The anti-apoptotic protein MCL1, a novel target of lung cancer therapy

Wataru Nakajima and Nobuyuki Tanaka*

Department of Molecular Oncology, Institute for Advanced Medical Sciences, Nippon Medical School, Japan


Evasion of apoptosis is one of the typical hallmarks of cancer and a major mechanism for cancer development, tumor growth, and acquisition of resistance to chemotherapy. The anti-apoptotic Bcl-2 protein family, particularly MCL1 and BCL-XL, play an important role in acquisition of apoptosis evasion. MCL1 is a highly unstable protein that is constantly degraded by the ubiquitin-proteasome system. An increase in MCL1 protein has been reported in many cancers, including lung cancer, through high mRNA expression or impairment of its degradation systems. To date, much evidence has shown that MCL1 is important for cancer cell survival and drug resistance in lung cancers. In this review, we discuss the role and mechanism of high MCL1 expression in lung cancer.


MCL1 was originally identified as an up-regulated gene in a human myeloid leukemia cell line, and its amino acid sequence has similarity to the anti-apoptotic protein BCL21. BCL-2 family proteins are critical regulators of mitochondrial apoptosis, a major regulatory pathway of mammalian apoptosis and a typical target to induce cell death by anti-cancer drugs2. The BCL-2 protein family has conserved BCL-2 homology (BH) domains and are classified as pro- or anti-apoptotic proteins. The pro-apoptotic “multi-domain” members that contain several conserved BH domains, BAX and BAK, function as apoptosis executors in mitochondria, while the anti-apoptotic “multi-domain” members, such as BCL-2, BCL-XL, and MCL1, inhibit BAX/BAK-mediated apoptosis. Among the BCL-2 homology domains, the BH3 domain directly associates with anti-apoptotic BCL-2 members, and BH3-only members of the BCL-2 protein family trigger mitochondrial apoptosis by activation of BAX/BAK and inhibition of the anti-apoptotic BCL-2 members in the response to numerous stimuli such as developmental signals, stress signals, the DNA damage response, and various anticancer drugs3 (Figure 1). Because anti-apoptotic BCL-2 family proteins secure survival of many cancer cells, it is possible that suppression of their anti-apoptotic functions induce apoptosis of cancer cells. In this context, inhibitors of anti-apoptotic BCL-2 members have been extensively explored, and several candidate compounds are now being analyzed for their efficacy against various cancers including lung cancer3-5 (see below).

JCTD-17-1123-Fig1

Figure 1. Mitochondrial apoptotic pathway. In response to cytotoxic stimuli, pro-apoptotic BH3-only proteins, such as BIM, BID, NOXA, and PUMA, inactivate anti-apoptotic multi-domain members, such as MCL1, BCL-XL, and BCL-2, through direct interaction with their BH3 domain. Inactivation of anti-apoptotic members changes the inactive form of BAX/BAK to the active form, resulting in oligomerization of BAX/BAK in the mitochondrial outer membrane. Oligomerized BAX/BAK form a pore to release mitochondrial protein cytochrome c into the cytoplasm. Released cytochrome c activates the Apaf-1 apoptosome, and subsequent activation of caspases results in induction of apoptosis2,3.

Lung cancer is the leading cause of cancer mortality worldwide, and accumulating evidence has suggested that high expression of anti-apoptotic MCL1 protein by various mechanisms is important for oncogenesis, tumor development, and chemotherapeutic drug resistance of lung cancer cells. Indeed, in a mouse lung adenocarcinoma model, in which the oncogenic transcription factor Myc was targeted to pulmonary alveolar cells, it has been shown that MCL1 overexpression augments tumor progression by circumventing Myc-induced apoptosis6. This mini-review introduces the role and mechanism of MCL1 overexpression in lung cancer cells and discusses the possibility of treatments targeting MCL1.

High resolution analyses of somatic copy number alterations revealed gene amplification of BCLX and MCL1 in a substantial proportion of human cancers, especially lung and breast cancers7. Moreover, growth of MCL1 gene-amplified lung cancer cell lines is affected by inhibition of MCL1 expression using RNA interference7,8, suggesting a crucial role of MCL1 in lung cancer. This notion is also supported by results showing that copy number variation of MCL1 predicts overall survival of patients with non-small cell lung cancer (NSCLC)9.

MCL1 gene transcription is upregulated by cytokines, such as interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor, and growth factors such as epidermal growth factor and vascular endothelial growth factor10,11. Moreover, the MCL-1 promoter has been shown to be activated by the transcription factors STAT3, NF-κB, CREBP, PU.1, SP-1, ELK-1, ATF-6, and HIF-111. In lung cancer, activation of STAT3 and NF-κB are frequently observed and their roles have been analyzed experimentally12-16. Moreover, STAT3 and ELK-1 are activated by EGFR (epidermal growth factor receptor)13,14. In addition, a recent report has shown that expression of a microRNA, which directly suppresses MCL1 mRNA, is suppressed in lung cancer17, suggesting that MCL1 mRNA expression is activated by transcriptional and post-transcriptional regulation in lung cancer.

Accumulating evidence has shown that expression of MCL1 protein is tightly regulated by the ubiquitin-proteasome system10,18. In the course of these experiments, several ubiquitin ligases were identified. MULE (Mcl-1 ubiquitin ligase E3) is a HECT domain ubiquitin ligase and contains the BH3 domain that allows MULE to specifically interact with MCL119. Although it has been shown that inhibition of MULE expression induces hepatocarcinogenesis through stabilization of MCL120, it has not been reported whether it is involved in the onset of lung cancer. In addition, β-TrCP (β-transducin repeat-containing protein) degrades MCL1 via its phosphorylation at serine 155, serine 159, and threonine 163 by GSK-3β (glycogen synthase kinase-3β)21. β-TrCP is an F-box protein and functions as a substrate recognition component of the SCF (SKP1-cullin 1-F-box protein) family of ubiquitin ligases22. The inactive phosphorylation of GSK-3β at serine 9 and EGFR expression are both negatively linked to survival of lung cancer patients23. However, the relationship between lung cancer and GSK3 is currently under investigation24.

The F-box protein FBW7 is a well characterized tumor suppressor acting as an ubiquitin ligase that targets MCL1 and well characterized as a tumor suppressor gene25-27. The FBXW7 gene, which encodes FBW7, is frequently mutated in diverse cancer types including leukemia and breast, colon, liver, ovarian, and lung cancers26-28. FBW7 expression is transcriptionally regulated by the tumor suppressor p5329, and loss-of-function of p53 reduces the expression of FBW730. Therefore, the tumor-suppressive function of FBW7 may be considered only in the context of p53. Since, FBW7 degrades several proto-oncogenes that function in cell growth, such as c-MYC, cyclin E, Notch, and c-JUN26,28, its tumor-suppressing function of FBW7 is not only limited to MCL1 degradation. In addition to these mechanisms, we recently found that chaperone-mediated autophagy31, a specific protein degradation system, promotes survival of several lung cancer cell lines through the selective stabilization of MCL1 by degradation of the ubiquitin ligase that targets MCL132. Despite these findings, much is still unknown about the regulation of MCL1 protein stability in healthy and malignant cells.

The deubiquitinase USP9X binds to MCL1 and removes polyubiquitin chains that mark MCL1 for proteasomal degradation33. Moreover, increased USP9X expression correlates with increased MCL1 protein in human cancers, and knockdown of USP9X enhances MCL1 turnover, suggesting that USP9X stabilizes MCL1 and promotes cancer cell survival. In lung cancer, it has been reported that USP9X expression in NSCLC tissue is significantly higher than in normal lung tissue, and that an elevated expression level of USP9X is associated with a poor prognosis34. A global map of p53 transcription factor-binding sites revealed that USP9X might be a p53 target gene35. Induction of USP9X by radiation renders cancer cells more therapy resistant as high MCL1 protein levels prevent apoptosis36. These findings suggest that USP9X performs its oncogenic activity through stabilization of MCL1.

During the process of oncogenic transformation, cells show higher expression of pro-apoptotic proteins resulting from cell cycle checkpoint activation, DNA replication stress, and/or many other stresses37. However, cancer cells survive by adapting to the effects of increasing levels of MCL1 and other anti-apoptotic BCL-2 family proteins. Although some lung cancer cells do not depend on MCL1 for survival38, the expression of MCL1 is elevated in most lung cancer cells by various mechanisms (Figure 2). Many reports have shown that suppression of MCL1 increases the sensitivity of lung cancer cells to anticancer drugs10,18,27. Considering these facts, BH3 mimetics that are selective inhibitors of MCL1 and other anti-apoptotic BCL-2 proteins may be potential therapeutic agents for lung cancer.

JCTD-17-1123-Fig2

Figure 2. Schematic representation of the mechanism of high MCL1 expression in cancer cells. In cancer cells, MCL1 protein is highly expressed, mainly by three mechanisms: gene amplification, enhanced gene expression by transcription factors involved in cell proliferation, and protein stabilization by decreased expression of the ubiquitin-ligase complex or high expression of deubiquitinating enzymes. See main text for details.

BH3 mimetics are small compounds that antagonize anti-apoptotic BCL-2 family proteins, leading to apoptosis induction in cancer cells3,4,39. Similar to the BH3 domain in BH3-only proteins, BH3 mimetics specifically interact with anti-apoptotic BCL-2 family proteins and disrupt their ability to interact with pro-apoptotic BCL-2 proteins to induce BAX/BAK-dependent apoptosis2,39. Among such compounds, ABT-263 (navitoclax), a dual inhibitor of BCL-XL and BCL-2, has been shown to be significantly effective in most chronic lymphocytic leukemia (CLL) patients in clinical trials, and ABT-199 (venetoclax), a selective BCL-2 inhibitor, is also effective in patients with relapsed or refractory CLL2,40. The three-dimensional structures of anti-apoptotic BCL-2 proteins, such as BCL-2 and BCL-XL, share a common motif consisting of four amphipathic helices that form a hydrophobic groove serving as the binding site for pro-apoptotic BH3 domains. High- affinity binding of BH3 peptides to both BCL-2 and BCL-XL is mediated primarily by interactions in two hydrophobic pockets, termed P2 and P4, and navitoclax specifically binds to these P2 and P4 pockets39. The efficacy of ABT-263 and its related compound, ABT-737, has been demonstrated in lung cancer8,41-46. Because of the key role of MCL1 in protecting malignant cells against anti-cancer treatments, combinatorial therapy with BH3-binding molecules such as navitoclax may enhance the therapeutic effects of radiotherapy and other treatments. Multiple approaches have been undertaken to directly target MCL1, and several MCL1-specific BH3 mimetics have been identified39,47. For example, S63845 binds with high affinity to human MCL1 without appreciable binding to BCL-2 or BCL-XL47. Analyses of these compounds and further molecular development are expected to lead to effective treatments for lung cancer.

The authors declare no conflict of interest.

We thank Mitchell Arico from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

  1. Kozopas KM, Yang T, Buchan HL, et al. MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci U S A. 1993; 90: 3516-3520
  2. Delbridge AR, Grabow S, Strasser A, et al. Thirty years of BCL-2: translating cell death discoveries into novel cancer therapies. Nat Rev Cancer. 2016; 16: 99-109. doi:10.1038/nrc.2015.17
  3. Czabotar PE, Lessene G, Strasser A, et al. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nature reviews. Molecular cell biology. 2014; 15: 49-63. doi:10.1038/nrm3722
  4. Cragg MS, Harris C, Strasser A, et al. Unleashing the power of inhibitors of oncogenic kinases through BH3 mimetics. Nat Rev Cancer. 2009; 9: 321-326. doi:10.1038/nrc2615
  5. Nakajima W, Tanaka N. BH3 mimetics: their action and efficacy in cancer chemotherapy. Integr Cancer Sci Ther. 2016; 3: 437-441. doi:DOI: 10.15761/ICST.1000184
  6. Allen TD, Zhu CQ, Jones KD, et al. Interaction between MYC and MCL1 in the genesis and outcome of non-small-cell lung cancer. Cancer Res. 2011; 71: 2212-2221. doi:10.1158/0008-5472.CAN-10-3590
  7. Beroukhim R, Mermel CH, Porter D, et al. The landscape of somatic copy-number alteration across human cancers. Nature. 2010; 463: 899-905. doi:10.1038/nature08822
  8. Lin X, Morgan-Lappe S, Huang X, et al. 'Seed' analysis of off-target siRNAs reveals an essential role of Mcl-1 in resistance to the small-molecule Bcl-2/Bcl-XL inhibitor ABT-737. Oncogene. 2007; 26: 3972-3979. doi:10.1038/sj.onc.1210166
  9. Yin J, Li Y, Zhao H, et al. Copy-number variation of MCL1 predicts overall survival of non-small-cell lung cancer in a Southern Chinese population. Cancer Med. 2016; 5: 2171-2179. doi:10.1002/cam4.774
  10. Ertel F, Nguyen M, Roulston A, et al. Programming cancer cells for high expression levels of Mcl1. EMBO Rep. 2013; 14: 328-336. doi:10.1038/embor.2013.20
  11. Thomas LW, Lam C, Edwards SW. Mcl-1; the molecular regulation of protein function. FEBS Lett. 2010; 584: 2981-2989. doi:10.1016/j.febslet.2010.05.061
  12. Chen W, Li Z, Bai L, et al. NF-kappaB in lung cancer, a carcinogenesis mediator and a prevention and therapy target. Front Biosci (Landmark Ed). 2011; 16: 1172-1185
  13. Dutta P, Sabri N, Li J, et al. Role of STAT3 in lung cancer. JAKSTAT. 2014; 3: e999503. doi:10.1080/21623996.2014.999503
  14. Booy EP, Henson ES, Gibson SB. Epidermal growth factor regulates Mcl-1 expression through the MAPK-Elk-1 signalling pathway contributing to cell survival in breast cancer. Oncogene. 2011; 30: 2367-2378. doi:10.1038/onc.2010.616
  15. Jiang CC, Lucas K, Avery-Kiejda KA, et al. Up-regulation of Mcl-1 is critical for survival of human melanoma cells upon endoplasmic reticulum stress. Cancer Res. 2008; 68: 6708-6717. doi:10.1158/0008-5472.CAN-08-0349
  16. Piret JP, Minet E, Cosse JP, et al. Hypoxia-inducible factor-1-dependent overexpression of myeloid cell factor-1 protects hypoxic cells against tert-butyl hydroperoxide-induced apoptosis. J Biol Chem. 2005; 280: 9336-9344. doi:10.1074/jbc.M411858200
  17. Crawford M, Batte K, Yu L, et al. MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer. Biochem Biophys Res Commun. 2009; 388: 483-489. doi:10.1016/j.bbrc.2009.07.143
  18. Vucic D, Dixit VM, Wertz IE. Ubiquitylation in apoptosis: a post-translational modification at the edge of life and death. Nature reviews. Molecular cell biology. 2011; 12: 439-452. doi:10.1038/nrm3143
  19. Zhong Q, Gao W, Du F, et al. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis. Cell. 2005; 121: 1085-1095. doi:10.1016/j.cell.2005.06.009
  20. Gruber S, Straub BK, Ackermann PJ, et al. Obesity promotes liver carcinogenesis via Mcl-1 stabilization independent of IL-6Ralpha signaling. Cell Rep. 2013; 4: 669-680. doi:10.1016/j.celrep.2013.07.023
  21. Ding Q, He X, Hsu JM, et al. Degradation of Mcl-1 by beta-TrCP mediates glycogen synthase kinase 3-induced tumor suppression and chemosensitization. Mol Cell Biol. 2007; 27: 4006-4017. doi:10.1128/MCB.00620-06
  22. Lipkowitz S, Weissman AM. RINGs of good and evil: RING finger ubiquitin ligases at the crossroads of tumour suppression and oncogenesis. Nat Rev Cancer. 2011; 11: 629-643. doi:10.1038/nrc3120
  23. Zheng H, Saito H, Masuda S, et al. Phosphorylated GSK3beta-ser9 and EGFR are good prognostic factors for lung carcinomas. Anticancer Res. 2007; 27: 3561-3569
  24. Rapp J, Jaromi L, Kvell K, et al. WNT signaling - lung cancer is no exception. Respir Res. 2017; 18: 167. doi:10.1186/s12931-017-0650-6
  25. Inuzuka H, Shaik S, Onoyama I, et al. SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction. Nature. 2011; 471: 104-109. doi:10.1038/nature09732
  26. Welcker M, Clurman BE. FBW7 ubiquitin ligase: a tumour suppressor at the crossroads of cell division, growth and differentiation. Nat Rev Cancer. 2008; 8: 83-93. doi:10.1038/nrc2290
  27. Wertz IE, Kusam S, Lam C, et al. Sensitivity to antitubulin chemotherapeutics is regulated by MCL1 and FBW7. Nature. 2011; 471: 110-114. doi:10.1038/nature09779
  28. Davis RJ, Welcker M, Clurman BE. Tumor suppression by the Fbw7 ubiquitin ligase: mechanisms and opportunities. Cancer Cell. 2014; 26: 455-464. doi:10.1016/j.ccell.2014.09.013
  29. Perez-Losada J, Mao JH, Balmain A. Control of genomic instability and epithelial tumor development by the p53-Fbxw7/Cdc4 pathway. Cancer Res. 2005; 65: 6488-6492. doi:10.1158/0008-5472.CAN-05-1294
  30. Cao J, Ge MH, Ling ZQ. Fbxw7 Tumor Suppressor: A Vital Regulator Contributes to Human Tumorigenesis. Medicine (Baltimore). 2016; 95: e2496. doi:10.1097/MD.0000000000002496
  31. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 2011; 147: 728-741. doi:10.1016/j.cell.2011.10.026
  32. Suzuki J, Nakajima W, Suzuki H, et al. Chaperone-mediated autophagy promotes lung cancer cell survival through selective stabilization of the pro-survival protein, MCL1. Biochem Biophys Res Commun. 2017; 482: 1334-1340. doi:10.1016/j.bbrc.2016.12.037
  33. Schwickart M, Huang X, Lill JR, et al. Deubiquitinase USP9X stabilizes MCL1 and promotes tumour cell survival. Nature. 2010; 463: 103-107. doi:10.1038/nature08646
  34. Wang Y, Liu Y, Yang B, et al. Elevated expression of USP9X correlates with poor prognosis in human non-small cell lung cancer. J Thorac Dis. 2015; 7: 672-679. doi:10.3978/j.issn.2072-1439.2015.04.28
  35. Wei CL, Wu Q, Vega VB, et al. A global map of p53 transcription-factor binding sites in the human genome. Cell. 2006; 124: 207-219. doi:10.1016/j.cell.2005.10.043
  36. Trivigno D, Essmann F, Huber SM, et al. Deubiquitinase USP9x confers radioresistance through stabilization of Mcl-1. Neoplasia. 2012; 14: 893-904
  37. Montero J, Letai A. Why do BCL-2 inhibitors work and where should we use them in the clinic? Cell Death Differ. 2018; 25: 56-64. doi:10.1038/cdd.2017.183
  38. Matsumoto M, Nakajima W, Seike M, et al. Cisplatin-induced apoptosis in non-small-cell lung cancer cells is dependent on Bax- and Bak-induction pathway and synergistically activated by BH3-mimetic ABT-263 in p53 wild-type and mutant cells. Biochem Biophys Res Commun. 2016; 473: 490-496. doi:10.1016/j.bbrc.2016.03.053
  39. Ashkenazi A, Fairbrother WJ, Leverson JD, et al. From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors. Nat Rev Drug Discov. 2017; 16: 273-284. doi:10.1038/nrd.2016.253
  40. Vela L, Marzo I. Bcl-2 family of proteins as drug targets for cancer chemotherapy: the long way of BH3 mimetics from bench to bedside. Curr Opin Pharmacol. 2015; 23: 74-81. doi:10.1016/j.coph.2015.05.014
  41. Hann CL, Daniel VC, Sugar EA, et al. Therapeutic efficacy of ABT-737, a selective inhibitor of BCL-2, in small cell lung cancer. Cancer Res. 2008; 68: 2321-2328. doi:10.1158/0008-5472.CAN-07-5031
  42. Hauck P, Chao BH, Litz J, et al. Alterations in the Noxa/Mcl-1 axis determine sensitivity of small cell lung cancer to the BH3 mimetic ABT-737. Mol Cancer Ther. 2009; 8: 883-892. doi:10.1158/1535-7163.MCT-08-1118
  43. Rudin CM, Hann CL, Garon EB, et al. Phase II study of single-agent navitoclax (ABT-263) and biomarker correlates in patients with relapsed small cell lung cancer. Clin Cancer Res. 2012; 18: 3163-3169. doi:10.1158/1078-0432.CCR-11-3090
  44. Tahir SK, Yang X, Anderson MG et al. Influence of Bcl-2 family members on the cellular response of small-cell lung cancer cell lines to ABT-737. Cancer Res. 2007; 67: 1176-1183. doi:10.1158/0008-5472.CAN-06-2203
  45. Nakajima W, Hicks MA, Tanaka N, et al. Noxa determines localization and stability of MCL-1 and consequently ABT-737 sensitivity in small cell lung cancer. Cell Death Dis. 2014; 5: e1052. doi:10.1038/cddis.2014.6
  46. Nakajima W, Sharma K, Hicks MA, et al. Combination with vorinostat overcomes ABT-263 (navitoclax) resistance of small cell lung cancer. Cancer Biol Ther. 2016; 17: 27-35. doi:10.1080/15384047.2015.1108485
  47. Kotschy A, Szlavik Z, Murray J, et al. The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature. 2016; 538: 477-482. doi:10.1038/nature19830
 

Article Info

Article Notes

  • Published on: February 07, 2018

Keywords

  • Lung cancer

  • MCL1
  • Apoptosis evasion
  • Chemotherapy
  • Protein stabilization
  • Drug resistance

*Correspondence:

Dr. Nobuyuki Tanaka
Department of Molecular Oncology
Institute for Advanced Medical Sciences, Nippon Medical School, Japan
Email: nobuta@nms.ac.jp