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A large-scale RNAi screen identifies LCMR1 as a critical regulator of Tspan8-mediated melanoma invasion

Oncogene volume 36, pages 446–457 (2017)Cite this article

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An Erratum to this article was published on 19 June 2017

Abstract

Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3β, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell–matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.

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References

  1. Miller AJ, Mihm MC Jr . Melanoma. N Engl J Med 2006; 355: 51–65.

    Article  CAS  PubMed  Google Scholar 

  2. Ascierto PA, Marincola FM, Atkins MB . What's new in melanoma? Combination!. J Transl Med 2015; 13: 213.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Spagnolo F, Ghiorzo P, Queirolo P . Overcoming resistance to BRAF inhibition in BRAF-mutated metastatic melanoma. Oncotarget 2014; 5: 10206–10221.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Berthier-Vergnes O, Zebda N, Bailly M, Bailly C, Dore JF, Thomas L et al. Expression of peanut agglutinin-binding glycoconjugates in primary melanomas with high risk of metastases. Lancet 1993; 341: 1292.

    Article  CAS  PubMed  Google Scholar 

  5. Cochran AJ, Wen DR, Berthier-Vergnes O, Bailly C, Dore JF, Berard F et al. Cytoplasmic accumulation of peanut agglutinin-binding glycoconjugates in the cells of primary melanoma correlates with clinical outcome. Hum Pathol 1999; 30: 556–561.

    Article  CAS  PubMed  Google Scholar 

  6. Berthier-Vergnes O, Kharbili ME, de la Fouchardiere A, Pointecouteau T, Verrando P, Wierinckx A et al. Gene expression profiles of human melanoma cells with different invasive potential reveal TSPAN8 as a novel mediator of invasion. Br J Cancer 2011; 104: 155–165.

    Article  CAS  PubMed  Google Scholar 

  7. Yanez-Mo M, Barreiro O, Gordon-Alonso M, Sala-Valdes M, Sanchez-Madrid F . Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes. Trends Cell Biol 2009; 19: 434–446.

    Article  CAS  PubMed  Google Scholar 

  8. Hemler ME . Tetraspanin proteins promote multiple cancer stages. Nat Rev Cancer 2014; 14: 49–60.

    Article  CAS  PubMed  Google Scholar 

  9. Kanetaka K, Sakamoto M, Yamamoto Y, Yamasaki S, Lanza F, Kanematsu T et al. Overexpression of tetraspanin CO-029 in hepatocellular carcinoma. J Hepatol 2001; 35: 637–642.

    Article  CAS  PubMed  Google Scholar 

  10. Gesierich S, Paret C, Hildebrand D, Weitz J, Zgraggen K, Schmitz-Winnenthal FH et al. Colocalization of the tetraspanins, CO-029 and CD151, with integrins in human pancreatic adenocarcinoma: impact on cell motility. Clin Cancer Res 2005; 11: 2840–2852.

    Article  CAS  PubMed  Google Scholar 

  11. Zhou Z, Ran YL, Hu H, Pan J, Li ZF, Chen LZ et al. TM4SF3 promotes esophageal carcinoma metastasis via upregulating ADAM12m expression. Clin Exp Metastasis 2008; 25: 537–548.

    Article  CAS  PubMed  Google Scholar 

  12. Greco C, Bralet MP, Ailane N, Dubart-Kupperschmitt A, Rubinstein E, Le Naour F et al. E-cadherin/p120-catenin and tetraspanin Co-029 cooperate for cell motility control in human colon carcinoma. Cancer Res 2010; 70: 7674–7683.

    Article  CAS  PubMed  Google Scholar 

  13. Nazarenko I, Rana S, Baumann A, McAlear J, Hellwig A, Trendelenburg M et al. Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation. Cancer Res 2010; 70: 1668–1678.

    Article  CAS  PubMed  Google Scholar 

  14. Yue S, Mu W, Erb U, Zoller M . The tetraspanins CD151 and Tspan8 are essential exosome components for the crosstalk between cancer initiating cells and their surrounding. Oncotarget 2015; 6: 2366–2384.

    PubMed  Google Scholar 

  15. Felding-Habermann B, Fransvea E, O'Toole TE, Manzuk L, Faha B, Hensler M . Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells. Clin Exp Metastasis 2002; 19: 427–436.

    Article  CAS  PubMed  Google Scholar 

  16. Saalbach A, Wetzel A, Haustein UF, Sticherling M, Simon JC, Anderegg U . Interaction of human Thy-1 (CD 90) with the integrin alphavbeta3 (CD51/CD61): an important mechanism mediating melanoma cell adhesion to activated endothelium. Oncogene 2005; 24: 4710–4720.

    Article  CAS  PubMed  Google Scholar 

  17. Kubic JD, Mascarenhas JB, Iizuka T, Wolfgeher D, Lang D . GSK-3 promotes cell survival, growth, and PAX3 levels in human melanoma cells. Mol Cancer Res 2012; 10: 1065–1076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. John JK, Paraiso KH, Rebecca VW, Cantini LP, Abel EV, Pagano N et al. GSK3beta inhibition blocks melanoma cell/host interactions by downregulating N-cadherin expression and decreasing FAK phosphorylation. J Invest Dermatol 2012; 132: 2818–2827.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Conde-Perez A, Larue L . PTEN and melanomagenesis. Future Oncol 2012; 8: 1109–1120.

    Article  CAS  PubMed  Google Scholar 

  20. Jameson KL, Mazur PK, Zehnder AM, Zhang J, Zarnegar B, Sage J et al. IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors. Nat Med 2013; 19: 626–630.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Chen L, Liang Z, Tian Q, Li C, Ma X, Zhang Y et al. Overexpression of LCMR1 is significantly associated with clinical stage in human NSCLC. J Exp Clin Cancer Res 2011; 30: 18.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Sinha P, Kohl S, Fischer J, Hutter G, Kern M, Kottgen E et al. Identification of novel proteins associated with the development of chemoresistance in malignant melanoma using two-dimensional electrophoresis. Electrophoresis 2000; 21: 3048–3057.

    Article  CAS  PubMed  Google Scholar 

  23. Tikhmyanova N, Golemis EA . NEDD9 and BCAR1 negatively regulate E-cadherin membrane localization, and promote E-cadherin degradation. PLoS One 2011; 6: e22102.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Byers HR, Etoh T, Vink J, Franklin N, Gattoni-Celli S, Mihm MC Jr . Actin organization and cell migration of melanoma cells relate to differential expression of integrins and actin-associated proteins. J Dermatol 1992; 19: 847–852.

    Article  CAS  PubMed  Google Scholar 

  25. Kim JH, Kim B, Cai L, Choi HJ, Ohgi KA, Tran C et al. Transcriptional regulation of a metastasis suppressor gene by Tip60 and beta-catenin complexes. Nature 2005; 434: 921–926.

    Article  CAS  PubMed  Google Scholar 

  26. Le Naour F, Prenant M, Francastel C, Rubinstein E, Uzan G, Boucheix C . Transcriptional regulation of the human CD9 gene: characterization of the 5'-flanking region. Oncogene 1996; 13: 481–486.

    CAS  PubMed  Google Scholar 

  27. Lu Z, Luo T, Nie M, Pang T, Zhang X, Shen X et al. TSPAN1 functions as an oncogene in gastric cancer and is downregulated by miR-573. FEBS Lett 2015; 589: 1988–1994.

    Article  CAS  PubMed  Google Scholar 

  28. Mekky RY, El-Ekiaby NM, Hamza MT, Elemam NM, El-Sayed M, Esmat G et al. Mir-194 is a hepatocyte gate keeper hindering HCV entry through targeting CD81 receptor. J Infect 2015; 70: 78–87.

    Article  PubMed  Google Scholar 

  29. Xu Y, Li C, Tian Q, Li Y, Yang Z, Liang Z et al. Suppression of lung cancer metastasis-related protein 1 promotes apoptosis in lung cancer cells. Int J Mol Med 2012; 30: 1481–1486.

    Article  CAS  PubMed  Google Scholar 

  30. Ji-Fu E, Xing JJ, Hao LQ, Fu CG . Suppression of lung cancer metastasis-related protein 1 (LCMR1) inhibits the growth of colorectal cancer cells. Mol Biol Rep 2012; 39: 3675–3681.

    Article  CAS  PubMed  Google Scholar 

  31. Zou SW, Ai KX, Wang ZG, Yuan Z, Yan J, Zheng Q . The role of Med19 in the proliferation and tumorigenesis of human hepatocellular carcinoma cells. Acta Pharmacol Sin 2011; 32: 354–360.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Li LH, He J, Hua D, Guo ZJ, Gao Q . Lentivirus-mediated inhibition of Med19 suppresses growth of breast cancer cells in vitro. Cancer Chemother Pharmacol 2011; 68: 207–215.

    Article  PubMed  Google Scholar 

  33. Li XH, Fang DN, Zeng CM . Knockdown of MED19 by short hairpin RNA-mediated gene silencing inhibits pancreatic cancer cell proliferation. Cancer Biother Radiopharm 2011; 26: 495–501.

    Article  CAS  PubMed  Google Scholar 

  34. Wang T, Hao L, Feng Y, Wang G, Qin D, Gu G . Knockdown of MED19 by lentivirus-mediated shRNA in human osteosarcoma cells inhibits cell proliferation by inducing cell cycle arrest in the G0/G1 phase. Oncol Res 2011; 19: 193–201.

    Article  PubMed  Google Scholar 

  35. Yu W, Zhang Z, Min D, Yang Q, Du X, Tang L et al. Mediator of RNA polymerase II transcription subunit 19 promotes osteosarcoma growth and metastasis and associates with prognosis. Eur J Cancer 2014; 50: 1125–1136.

    Article  CAS  PubMed  Google Scholar 

  36. Sun M, Jiang R, Li JD, Luo SL, Gao HW, Jin CY et al. MED19 promotes proliferation and tumorigenesis of lung cancer. Mol Cell Biochem 2011; 355: 27–33.

    Article  CAS  PubMed  Google Scholar 

  37. Cui X, Xu D, Lv C, Qu F, He J, Chen M et al. Suppression of MED19 expression by shRNA induces inhibition of cell proliferation and tumorigenesis in human prostate cancer cells. BMB Rep 2011; 44: 547–552.

    Article  CAS  PubMed  Google Scholar 

  38. Ding XF, Huang GM, Shi Y, Li JA, Fang XD . Med19 promotes gastric cancer progression and cellular growth. Gene 2012; 504: 262–267.

    Article  CAS  PubMed  Google Scholar 

  39. Liu Y, Tao X, Fan L, Jia L, Gu C, Feng Y . Knockdown of mediator complex subunit 19 inhibits the growth of ovarian cancer. Mol Med Rep 2012; 6: 1050–1056.

    Article  CAS  PubMed  Google Scholar 

  40. Zhang H, Jiang H, Wang W, Gong J, Zhang L, Chen Z et al. Expression of Med19 in bladder cancer tissues and its role on bladder cancer cell growth. Urol Oncol 2012; 30: 920–927.

    Article  CAS  PubMed  Google Scholar 

  41. Wen H, Feng CC, Ding GX, Meng DL, Ding Q, Fang ZJ et al. Med19 promotes bone metastasis and invasiveness of bladder urothelial carcinoma via bone morphogenetic protein 2. Ann Diagn Pathol 2013; 17: 259–264.

    Article  PubMed  Google Scholar 

  42. Zhu LJ, Yan WX, Chen ZW, Chen Y, Chen D, Zhang TH et al. Disruption of mediator complex subunit 19 (Med19) inhibits cell growth and migration in tongue cancer. World J Surg Oncol 2013; 11: 116.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Guglielmi B, van Berkum NL, Klapholz B, Bijma T, Boube M, Boschiero C et al. A high resolution protein interaction map of the yeast Mediator complex. Nucleic Acids Res 2004; 32: 5379–5391.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Sato S, Tomomori-Sato C, Parmely TJ, Florens L, Zybailov B, Swanson SK et al. A set of consensus mammalian mediator subunits identified by multidimensional protein identification technology. Mol Cell 2004; 14: 685–691.

    Article  CAS  PubMed  Google Scholar 

  45. Allen BL, Taatjes DJ . The Mediator complex: a central integrator of transcription. Nat Rev Mol Cell Biol 2015; 16: 155–166.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Plaschka C, Lariviere L, Wenzeck L, Seizl M, Hemann M, Tegunov D et al. Architecture of the RNA polymerase II-Mediator core initiation complex. Nature 2015; 518: 376–380.

    Article  CAS  PubMed  Google Scholar 

  47. Tsai J, Lee JT, Wang W, Zhang J, Cho H, Mamo S et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc Natl Acad Sci USA 2008; 105: 3041–3046.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Paraiso KH, Xiang Y, Rebecca VW, Abel EV, Chen YA, Munko AC et al. PTEN loss confers BRAF inhibitor resistance to melanoma cells through the suppression of BIM expression. Cancer Res 2011; 71: 2750–2760.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Colombino M, Sperlongano P, Izzo F, Tatangelo F, Botti G, Lombardi A et al. BRAF and PIK3CA genes are somatically mutated in hepatocellular carcinoma among patients from South Italy. Cell Death Dis 2012; 3: e259.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Phipps AI, Buchanan DD, Makar KW, Win AK, Baron JA, Lindor NM et al. KRAS-mutation status in relation to colorectal cancer survival: the joint impact of correlated tumour markers. Br J Cancer 2013; 108: 1757–1764.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Wei L, Li Y, Suo Z . TSPAN8 promotes gastric cancer growth and metastasis via ERK MAPK pathway. Int J Clin Exp Med 2015; 8: 8599–8607.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Ishimura N, Yamasawa K, Karim Rumi MA, Kadowaki Y, Ishihara S, Amano Y et al. BRAF and K-ras gene mutations in human pancreatic cancers. Cancer Lett 2003; 199: 169–173.

    Article  CAS  PubMed  Google Scholar 

  53. Gesierich S, Berezovskiy I, Ryschich E, Zoller M . Systemic induction of the angiogenesis switch by the tetraspanin D6.1 A/CO-029. Cancer Res 2006; 66: 7083–7094.

    Article  CAS  PubMed  Google Scholar 

  54. Ailane N, Greco C, Zhu Y, Sala-Valdes M, Billard M, Casal I et al. Effect of an anti-human Co-029/tspan8 mouse monoclonal antibody on tumor growth in a nude mouse model. Front Physiol 2014; 5: 364.

    Article  PubMed  PubMed Central  Google Scholar 

  55. Park CS, Kim TK, Kim HG, Kim YJ, Jeoung MH, Lee WR et al. Therapeutic targeting of tetraspanin8 in epithelial ovarian cancer invasion and metastasis. Oncogene e-pub ahead of print; 2016.

  56. Le Naour F, Andre M, Greco C, Billard M, Sordat B, Emile JF et al. Profiling of the tetraspanin web of human colon cancer cells. Mol Cell Proteomics 2006; 5: 845–857.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Elise Malandain for technical assistance and Cyril Py for immunohistochemical processing, Stéphanie Combe and Patricia Obeid for RNAi screening, Alain Géloën for adhesion assays, and Tiffany Witkowski and Janine Papon for experiments on mice. This work was supported by a grant from the French Society for Dermatological Research (SRD) and a grant from La Ligue Contre le Cancer (Comité Ardèche) and a grant from the association ‘Vaincre le Mélanome’. We acknowledge the Roche Company for financial support for manuscript writing and publication.

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Author notes

  1. M E l Kharbili

    Present address: 13Current address: Department of Dermatology, University of Colorado Anschutz Medical Campus, 12800 E. 19th Avenue, P18-8132, Aurora, CO 80045, USA.,

  2. G Agaësse and L Barbollat-Boutrand: These authors contributed equally to this work.

Authors and Affiliations

  1. Université de Lyon, Lyon, France

    G Agaësse, L Barbollat-Boutrand, M E l Kharbili, O Berthier-Vergnes, J Lamartine & I Masse

  2. Université Lyon 1, Lyon, France

    G Agaësse, L Barbollat-Boutrand, M E l Kharbili, O Berthier-Vergnes, J Lamartine & I Masse

  3. CNRS, UMR5534, Centre de Génétique et de Physiologie Moléculaires et Cellulaires, Villeurbanne, France

    G Agaësse, L Barbollat-Boutrand, M E l Kharbili, O Berthier-Vergnes, J Lamartine & I Masse

  4. Université Grenoble-Alpes, Grenoble, France

    E Sulpice, R Bhajun & X Gidrol

  5. CEA, BIG-BGE, Biomics, Grenoble, France

    E Sulpice, R Bhajun & X Gidrol

  6. Inserm, BGE, Grenoble, France

    E Sulpice, R Bhajun & X Gidrol

  7. Clermont Université, Université d'Auvergne, Imagerie Moléculaire et Thérapie Vectorisée, BP,

    F Degoul

  8. Clermont-Ferrand, France

    F Degoul

  9. Inserm, U 990, Clermont-Ferrand, France

    F Degoul

  10. Département de Biopathologie, Centre Léon Bérard, Lyon, France

    A de la Fouchardière

  11. Laboratoire CarMeN (INSERM 1060, INRA 1397, INSA), Université de Lyon, Lyon, France

    E Berger

  12. Centre de Recherche en Cancérologie de Lyon, CNRS UMR5286, Inserm U1052, Université de Lyon, Université Lyon 1, Lyon, France

    T Voeltzel

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Agaësse, G., Barbollat-Boutrand, L., Sulpice, E. et al. A large-scale RNAi screen identifies LCMR1 as a critical regulator of Tspan8-mediated melanoma invasion. Oncogene 36, 446–457 (2017). https://doi.org/10.1038/onc.2016.219

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