Abstract
In theory, pharmacological inhibition of oncogenic signaling is an effective strategy to halt cellular proliferation, induce apoptosis and eliminate cancer cells. In practice, drugs (for example, PLX-4032) that inhibit oncogenes like B-RAFV600E provide relatively short-term success in patients, owing to a combination of incomplete cellular responses and the development of resistance. To define the relationship between PLX-4032-induced responses and resistance, we interrogated the contributions of anti-apoptotic BCL-2 proteins in determining the fate of B-RAFV600E-inhibited melanoma cells. Although PLX-4032 eliminated B-RAFV600E signaling leading to marked cell cycle arrest, only a fraction of cells eventually underwent apoptosis. These data proposed two hypotheses regarding B-RAFV600E inhibition: (1) only a few cells generate a pro-apoptotic signal, or (2) all the cells generate a pro-apoptotic signal but the majority silences this pathway to ensure survival. Indeed, the latter hypothesis is supported by our observations as the addition of ABT-737, an inhibitor to anti-apoptotic BCL-2 proteins, revealed massive apoptosis following PLX-4032 exposure. B-RAFV600E inhibition alone sensitized cells to the mitochondrial pathway of apoptosis characterized by the rapid accumulation of BIM on the outer mitochondrial membrane, which could be functionally revealed by ABT-737 to promote apoptosis and loss of clonogenic survival. Furthermore, PLX-4032-resistant cells demonstrated collateral resistance to conventional chemotherapy, yet could be re-sensitized to PLX-4032 by BCL-2 family inhibition in vivo and conventional chemotherapies in vitro. Our data suggest that inhibiting anti-apoptotic BCL-2 proteins will enhance primary responses to PLX-4032, along with reducing the development of resistance to both targeted and conventional therapies.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 50 print issues and online access
$259.00 per year
only $5.18 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Hanahan D, Weinberg RA . Hallmarks of cancer: the next generation. Cell 2011; 144: 646–674.
Whittaker S, Kirk R, Hayward R, Zambon A, Viros A, Cantarino N et al Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci Transl Med 2010; 2: 35ra41.
Young A, Lyons J, Miller AL, Phan VT, Alarcon IR, McCormick F . Ras signaling and therapies. Adv Cancer Res 2009; 102: 1–17.
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: 949–954.
Gray-Schopfer V, Wellbrock C, Marais R . Melanoma biology and new targeted therapy. Nature 2007; 445: 851–857.
Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA et al Inhibition of mutated, activated BRAF in metastatic melanoma. New Engl J Med 2010; 363: 809–819.
Montagut C, Settleman J . Targeting the RAF-MEK-ERK pathway in cancer therapy. Cancer Lett 2009; 283: 125–134.
Joseph EW, Pratilas CA, Poulikakos PI, Tadi M, Wang W, Taylor BS et al The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF-selective manner. Proc Natl Acad Sci USA 2010; 107: 14903–14908.
Poulikakos PI, Persaud Y, Janakiraman M, Kong X, Ng C, Moriceau G et al RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 2011; 480: 387–390.
Poulikakos PI, Rosen N . Mutant BRAF melanomas—dependence and resistance. Cancer Cell 2011; 19: 11–15.
Letai AG . Diagnosing and exploiting cancer's addiction to blocks in apoptosis. Nat Rev Cancer 2008; 8: 121–132.
Chipuk JE, Green DR . How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol 2008; 18: 157–164.
Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR . The BCL-2 family reunion. Mol Cell 2010; 37: 299–310.
Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R et al Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002; 111: 331–342.
Letai A, Bassik MC, Walensky LD, Sorcinelli MD, Weiler S, Korsmeyer SJ . Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2002; 2: 183–192.
Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR et al BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell 2005; 17: 525–535.
Chipuk JE, Fisher JC, Dillon CP, Kriwacki RW, Kuwana T, Green DR . Mechanism of apoptosis induction by inhibition of the anti-apoptotic BCL-2 proteins. Proc Natl Acad Sci USA 2008; 105: 20327–20332.
Souers AJ, Leverson JD, Boghaert ER, Ackler SL, Catron ND, Chen J et al ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med 2013; 19: 202–208.
Anvekar RA, Asciolla JJ, Missert DJ, Chipuk JE . Born to be alive: a role for the BCL-2 family in melanoma tumor cell survival, apoptosis, and treatment. Front Oncol 2011; 1: 34.
Tse C, Shoemaker AR, Adickes J, Anderson MG, Chen J, Jin S et al ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res 2008; 68: 3421–3428.
Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 2005; 435: 677–681.
Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS et al Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. New Engl J Med 2012; 366: 707–714.
McArthur GA, Puzanov I, Amaravadi R, Ribas A, Chapman P, Kim KB et al Marked, homogeneous, and early [18F]fluorodeoxyglucose-positron emission tomography responses to vemurafenib in BRAF-mutant advanced melanoma. J Clin Oncol 2012; 30: 1628–1634.
Mason KD, Carpinelli MR, Fletcher JI, Collinge JE, Hilton AA, Ellis S et al Programmed anuclear cell death delimits platelet life span. Cell 2007; 128: 1173–1186.
Hsu YT, Youle RJ . Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 1997; 272: 13829–13834.
Asciolla JJ, Renault TT, Chipuk JE . Examining BCL-2 Family Function with large unilamellar vesicles. J Vis Exp 2012; 68: 1–6.
Anvekar RA, Asciolla JJ, Lopez-Rivera E, Floros KV, Izadmehr S, Elkholi R et al Sensitization to the mitochondrial pathway of apoptosis augments melanoma tumor cell responses to conventional chemotherapeutic regimens. Cell Death Dis 2012; 3: e420.
Cragg MS, Jansen ES, Cook M, Harris C, Strasser A, Scott CL . Treatment of B-RAF mutant human tumor cells with a MEK inhibitor requires Bim and is enhanced by a BH3 mimetic. J Clin Invest 2008; 118: 3651–3659.
Suzuki M, Youle RJ, Tjandra N . Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 2000; 103: 645–654.
Logue SE, Elgendy M, Martin SJ . Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Nat Protoc 2009; 4: 1383–1395.
Acknowledgements
We thank everyone in the Chipuk Laboratory for their assistance and support; Dr Stuart Aaronson, Dr Mark Lebwohl, Dr Emily Bernstein, Dr E Premkumar Reddy and Dr Poulikos Poulikakos for mentorship, guidance and/or discussion about the melanoma field; and Dr Suvendu Das, Eliana Sarrou, Rana Elkholi and Andrew Cruz for technical assistance. This work was supported by: NIH CA157740 (to JEC), NIH KL2TR000069 (to SI), the JJR Foundation (to JEC), the Breast Cancer Alliance (MS), the William A. Spivak Fund (to JEC), and the Fridolin Charitable Trust (to JEC). This work was also supported in part by a Research Grant FY13-238 from the March of Dimes Foundation (to JEC), a developmental PO1 grant from the Department of Oncological Sciences at Mount Sinai (to JEC), an Albert Einstein College of Medicine Research Fellowship (to SYW) and an American Skin Association Medical Students Grant (to SYW).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on the Oncogene website
Rights and permissions
About this article
Cite this article
Serasinghe, M., Missert, D., Asciolla, J. et al. Anti-apoptotic BCL-2 proteins govern cellular outcome following B-RAFV600E inhibition and can be targeted to reduce resistance. Oncogene 34, 857–867 (2015). https://doi.org/10.1038/onc.2014.21
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/onc.2014.21
This article is cited by
-
Bcl-2 family inhibitors sensitize human cancer models to therapy
Cell Death & Disease (2023)
-
Targeting the RAS/RAF/MAPK pathway for cancer therapy: from mechanism to clinical studies
Signal Transduction and Targeted Therapy (2023)
-
Pharmacological inhibition of Ref-1 enhances the therapeutic sensitivity of papillary thyroid carcinoma to vemurafenib
Cell Death & Disease (2022)
-
Increased apoptotic sensitivity of glioblastoma enables therapeutic targeting by BH3-mimetics
Cell Death & Differentiation (2022)
-
Targeting melanoma’s MCL1 bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway inhibitors
Nature Communications (2019)