institute of Molecular Genetics, National Research Council, 27100 Pavia, Italy
| | - Neetu Singh
- Advanced Molecular Science Research Centre, King George’s Medical University, Lucknow, UP 226003, India
| | - Monica Vaccari
- Center for Environmental Carcinogenesis and Risk Assessment, Environmental Protection and Health Prevention Agency, Bologna 40126, Italy
| | - Renza Vento
- Department of Biological, Chemical, and Pharmaceutical Sciences and Technologies, Polyclinic Plexus, University of Palermo, 90127 Palermo, Italy
- Sbarro Institute for Cancer Research and Molecular Medicine, College of Science and Technology, Temple University, Philadelphia, PA 19122, USA
| | - Jan Vondráček
- Department of Cytokinetics, Institute of Biophysics AS CR, Brno 612 65, Czech Republic
| | - Mark Wade
- Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia (IIT), Milan 16163, Italy and
| | - Jordan Woodrick
- Molecular Oncology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20057, USA
| | - William H. Bisson
- Environmental and Molecular Toxicology, Environmental Health Sciences Center, Oregon State University, Corvallis, OR 97331, USA
| |
Collapse
|
|
13
|
Humbert L, Ghozlan M, Canaff L, Tian J, Lebrun JJ. The leukemia inhibitory factor (LIF) and p21 mediate the TGFβ tumor suppressive effects in human cutaneous melanoma. BMC Cancer 2015; 15:200. [PMID: 25885043 PMCID: PMC4389797 DOI: 10.1186/s12885-015-1177-1] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2014] [Accepted: 03/06/2015] [Indexed: 12/26/2022] Open
Abstract
BACKGROUND Cutaneous melanoma is the most lethal skin cancer and its incidence in developed countries has dramatically increased over the past decades. Localized tumors are easily treated by surgery, but advanced melanomas lack efficient treatment and are associated with very poor outcomes. Thus, understanding the processes underlying melanoma development and progression is critical. The Transforming Growth Factor beta (TGFβ) acts as a potent tumor suppressor in human melanoma, by inhibiting cell growth and preventing cellular migration and invasion. METHODS In this study, we aimed at elucidating the molecular mechanisms underlying TGFβ-mediated tumor suppression. Human cutaneous melanoma cell lines, derived from different patients, were used to assess for cell cycle analysis, apoptosis/caspase activity and cell migration. Techniques involved immunoblotting, immunohistochemistry, real time PCR and luciferase reporter assays. RESULTS We found the leukemia inhibitory factor (LIF) to be strongly up-regulated by TGFβ in melanoma cells, defining LIF as a novel TGFβ downstream target gene in cutaneous melanoma. Interestingly, we also showed that TGFβ-mediated LIF expression is required for TGFβ-induced cell cycle arrest and caspase-mediated apoptosis, as well as for TGFβ-mediated inhibition of cell migration. Moreover, we found that TGFβ-mediated LIF expression leads to activation of transcription of the cell cycle inhibitor p21 in a STAT3-dependent manner, and further showed that p21 is required for TGFβ/LIF-mediated cell cycle arrest and TGFβ-induced gene activation of several pro-apoptotic genes. CONCLUSIONS Together, our results define the LIF/p21 signaling cascade as a novel tumor suppressive-like pathway in melanoma, acting downstream of TGFβ to regulate cell cycle arrest and cell death, further highlight new potential therapeutic strategies for the treatment of cutaneous melanoma.
Collapse
Affiliation(s)
- Laure Humbert
- Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada.
| | - Mostafa Ghozlan
- Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada.
| | - Lucie Canaff
- Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada.
| | - Jun Tian
- Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada.
| | - Jean-Jacques Lebrun
- Division of Medical Oncology, Department of Medicine, McGill University Health Centre, Montreal, QC, Canada.
- Department of Medicine, Royal Victoria Hospital, Suite H7.66, 687 Pine Avenue West, H3A 1A1, Montreal, QC, Canada.
| |
Collapse
|
|
14
|
Guo X, Xu Y, Zhao Z. In-depth genomic data analyses revealed complex transcriptional and epigenetic dysregulations of BRAFV600E in melanoma. Mol Cancer 2015; 14:60. [PMID: 25890285 PMCID: PMC4373107 DOI: 10.1186/s12943-015-0328-y] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2014] [Accepted: 02/26/2015] [Indexed: 12/30/2022] Open
Abstract
BACKGROUND The recurrent BRAF driver mutation V600E (BRAF (V600E)) is currently one of the most clinically relevant mutations in melanoma. However, the genome-wide transcriptional and epigenetic dysregulations induced by BRAF (V600E) are still unclear. The investigation of this driver mutation's functional consequences is critical to the understanding of tumorigenesis and the development of therapeutic strategies. METHODS AND RESULTS We performed an integrative analysis of transcriptomic and epigenomic changes disturbed by BRAF (V600E) by comparing the gene expression and methylation profiles of 34 primary cutaneous melanoma tumors harboring BRAF (V600E) with those of 27 BRAF (WT) samples available from The Cancer Genome Atlas (TCGA). A total of 711 significantly differentially expressed genes were identified as putative BRAF (V600E) target genes. Functional enrichment analyses revealed the transcription factor MITF (p < 3.6 × 10(-16)) and growth factor TGFB1 (p < 3.1 × 10(-9)) were the most significantly enriched up-regulators, with MITF being significantly up-regulated, whereas TGFB1 was significantly down-regulated in BRAF (V600E), suggesting that they may mediate tumorigenesis driven by BRAF (V600E). Further investigation using the MITF ChIP-Seq data confirmed that BRAF (V600E) led to an overall increased level of gene expression for the MITF targets. Furthermore, DNA methylation analysis revealed a global DNA methylation loss in BRAF (V600E) relative to BRAF (WT). This might be due to BRAF dysregulation of DNMT3A, which was identified as a potential target with significant down-regulation in BRAF (V600E). Finally, we demonstrated that BRAF (V600E) targets may play essential functional roles in cell growth and proliferation, measured by their effects on melanoma tumor growth using a short hairpin RNA silencing experimental dataset. CONCLUSIONS Our integrative analysis identified a set of BRAF (V600E) target genes. Further analyses suggested a complex mechanism driven by mutation BRAF (V600E) on melanoma tumorigenesis that disturbs specific cancer-related genes, pathways, and methylation modifications.
Collapse
Affiliation(s)
- Xingyi Guo
- Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN, 37203, USA.
- Division of Epidemiology, Department of Medicine, Vanderbilt Epidemiology Center, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
| | - Yaomin Xu
- Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN, 37203, USA.
- Department of Biostatistics, Vanderbilt University School of Medicine, Nashville, TN, 37203, USA.
- Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, TN, 37232, USA.
| | - Zhongming Zhao
- Department of Biomedical Informatics, Vanderbilt University School of Medicine, Nashville, TN, 37203, USA.
- Center for Quantitative Sciences, Vanderbilt University Medical Center, Nashville, TN, 37232, USA.
- Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA.
| |
Collapse
|
|
15
|
EWI-2 negatively regulates TGF-β signaling leading to altered melanoma growth and metastasis. Cell Res 2015; 25:370-85. [PMID: 25656846 DOI: 10.1038/cr.2015.17] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2014] [Revised: 09/24/2014] [Accepted: 11/07/2014] [Indexed: 01/02/2023] Open
Abstract
In normal melanocytes, TGF-β signaling has a cytostatic effect. However, in primary melanoma cells, TGF-β-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-β signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these "present-absent-present" TGF-β signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is "absent-present-absent" in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-β signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-β signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 - molecules not previously linked to TGF-β signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TβR2-TβR1 association or TGF-β signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TβR2-TβR1 association, thus markedly elevating TGF-β signaling. Consequently, all of those TGF-β-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-β signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.
Collapse
|
|
16
|
TANG MINGRUI, WANG YUXIN, GUO SHU, HAN SIYUAN, LI HEHUAN, JIN SHIFENG. Prognostic significance of in situ and plasma levels of transforming growth factor β1, -2 and -3 in cutaneous melanoma. Mol Med Rep 2015; 11:4508-12. [DOI: 10.3892/mmr.2015.3250] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2014] [Accepted: 01/02/2015] [Indexed: 11/05/2022] Open
|
|
17
|
Xu Y, Guo X, Sun J, Zhao Z. Snowball: resampling combined with distance-based regression to discover transcriptional consequences of a driver mutation. ACTA ACUST UNITED AC 2014; 31:84-93. [PMID: 25192743 DOI: 10.1093/bioinformatics/btu603] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
MOTIVATION Large-scale cancer genomic studies, such as The Cancer Genome Atlas (TCGA), have profiled multidimensional genomic data, including mutation and expression profiles on a variety of cancer cell types, to uncover the molecular mechanism of cancerogenesis. More than a hundred driver mutations have been characterized that confer the advantage of cell growth. However, how driver mutations regulate the transcriptome to affect cellular functions remains largely unexplored. Differential analysis of gene expression relative to a driver mutation on patient samples could provide us with new insights in understanding driver mutation dysregulation in tumor genome and developing personalized treatment strategies. RESULTS Here, we introduce the Snowball approach as a highly sensitive statistical analysis method to identify transcriptional signatures that are affected by a recurrent driver mutation. Snowball utilizes a resampling-based approach and combines a distance-based regression framework to assign a robust ranking index of genes based on their aggregated association with the presence of the mutation, and further selects the top significant genes for downstream data analyses or experiments. In our application of the Snowball approach to both synthesized and TCGA data, we demonstrated that it outperforms the standard methods and provides more accurate inferences to the functional effects and transcriptional dysregulation of driver mutations. AVAILABILITY AND IMPLEMENTATION R package and source code are available from CRAN at http://cran.r-project.org/web/packages/DESnowball, and also available at http://bioinfo.mc.vanderbilt.edu/DESnowball/.
Collapse
Affiliation(s)
- Yaomin Xu
- Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA
| | - Xingyi Guo
- Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA
| | - Jiayang Sun
- Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA
| | - Zhongming Zhao
- Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA Department of Biomedical Informatics, Department of Biostatistics and Center for Quantitative Sciences, Vanderbilt University, Nashville, TN 37232, Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, OH 44106, Department of Psychiatry and Department of Cancer Biology, Vanderbilt University, Nashville, TN 37212, USA
| |
Collapse
|
|
18
|
Boregowda RK, Olabisi OO, Abushahba W, Jeong BS, Haenssen KK, Chen W, Chekmareva M, Lasfar A, Foran DJ, Goydos JS, Cohen-Solal KA. RUNX2 is overexpressed in melanoma cells and mediates their migration and invasion. Cancer Lett 2014; 348:61-70. [PMID: 24657655 DOI: 10.1016/j.canlet.2014.03.011] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2013] [Revised: 03/04/2014] [Accepted: 03/07/2014] [Indexed: 12/12/2022]
Abstract
In the present study, we investigated the role of the transcription factor RUNX2 in melanomagenesis. We demonstrated that the expression of transcriptionally active RUNX2 was increased in melanoma cell lines as compared with human melanocytes. Using a melanoma tissue microarray, we showed that RUNX2 levels were higher in melanoma cells as compared with nevic melanocytes. RUNX2 knockdown in melanoma cell lines significantly decreased Focal Adhesion Kinase expression, and inhibited their cell growth, migration and invasion ability. Finally, the pro-hormone cholecalciferol reduced RUNX2 transcriptional activity and decreased migration of melanoma cells, further suggesting a role of RUNX2 in melanoma cell migration.
Collapse
Affiliation(s)
- Rajeev K Boregowda
- Rutgers Cancer Institute of New Jersey, Department of Medicine, Division of Medical Oncology - Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Oyenike O Olabisi
- Rutgers Cancer Institute of New Jersey, Department of Medicine, Division of Medical Oncology - Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Walid Abushahba
- Rutgers Cancer Institute of New Jersey, Department of Medicine, Division of Medical Oncology - Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Byeong-Seon Jeong
- Rutgers Cancer Institute of New Jersey, Department of Surgery, Division of Surgical Oncology, Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Keneshia K Haenssen
- Rutgers Cancer Institute of New Jersey, Department of Surgery, Division of Surgical Oncology, Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Wenjin Chen
- Center for Biomedical Imaging & Informatics - Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Marina Chekmareva
- Rutgers Cancer Institute of New Jersey - Department of Pathology and Laboratory Medicine, Robert Wood Johnson University Hospital, 1 RWJ Place, New Brunswick, NJ 08901, USA
| | - Ahmed Lasfar
- Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854, USA
| | - David J Foran
- Center for Biomedical Imaging & Informatics - Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - James S Goydos
- Rutgers Cancer Institute of New Jersey, Department of Surgery, Division of Surgical Oncology, Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA
| | - Karine A Cohen-Solal
- Rutgers Cancer Institute of New Jersey, Department of Medicine, Division of Medical Oncology - Rutgers, The State University of New Jersey, Robert Wood Johnson Medical School, 195 Little Albany Street, New Brunswick, NJ 08903, USA.
| |
Collapse
|
|
19
|
FKBP51 increases the tumour-promoter potential of TGF-beta. Clin Transl Med 2014; 3:1. [PMID: 24460977 PMCID: PMC3906759 DOI: 10.1186/2001-1326-3-1] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2013] [Accepted: 12/26/2013] [Indexed: 12/17/2022] Open
Abstract
Background FKBP51 (FKBP5 Official Symbol) is a large molecular weight component of the family of FK506 binding proteins (FKBP). In recent years, research studies from our laboratory highlighted functions for FKBP51 in the control of apoptosis and melanoma progression. FKBP51 expression correlated with the invasiveness and aggressiveness of melanoma. Since a role for TGF-β in the enhanced tumorigenic potential of melanoma cells is widely described, we hypothesized a cooperative effect between FKBP51 and TGF-β in melanoma progression. Methods SAN and A375 melanoma cell lines were utilized for this study. Balb/c IL2γ NOD SCID served to assess the ability to colonize organs and metastasize of different cell lines, which was evaluated by in vivo imaging. Realtime PCR and western blot served for measurement of mRNA and protein expression, respectively. Results By comparing the metastatic potential of two melanoma cell lines, namely A375 and SAN, we confirmed that an increased capability to colonize murine organs was associated with increased levels of FKBP51. A375 melanoma cell line expressed FKBP51 mRNA levels 30-fold higher in comparison to the SAN mRNA level and appeared more aggressive than SAN melanoma cell line in an experimental metastasis model. In addition, A375 expressed, more abundantly than SAN, the TGF-β and the pro angiogenic TGF-β receptor type III (TβRIII) factors. FKBP51 silencing produced a reduction of TGF-β and TβRIII gene expression in A375 cell line, in accordance with previous studies. We found that the inducing effect of TGF-β on Sparc and Vimentin expression was impaired in condition of FKBP51 depletion, suggesting that FKBP51 is an important cofactor in the TGF-β signal. Such a hypothesis was supported by co immunoprecipitation assays, showing that FKBP51 interacted with either Smad2,3 and p300. In normal melanocytes, FKBP51 potentiated the effect of TGF-β on N-cadherin expression and conferred a mesenchymal-like morphology to such round-shaped cells. Conclusions Overall, our findings show that FKBP51 enhances some pro oncogenic functions of TGF-β, suggesting that FKBP51-overexpression may help melanoma to take advantage of the tumor promoting activities of the cytokine.
Collapse
|
|
20
|
Hanks BA, Holtzhausen A, Evans KS, Jamieson R, Gimpel P, Campbell OM, Hector-Greene M, Sun L, Tewari A, George A, Starr M, Nixon A, Augustine C, Beasley G, Tyler DS, Osada T, Morse MA, Ling L, Lyerly HK, Blobe GC. Type III TGF-β receptor downregulation generates an immunotolerant tumor microenvironment. J Clin Invest 2013; 123:3925-40. [PMID: 23925295 PMCID: PMC3754240 DOI: 10.1172/jci65745] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2012] [Accepted: 06/13/2013] [Indexed: 01/02/2023] Open
Abstract
Cancers subvert the host immune system to facilitate disease progression. These evolved immunosuppressive mechanisms are also implicated in circumventing immunotherapeutic strategies. Emerging data indicate that local tumor-associated DC populations exhibit tolerogenic features by promoting Treg development; however, the mechanisms by which tumors manipulate DC and Treg function in the tumor microenvironment remain unclear. Type III TGF-β receptor (TGFBR3) and its shed extracellular domain (sTGFBR3) regulate TGF-β signaling and maintain epithelial homeostasis, with loss of TGFBR3 expression promoting progression early in breast cancer development. Using murine models of breast cancer and melanoma, we elucidated a tumor immunoevasion mechanism whereby loss of tumor-expressed TGFBR3/sTGFBR3 enhanced TGF-β signaling within locoregional DC populations and upregulated both the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in plasmacytoid DCs and the CCL22 chemokine in myeloid DCs. Alterations in these DC populations mediated Treg infiltration and the suppression of antitumor immunity. Our findings provide mechanistic support for using TGF-β inhibitors to enhance the efficacy of tumor immunotherapy, indicate that sTGFBR3 levels could serve as a predictive immunotherapy biomarker, and expand the mechanisms by which TGFBR3 suppresses cancer progression to include effects on the tumor immune microenvironment.
Collapse
MESH Headings
- Animals
- Cell Line, Tumor
- Chemokine CCL22/metabolism
- Dendritic Cells/immunology
- Dendritic Cells/metabolism
- Down-Regulation
- Female
- Humans
- Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism
- Mammary Neoplasms, Experimental/immunology
- Mammary Neoplasms, Experimental/metabolism
- Mammary Neoplasms, Experimental/pathology
- Melanoma, Experimental/immunology
- Melanoma, Experimental/metabolism
- Melanoma, Experimental/pathology
- Mice
- Mice, Inbred BALB C
- Mice, Inbred C57BL
- Mice, Transgenic
- Neoplasm Transplantation
- Proteoglycans/genetics
- Proteoglycans/metabolism
- Receptors, Transforming Growth Factor beta/genetics
- Receptors, Transforming Growth Factor beta/metabolism
- Transforming Growth Factor beta/metabolism
- Tumor Escape
- Tumor Microenvironment/immunology
Collapse
Affiliation(s)
- Brent A. Hanks
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Alisha Holtzhausen
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Katherine S. Evans
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Rebekah Jamieson
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Petra Gimpel
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Olivia M. Campbell
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Melissa Hector-Greene
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Lihong Sun
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Alok Tewari
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Amanda George
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Mark Starr
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Andrew Nixon
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Christi Augustine
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Georgia Beasley
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Douglas S. Tyler
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Takayu Osada
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Michael A. Morse
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Leona Ling
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - H. Kim Lyerly
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| | - Gerard C. Blobe
- Department of Medicine and
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina, USA.
Freie Universität Berlin, Institut für Chemie und Biochemie, Berlin, Germany.
Medical Scientist Training Program, Duke University Medical Center, Durham, North Carolina, USA.
Biogen Idec Inc., Cambridge, Massachusetts, USA.
Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
| |
Collapse
|
|
21
|
Perrot CY, Javelaud D, Mauviel A. Insights into the Transforming Growth Factor-β Signaling Pathway in Cutaneous Melanoma. Ann Dermatol 2013; 25:135-44. [PMID: 23717002 PMCID: PMC3662904 DOI: 10.5021/ad.2013.25.2.135] [Citation(s) in RCA: 68] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Transforming growth factor-β (TGF-β) is a pleiotropic growth factor with broad tissue distribution that plays critical roles during embryonic development, normal tissue homeostasis, and cancer. While its cytostatic activity on normal epithelial cells initially defined TGF-β signaling as a tumor suppressor pathway, there is ample evidence indicating that TGF-β is a potent pro-tumorigenic agent, acting via autocrine and paracrine mechanisms to promote peri-tumoral angiogenesis, together with tumor cell migration, immune escape, and dissemination to metastatic sites. This review summarizes the current knowledge on the implication of TGF-β signaling in melanoma.
Collapse
Affiliation(s)
- Carole Yolande Perrot
- Institut Curie, Team "TGF-β and Oncogenesis", Equipe Labellisée Ligue Contre le Cancer, Orsay, France
- INSERM U1021 Orsay, France
- CNRS UMR 3347, Orsay, France
| | - Delphine Javelaud
- Institut Curie, Team "TGF-β and Oncogenesis", Equipe Labellisée Ligue Contre le Cancer, Orsay, France
- INSERM U1021 Orsay, France
- CNRS UMR 3347, Orsay, France
| | - Alain Mauviel
- Institut Curie, Team "TGF-β and Oncogenesis", Equipe Labellisée Ligue Contre le Cancer, Orsay, France
- INSERM U1021 Orsay, France
- CNRS UMR 3347, Orsay, France
| |
Collapse
|
|
22
|
Abushahba W, Olabisi OO, Jeong BS, Boregowda RK, Wen Y, Liu F, Goydos JS, Lasfar A, Cohen-Solal KA. Non-canonical Smads phosphorylation induced by the glutamate release inhibitor, riluzole, through GSK3 activation in melanoma. PLoS One 2012; 7:e47312. [PMID: 23077590 PMCID: PMC3470581 DOI: 10.1371/journal.pone.0047312] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2012] [Accepted: 09/11/2012] [Indexed: 12/25/2022] Open
Abstract
Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.
Collapse
Affiliation(s)
- Walid Abushahba
- Department of Medicine, Division of Medical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
| | - Oyenike O. Olabisi
- Department of Medicine, Division of Medical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
| | - Byeong-Seon Jeong
- Department of Surgery, Division of Surgical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
| | - Rajeev K. Boregowda
- Department of Medicine, Division of Medical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
| | - Yu Wen
- Department of Surgery, Division of Surgical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
| | - Fang Liu
- Center for Advanced Biotechnology and Medicine, Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America
| | - James S. Goydos
- Department of Surgery, Division of Surgical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
| | - Ahmed Lasfar
- Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America
| | - Karine A. Cohen-Solal
- Department of Medicine, Division of Medical Oncology, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, The Cancer Institute of New Jersey, New Brunswick, New Jersey, United States of America
- * E-mail:
| |
Collapse
|
|
23
|
Wulfänger J, Schneider H, Wild P, Ikenberg K, Rodolfo M, Rivoltini L, Meyer S, Riemann D, Seliger B. Promoter methylation of aminopeptidase N/CD13 in malignant melanoma. Carcinogenesis 2012; 33:781-90. [DOI: 10.1093/carcin/bgs091] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
|
|
24
|
Interferon lambda: a new sword in cancer immunotherapy. Clin Dev Immunol 2011; 2011:349575. [PMID: 22190970 PMCID: PMC3235441 DOI: 10.1155/2011/349575] [Citation(s) in RCA: 58] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2011] [Accepted: 09/16/2011] [Indexed: 12/14/2022]
Abstract
The discovery of the interferon-lambda (IFN-λ) family has considerably contributed to our understanding of the role of interferon not only in viral infections but also in cancer. IFN-λ proteins belong to the new type III IFN group. Type III IFN is structurally similar to type II IFN (IFN-γ) but functionally identical to type I IFN (IFN-α/β). However, in contrast to type I or type II IFNs, the response to type III IFN is highly cell-type specific. Only epithelial-like cells and to a lesser extent some immune cells respond to IFN-λ. This particular pattern of response is controlled by the differential expression of the IFN-λ receptor, which, in contrast to IFN-α, should result in limited side effects in patients. Recently, we and other groups have shown in several animal models a potent antitumor role of IFN-λ that will open a new challenging era for the current IFN therapy.
Collapse
|
|
25
|
|
|
26
|
Abstract
BACKGROUND Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes most likely favour tumour growth and progression, and influence tumour environment. The induction of transforming growth factor beta1, 2 and 3 as well as BMP4 and BMP7 expression in malignant melanoma has been reported before, whereas the expression of an important modulator of these molecules, connective tissue growth factor (CTGF), has not been investigated in melanomas until now. METHODS Expression of CTGF was analysed in melanoma cell lines and tissue samples by qRT-PCR and immunohistochemistry. To determine the regulation of CTGF expression in malignant melanoma, specific siRNA was used. Additionally, migration, invasion and attachment assays were carried out. RESULTS We were able to demonstrate that CTGF expression is upregulated in nine melanoma cell lines and in primary and metastatic melanoma in situ. The transcription factor HIF-1α was revealed as a positive regulator for CTGF expression. Melanoma cells, in which CTGF expression is diminished, show a strong reduction of migratory and invasive properties when compared with controls. Further, treatment of normal human epidermal melanocytes with recombinant CTGF leads to an increase of migratory and invasive behaviour of these cells. CONCLUSION These results suggest that CTGF promotes melanoma cell invasion and migration and, therefore, has an important role in the progression of malignant melanoma.
Collapse
|
|
27
|
Abstract
Snail1 is a master regulator of the epithelial-mesenchymal transition (EMT) and has been implicated in key tumor biological processes such as invasion and metastasis. It has been previously shown that poly(ADP-ribose) polymerase-1 (PARP-1) knockdown, but not PARP inhibition, downregulates the expression of Snail1. In this study we have characterized a novel regulatory mechanism controlling Snail1 protein expression through poly(ADP-ribosyl)ation. The effect is not only limited to repression of Snail1 transcription but also to downregulated Snail1 protein stability. PARP-1 (but not PARP-2) poly(ADP) ribosylates Snail1, both in vivo and in vitro, and interacts with Snail1, an association that is sensitive to PARP inhibitors. PARP inhibition has also clear effects on EMT phenotype of different tumor cells, including Snail1 downregulation, E-cadherin upregulation, decreased cell elongation and invasiveness. Therefore, this study reveals a new regulatory mechanism of Snail1 activation through poly(ADP-ribosyl)ation with consequences in malignant transformation through EMT.
Collapse
|
|
28
|
Cohen-Solal KA, Merrigan KT, Chan JLK, Goydos JS, Chen W, Foran DJ, Liu F, Lasfar A, Reiss M. Constitutive Smad linker phosphorylation in melanoma: a mechanism of resistance to transforming growth factor-β-mediated growth inhibition. Pigment Cell Melanoma Res 2011; 24:512-24. [PMID: 21477078 DOI: 10.1111/j.1755-148x.2011.00858.x] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Melanoma cells are resistant to transforming growth factor-β (TGFβ)-induced cell-cycle arrest. In this study, we investigated a mechanism of resistance involving a regulatory domain, called linker region, in Smad2 and Smad3, main downstream effectors of TGFβ. Melanoma cells in culture and tumor samples exhibited constitutive Smad2 and Smad3 linker phosphorylation. Treatment of melanoma cells with the MEK1/2 inhibitor, U0126, or the two pan-CDK and GSK3 inhibitors, Flavopiridol and R547, resulted in decreased linker phosphorylation of Smad2 and Smad3. Overexpression of the linker phosphorylation-resistant Smad3 EPSM mutant in melanoma cells resulted in an increase in expression of p15(INK4B) and p21(WAF1) , as compared with cells transfected with wild-type (WT) Smad3. In addition, the cell numbers of EPSM Smad3-expressing melanoma cells were significantly reduced compared with WT Smad3-expressing cells. These results suggest that the linker phosphorylation of Smad3 contributes to the resistance of melanoma cells to TGFβ-mediated growth inhibition.
Collapse
Affiliation(s)
- Karine A Cohen-Solal
- Department of Medicine, Division of Medical Oncology, UMDNJ-Robert Wood Johnson Medical School, the Cancer Institute of New Jersey, New Brunswick, NJ, USA.
| | | | | | | | | | | | | | | | | |
Collapse
|