Abstract
BRAF and MEK inhibitors have shown remarkable clinical efficacy in BRAF-mutant melanoma; however, most patients develop resistance, which limits the clinical benefit of these agents. In this study, we found that the human melanoma cell clones, A375-DR and A375-TR, with acquired resistance to BRAF inhibitor dabrafenib and MEK inhibitor trametinib, were cross resistant to other MAPK pathway inhibitors. In these resistant cells, phosphorylation of ribosomal protein S6 (rpS6) but not phosphorylation of ERK or p90 ribosomal S6 kinase (RSK) were unable to be inhibited by MAPK pathway inhibitors. Notably, knockdown of rpS6 in these cells effectively downregulated G1 phase-related proteins, including RB, cyclin D1, and CDK6, induced cell cycle arrest, and inhibited proliferation, suggesting that aberrant modulation of rpS6 phosphorylation contributed to the acquired resistance. Interestingly, RSK inhibitor had little effect on rpS6 phosphorylation and cell proliferation in resistant cells, whereas P70S6K inhibitor showed stronger inhibitory effects on rpS6 phosphorylation and cell proliferation in resistant cells than in parental cells. Thus regulation of rpS6 phosphorylation, which is predominantly mediated by BRAF/MEK/ERK/RSK signaling in parental cells, was switched to mTOR/P70S6K signaling in resistant cells. Furthermore, mTOR inhibitors alone overcame acquired resistance and rescued the sensitivity of the resistant cells when combined with BRAF/MEK inhibitors. Taken together, our findings indicate that RSK-independent phosphorylation of rpS6 confers resistance to MAPK pathway inhibitors in BRAF-mutant melanoma, and that mTOR inhibitor-based regimens may provide alternative strategies to overcome this acquired resistance.
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References
Lo JenniferA, Fisher DE. The melanoma revolution: from UV carcinogenesis to a new era in therapeutics. Science. 2014;346:945–9.
Abildgaard C, Guldberg P. Molecular drivers of cellular metabolic repro-gramming in melanoma. Trends Mol Med. 2015;21:164–71.
Welsh SJ, Rizos H, Scolyer RA, Long GV. Resistance to combination BRAF and MEK inhibition in metastatic melanoma: Where to next? Eur J Cancer. 2016;62:76–85.
Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417:949–54.
Kolch. W. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J. 2000;351:289–305.
Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364:2507–16.
Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380:358–65.
Kim KB, Kefford R, Pavlick AC, Infante JR, Ribas A, Sosman JA, et al. Phase II study of the MEK1/MEK2 inhibitor Trametinib in patients with metastatic BRAF-mutant cutaneous melanoma previously treated with or without a BRAF inhibitor. J Clin Oncol. 2013;31:482–9.
Rosen LS, LoRusso P, Ma WW, Goldman JW, Weise A, Colevas AD, et al. A first-in-human phase I study to evaluate the MEK1/2 inhibitor, cobimetinib, administered daily in patients with advanced solid tumors. Invest New Drugs. 2016;34:604–13.
Watanabe M, Sowa Y, Yogosawa M, Sakai T. Novel MEK inhibitor trametinib and other retinoblastoma gene (RB)-reactivating agents enhance efficacy of 5-fluorouracil on human colon cancer cells. Cancer Sci. 2013;104:687–93.
Menzies AM, Long GV. Systemic treatment for BRAF-mutant melanoma: where do we go next? Lancet Oncol. 2014;15:e371–81.
Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N Engl J Med. 2015;372:30–9.
Ascierto PA, McArthur GA, Dréno B, Atkinson V, Liszkay G, Di Giacomo AM, et al. Cobimetinib combined with vemurafenib in advanced BRAFV600-mutant melanoma (coBRIM): updated efficacy results from a randomised, double-blind, phase 3 trial. Lancet Oncol. 2016;17:1248–60.
Long GV, Fung C, Menzies AM, Pupo GM, Carlino MS, Hyman J, et al. Increased MAPK reactivation in early resistance to dabrafenib/ trametinib combination therapy of BRAF-mutant metastatic melanoma. Nat Commun. 2014;5:5694.
Straussman R, Morikawa T, Shee K, Barzily-Rokni M, Qian ZR, Du J, et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature. 2012;487:500–4.
Villanueva J, Vultur A, Lee JT, Somasundaram R, Fukunaga-Kalabis M, Cipolla AK, et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/ PI3K. Cancer Cell. 2010;18:683–95.
Nazarian R, Shi H, Wang Q, Kong X, Koya RC, Lee H, et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature. 2010;468:973–7.
Frederick DT, Piris A, Cogdill AP, Cooper ZA, Lezcano C, Ferrone CR, et al. BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma. Clin Cancer Res. 2013;19:1225–31.
Wool. IG, Chan. Y-L, Gliick. A. Structure and evolution of mammalian ribosomal proteins. Cell Biol. 1995;73:933–47.
Ruvinsky I, Meyuhas O. Ribosomal protein S6 phosphorylation: from protein synthesis to cell size. Trends Biochem Sci. 2006;31:342–8.
Khalaileh A, Dreazen A, Khatib A, Apel R, Swisa A, Kidess-Bassir N, et al. Phosphorylation of ribosomal protein S6 attenuates DNA damage and tumor suppression during development of pancreatic cancer. Cancer Res. 2013;73:1811–20.
Roux PP, Shahbazian D, Vu H, Holz MK, Cohen MS, Taunton J, et al. RAS/ERK signaling promotes site-specific ribosomal protein S6 phosphorylation via RSK and stimulates cap-dependent translation. J Biol Chem. 2007;282:14056–64.
Pende M, Um SH, Mieulet V, Sticker M, Goss VL, Mestan J, et al. S6K1-/-/S6K2-/- mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway. Mol Cell Biol. 2004;24:3112–24.
Kim TS, Jang CY, Kim HD, Lee JY, Ahn BY, Kim J. Interaction of Hsp90 with ribosomal proteins protects from ubiquitination and proteasome-dependent degradation. Mol Biol Cell. 2006;17:824–33.
Schumacher. AM, Velentza AV, Watterson DM, Dresios J. Death-associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis. Biochemistry. 2006;45:13614–21.
Volarevic. S, Stewart. MJ, Ledermann. B, Zilberman. F, Terracciano. L, Montini. E, et al. Proliferation, but not growth, blocked by conditional deletion of 40S ribosomal protein S6. Science. 2000;288:2045–7.
Doerks T, Copley RR, Schultz J, Ponting CP, Bork P. Ribosomal protein S6 phosphorylation is a determinant of cell size and glucose homeostasis. Genome Res. 2002;12:47–56.
Meyuhas O. Chapter 1. Physiological roles of ribosomal protein S6: one of its kind. Int Rev Cel Mol Biol. 2008;268:1–37.
Xie CY, Chen XL, Zheng MY, Liu XH, Wang HB, Lou LG. Pharmacologic characterization of SHR8443, a novel dual inhibitor of phosphatidylinositol 3-kinase and mammalian target of rapamycin. Oncotarget. 2017;8:107977–90.
Zhao ZX, Zhu JM, Quan HT, Wang GM, Li B, Zhu WL, et al. X66, a novel N-terminal heat shock protein 90 inhibitor, exerts antitumor effects without induction of heat shock response. Oncotarget. 2016;7:29648–63.
Chen B, Tan Z, Gao J, Wu W, Liu L, Jin W, et al. Hyperphosphorylation of ribosomal protein S6 predicts unfavorable clinical survival in non-small cell lung cancer. J Exp Clin Cancer Res. 2015;34:126.
Gibney GT, Zager JS. Clinical development of dabrafenib in BRAF mutant melanoma and other malignancies. Expert Opin Drug Metab Toxicol. 2013;9:893–9.
Gilmartin AG, Bleam MR, Groy A, Moss KG, Minthorn EA, Kulkarni SG, et al. GSK1120212 (JTP-74057) is an inhibitor of MEK activity and activation with favorable pharmacokinetic properties for sustained in vivo pathway inhibition. Clin Cancer Res. 2011;17:989–1000.
Haq R, Yokoyama S, Hawryluk EB, Jonsson GB, Frederick DT, McHenry K, et al. BCL2A1 is a lineage-specific antiapoptotic melanoma oncogene that confers resistance to BRAF inhibition. Proc Natl Acad Sci U S A. 2013;110:4321–6.
Magnuson B, Ekim B, Fingar DC. Regulation and function of ribosomal protein S6 kinase (S6K) within mTOR signalling networks. Biochem J. 2012;441:1–21.
Sanchez-Hernandez I, Baquero P, Calleros L, Chiloeches A. Dual inhibition of (V600E)BRAF and the PI3K/AKT/mTOR pathway cooperates to induce apoptosis in melanoma cells through a MEK-independent mechanism. Cancer Lett. 2012;314:244–55.
Shimizu T, Tolcher AW, Papadopoulos KP, Beeram M, Rasco DW, Smith LS, et al. The clinical effect of the dual-targeting strategy involving PI3K/ AKT/mTOR and RAS/MEK/ERK pathways in patients with advanced cancer. Clin Cancer Res. 2012;18:2316–25.
Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, et al. Constitutive mitogen-activated protein kinase activation in melanoma is mediated by both BRAF mutations and autocrine growth factor stimulation. Cancer Res. 2003;63:756–9.
Menzies AM, Long GV. Dabrafenib and trametinib, alone and in combination for BRAF-mutant metastatic melanoma. Clin. Cancer Res. 2014;20:2035–43.
Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 2012;367:107–14.
Wagle N, Emery C, Berger MF, Davis MJ, Sawyer A, Pochanard P, et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumor genomic profiling. Cancer Res. 2011;29:3085–96.
Obaid NM, Bedard K, Huang WY. Strategies for overcoming resistance in tumours harboring BRAF mutations. Int J Mol Med. 2017;18:E585.
Shi H, Hugo W, Kong X, Hong A, Koya RC, Moriceau G, et al. Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov. 2014;4:80–93.
Kosnopfel C, Sinnberg T, Sauer B, Niessner H, Schmitt A, Makino E, et al. Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase. Oncotarget. 2017;8:35761–75.
Wilson TR, Fridlyand J, Yan Y, Penuel E, Burton L, Chan E, et al. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature. 2012;487:505–9.
Shi H, Hong A, Kong X, Koya RC, Song C, Moriceau G, et al. A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition. Cancer Discov. 2014;4:69–79.
Zheng Z, Zheng Y, Zhang M, Wang J, Yu G, Fang W. Reciprocal expression of p-AMPKa and p-S6 is strongly associated with the prognosis of gastric cancer. Tumour Biol. 2016;37:4803–11.
Yang-Kolodji G, Mumenthaler SM, Mehta A, Ji L, Tripathy D. Phosphory-lated ribosomal S6 (p-rpS6) as a post-treatment indicator of HER2 signalling targeted drug resistance. Biomarkers. 2015;20:313–22.
Corcoran RB, Rothenberg SM, Hata AN, Faber AC, Winokur D, Piris A., et al. TORC1 suppression predicts responsiveness to RAF and MEK inhibition in BRAF-mutant melanoma. Sci Transl Med. https://doi.org/10.1126/scitranslmed.3005753 (2013).
Knoll M, Macher-Goeppinger S, Kopitz J, Duensing S, Pahernik S, Hohenfellner M, et al. The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker. Oncotarget. 2016;7:418–32.
Gao J, Shi LZ, Zhao H, Chen J, Xiong L, He Q, et al. Loss of IFN-gamma pathway genes in tumor cells as a mechanism of resistance to anti-CTLA-4 therapy. Cell. 2016;167:397–404.
Liu L, Mayes PA, Eastman S, Shi H, Yadavilli S, Zhang TD, et al. The BRAF and MEK inhibitors dabrafenib and trametinib: effects on immune function and in combination with immunomodulatory antibodies targeting PD-1, PD-L1, and CTLA-4. Clin. Cancer Res. 2015;21:1639–51.
Kim H, Frederick DT, Levesque MP, Cooper ZA, Feng Y, Krepler C, et al. Downregulation of the ubiquitin ligase RNF125 underlies resistance of melanoma cells to BRAF inhibitors via JAK1 deregulation. Cell Rep. 2015;11:1458–73.
Karbowniczek M, Spittle CS, Morrison T, Wu H, Henske EP. mTOR is activated in the majority of malignant melanomas. J Invest Dermatol. 2008;128:980–7.
Acknowledgements
This research was supported by grants from the National Natural Science Foundation of China (No. 81273546) and the Shanghai Science and Technology Committee (No. 14DZ2294100).
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L.-g.L. and C.-y.X. conceived and designed the experiments; M.-z.G. wrote the manuscript, performed experiments, and analyzed the data; H.-b.W., X.-l.C., W.-t.C., L.F., Y.L., and H.-t.Q. performed the experiments.
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Gao, Mz., Wang, Hb., Chen, Xl. et al. Aberrant modulation of ribosomal protein S6 phosphorylation confers acquired resistance to MAPK pathway inhibitors in BRAF-mutant melanoma. Acta Pharmacol Sin 40, 268–278 (2019). https://doi.org/10.1038/s41401-018-0020-z
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DOI: https://doi.org/10.1038/s41401-018-0020-z
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