Beclin-1 expression is retained in high-grade serous ovarian cancer yet is not essential for autophagy induction in vitro

Journal of Ovarian Research

Gabriel E. DiMattia⁴  Trevor G. Shepherd³  Yudith Ramos Valdes¹  Rohann J. M. Correa² 

[1] Translational Ovarian Cancer Research Program, London Regional Cancer Program, London, Ontario, Canada;Department of Biochemistry, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada;Department of Anatomy & Cell Biology, Schulich School of Medicine & Dentistry, The University of Western Ontario, London, Ontario, Canada;London Regional Cancer Program, 790 Commissioners Road East, Room A4-919A, London N6A 4 L6Ontario, Canada

关键词: Beclin-1;    Autophagy;    Spheroid;    High-grade serous ovarian cancer;   

Others  :  1224169
DOI  :  10.1186/s13048-015-0182-y

 received in 2015-03-19, accepted in 2015-07-27,  发布年份 2015

【 摘 要 】

Background

Autophagy is a conserved cellular self-digestion mechanism that can either suppress or promote cancer in a context-dependent manner. In ovarian cancer, prevalent mono-allelic deletion of BECN1 (a canonical autophagy-inducer) suggests that autophagy is impaired to promote carcinogenesis and that Beclin-1 is a haploinsufficient tumor suppressor. Nonetheless, autophagy is known to be readily inducible in ovarian cancer cells. We sought to clarify whether Beclin-1 expression is in fact disrupted in ovarian cancer and whether this impacts autophagy regulation.

Methods

BECN1 expression levels were assessed using The Cancer Genome Atlas (TCGA) datasets from 398 ovarian high-grade serous cystadenocarcinomas (HGSC) and protein immunoblot data from HGSC samples obtained at our institution. Knockdown of BECN1 and other autophagy-related gene expression was achieved using siRNA in established human ovarian cancer cell lines (CaOV3, OVCAR8, SKOV3, and HeyA8) and a novel early-passage, ascites-derived cell line (iOvCa147-E2). LC3 immunoblot, autophagic flux assays, transmission electron microscopy and fluorescence microscopy were used to assess autophagy.

Results

We observed prevalent mono-allelic BECN1 gene deletion (76 %) in TCGA tumors, yet demonstrate for the first time that Beclin-1 protein expression remains relatively unaltered in these and additional samples generated at our institution. Surprisingly, efficient siRNA-mediated Beclin-1 knockdown did not attenuate autophagy induction, whereas knockdown of other autophagy-related genes blocked the process. Beclin-1 knockdown instead decreased cell viability without inducing apoptosis.

Conclusions

Taken together, these data demonstrate that despite its sustained expression, Beclin-1 is dispensable for autophagy induction in ovarian tumor cells in vitro, yet may be retained to promote cell viability by a mechanism independent of autophagy or apoptosis regulation. Overall, this work makes novel observations about tumor expression of Beclin-1 and challenges the accepted understanding of its role in regulating autophagy in ovarian cancer.

【 授权许可】

   
2015 Correa et al.

【 图 表 】

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

【 参考文献 】

• [1]Levine B, Kroemer G. Autophagy in the Pathogenesis of Disease. Cell. 2008; 132(1):27-42.
• [2]Leone RD, Amaravadi RK. Autophagy: a targetable linchpin of cancer cell metabolism. Trends in endocrinology and metabolism: TEM. 2013. doi:10.1016/j.tem.2013.01.008.
• [3]Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y et al.. Nutrient-dependent mTORC1 association with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell. 2009; 20(7):1981-91.
• [4]Yang Z, Klionsky DJ. Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010; 12(9):814-22.
• [5]Geng J, Klionsky DJ. The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series. EMBO reports. 2008; 9(9):859-64.
• [6]Kihara A, Kabeya Y, Ohsumi Y, Yoshimori T. Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network. EMBO reports. 2001; 2(4):330-5.
• [7]Panaretou C, Domin J, Cockcroft S, Waterfield MD. Characterization of p150, an adaptor protein for the human phosphatidylinositol (PtdIns) 3-kinase. Substrate presentation by phosphatidylinositol transfer protein to the p150.Ptdins 3-kinase complex. J Biol Chem. 1997; 272(4):2477-85.
• [8]Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H et al.. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999; 402(6762):672-6.
• [9]Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A et al.. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003; 112(12):1809-20.
• [10]Gao X, Zacharek A, Salkowski A, Grignon DJ, Sakr W, Porter AT et al.. Loss of heterozygosity of the BRCA1 and other loci on chromosome 17q in human prostate cancer. Cancer Res. 1995; 55(5):1002-5.
• [11]Saito H, Inazawa J, Saito S, Kasumi F, Koi S, Sagae S et al.. Detailed deletion mapping of chromosome 17q in ovarian and breast cancers: 2-cM region on 17q21.3 often and commonly deleted in tumors. Cancer Res. 1993; 53(14):3382-5.
• [12]Futreal PA, Soderkvist P, Marks JR, Iglehart JD, Cochran C, Barrett JC et al.. Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma using microsatellite length polymorphisms. Cancer Res. 1992; 52(9):2624-7.
• [13]Aita VM, Liang XH, Murty VV, Pincus DL, Yu W, Cayanis E et al.. Cloning and genomic organization of beclin 1, a candidate tumor suppressor gene on chromosome 17q21. Genomics. 1999; 59(1):59-65.
• [14]Cliby W, Ritland S, Hartmann L, Dodson M, Halling KC, Keeney G et al.. Human epithelial ovarian cancer allelotype. Cancer Res. 1993; 53(10 Suppl):2393-8.
• [15]Eccles DM, Russell SE, Haites NE, Atkinson R, Bell DW, Gruber L et al.. Early loss of heterozygosity on 17q in ovarian cancer. The Abe Ovarian Cancer Genetics Group. Oncogene. 1992; 7(10):2069-72.
• [16]Russell SE, Hickey GI, Lowry WS, White P, Atkinson RJ. Allele loss from chromosome 17 in ovarian cancer. Oncogene. 1990; 5(10):1581-3.
• [17]Tangir J, Muto MG, Berkowitz RS, Welch WR, Bell DA, Mok SC. A 400 kb novel deletion unit centromeric to the BRCA1 gene in sporadic epithelial ovarian cancer. Oncogene. 1996; 12(4):735-40.
• [18]Lazova R, Camp RL, Klump V, Siddiqui SF, Amaravadi RK, Pawelek JM. Punctate LC3B expression is a common feature of solid tumors and associated with proliferation, metastasis, and poor outcome. Clin Cancer Res. 2012; 18(2):370-9.
• [19]Guo JY, Chen HY, Mathew R, Fan J, Strohecker AM, Karsli-Uzunbas G et al.. Activated Ras requires autophagy to maintain oxidative metabolism and tumorigenesis. Genes Dev. 2011; 25(5):460-70.
• [20]Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY et al.. Autophagy suppresses tumorigenesis through elimination of p62. Cell. 2009; 137(6):1062-75.
• [21]Amaravadi RK, Yu D, Lum JJ, Bui T, Christophorou MA, Evan GI et al.. Autophagy inhibition enhances therapy-induced apoptosis in a Myc-induced model of lymphoma. J Clin Invest. 2007; 117(2):326-36.
• [22]Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouyssegur J et al.. Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains. Mol Cell Biol. 2009; 29(10):2570-81.
• [23]Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N et al.. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005; 25(3):1025-40.
• [24]Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T et al.. Growth Factor Regulation of Autophagy and Cell Survival in the Absence of Apoptosis. Cell. 2005; 120(2):237-48.
• [25]Lu Z, Luo RZ, Lu Y, Zhang X, Yu Q, Khare S et al.. The tumor suppressor gene ARHI regulates autophagy and tumor dormancy in human ovarian cancer cells. J Clin Invest. 2008; 118(12):3917-29.
• [26]Correa RJ, Valdes YR, Peart TM, Fazio EN, Bertrand M, McGee J et al. Combination of AKT inhibition with autophagy blockade effectively reduces ascites-derived ovarian cancer cell viability. Carcinogenesis. 2014. doi:10.1093/carcin/bgu049.
• [27]Lu Z, Baquero MT, Yang H, Yang M, Reger AS, Kim C et al.. DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells. Autophagy. 2014; 10(6):1071-92.
• [28]Shepherd TG, Theriault BL, Campbell EJ, Nachtigal MW. Primary culture of ovarian surface epithelial cells and ascites-derived ovarian cancer cells from patients. Nat Protoc. 2006; 1(6):2643-9.
• [29]Gao J, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO et al.. Integrative Analysis of Complex Cancer Genomics and Clinical Profiles Using the cBioPortal. Sci Signal. 2013; 6(269):l1.
• [30]Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB et al.. Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Molecular cancer therapeutics. 2006; 5(10):2512-21.
• [31]Smith DM, Patel S, Raffoul F, Haller E, Mills GB, Nanjundan M. Arsenic trioxide induces a beclin-1-independent autophagic pathway via modulation of SnoN/SkiL expression in ovarian carcinoma cells. Cell Death Differ. 2010; 17(12):1867-81.
• [32]Shield K, Ackland ML, Ahmed N, Rice GE. Multicellular spheroids in ovarian cancer metastases: Biology and pathology. Gynecol Oncol. 2009; 113(1):143-8.
• [33]Correa RJ, Peart T, Valdes YR, DiMattia GE, Shepherd TG. Modulation of AKT activity is associated with reversible dormancy in ascites-derived epithelial ovarian cancer spheroids. Carcinogenesis. 2012; 33(1):49-58.
• [34]Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S et al.. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012; 483(7391):603-7.
• [35]Mizushima N, Yoshimori T, Levine B. Methods in Mammalian Autophagy Research. Cell. 2010; 140(3):313-26.
• [36]Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011; 18(4):571-80.
• [37]Carduner L, Picot CR, Leroy-Dudal J, Blay L, Kellouche S, Carreiras F. Cell cycle arrest or survival signaling through alphav integrins, activation of PKC and ERK1/2 lead to anoikis resistance of ovarian cancer spheroids. Exp Cell Res. 2014; 320(2):329-42.
• [38]Xing H, Wang S, Hu K, Tao W, Li J, Gao Q et al.. Effect of the cyclin-dependent kinases inhibitor p27 on resistance of ovarian cancer multicellular spheroids to anticancer chemotherapy. J Cancer Res Clin Oncol. 2005; 131(8):511-9.
• [39]Cai M, Hu Z, Liu J, Gao J, Liu C, Liu D et al.. Beclin 1 expression in ovarian tissues and its effects on ovarian cancer prognosis. International journal of molecular sciences. 2014; 15(4):5292-303.
• [40]Zhao Y, Chen S, Gou WF, Xiao LJ, Takano Y, Zheng HC. Aberrant Beclin 1 expression is closely linked to carcinogenesis, differentiation, progression, and prognosis of ovarian epithelial carcinoma. Tumour Biol. 2013. doi:10.1007/s13277-013-1261-6.
• [41]Grishchuk Y, Ginet V, Truttmann AC, Clarke PG, Puyal J. Beclin 1-independent autophagy contributes to apoptosis in cortical neurons. Autophagy. 2011; 7(10):1115-31.
• [42]Zhu JH, Horbinski C, Guo F, Watkins S, Uchiyama Y, Chu CT. Regulation of autophagy by extracellular signal-regulated protein kinases during 1-methyl-4-phenylpyridinium-induced cell death. Am J Pathol. 2007; 170(1):75-86.
• [43]Seo G, Kim SK, Byun YJ, Oh E, Jeong SW, Chae GT et al.. Hydrogen peroxide induces Beclin 1-independent autophagic cell death by suppressing the mTOR pathway via promoting the ubiquitination and degradation of Rheb in GSH-depleted RAW 264.7 cells. Free Radic Res. 2011; 45(4):389-99.
• [44]Scarlatti F, Maffei R, Beau I, Codogno P, Ghidoni R. Role of non-canonical Beclin 1-independent autophagy in cell death induced by resveratrol in human breast cancer cells. Cell Death Differ. 2008; 15(8):1318-29.
• [45]Wen Y, Zand B, Ozpolat B, Szczepanski MJ, Lu C, Yuca E et al. Antagonism of Tumoral Prolactin Receptor Promotes Autophagy-Related Cell Death. Cell reports. 2014. doi:10.1016/j.celrep.2014.03.009.
• [46]Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A. 2003; 100(25):15077-82.
• [47]Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T et al.. The role of autophagy during the early neonatal starvation period. Nature. 2004; 432(7020):1032-6.
• [48]Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I et al.. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. The Journal of cell biology. 2005; 169(3):425-34.
• [49]Takamura A, Komatsu M, Hara T, Sakamoto A, Kishi C, Waguri S et al.. Autophagy-deficient mice develop multiple liver tumors. Genes Dev. 2011; 25(8):795-800.
• [50]Wirawan E, Lippens S, Vanden Berghe T, Romagnoli A, Fimia GM, Piacentini M et al.. Beclin1: a role in membrane dynamics and beyond. Autophagy. 2012; 8(1):6-17.
• [51]Sanjuan MA, Dillon CP, Tait SW, Moshiach S, Dorsey F, Connell S et al.. Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature. 2007; 450(7173):1253-7.
• [52]Thoresen SB, Pedersen NM, Liestol K, Stenmark H. A phosphatidylinositol 3-kinase class III sub-complex containing VPS15, VPS34, Beclin 1, UVRAG and BIF-1 regulates cytokinesis and degradative endocytic traffic. Exp Cell Res. 2010; 316(20):3368-78.
• [53]Fremont S, Gerard A, Galloux M, Janvier K, Karess RE, Berlioz-Torrent C. Beclin-1 is required for chromosome congression and proper outer kinetochore assembly. EMBO reports. 2013. doi:10.1038/embor.2013.23.