Superenhancers activate the autophagy-related genes Beclin1 and LC3B to drive metastasis and drug resistance in osteosarcoma

Abstract

Metastasis and drug resistance are the leading causes of poor prognosis in patients with osteosarcoma. Identifying the relevant factors that drive metastasis and drug resistance is the key to improving the therapeutic outcome of osteosarcoma. Here, we reported that autophagy was highly activated in metastatic osteosarcoma. We found increased autophagolysosomes in metastatic osteosarcoma cell lines by using electron microscopy, Western blot, and immunofluorescence experiments. We further examined the expression of the autophagy-related genes Beclin1 and LC3B in 82 patients through immunohistochemistry and found that Beclin1 and LC3B were highly related to unfavorable prognosis of osteosarcoma. Knockdown of Beclin1 and LC3B reduced invasion, metastasis, and proliferation in metastatic osteosarcoma cells. In vitro and in vivo studies also demonstrated that inhibiting by 3-MA inhibited cell growth and metastasis. Moreover, we demonstrated that autophagy-related genes were activated by SEs and that the inhibition of SEs by JQ-1 decreased the metastasis of osteosarcoma. Overall, our findings highlighted the association of autophagy with osteosarcoma progression and shed new light on autophagy-targeting therapy for osteosarcoma.

Keywords

osteosarcoma / autophagy / metastasis / drug resistance / Beclin1 / LC3B

References

[1] Kelley LM, Schlegel M, Hecker-Nolting S, Kevric M, Haller B, Rössig C, Reichardt P, Kager L, Kühne T, Gosheger G, Windhager R, Specht K, Rechl H, Tunn PU, Baumhoer D, Wirth T, Werner M, von Kalle T, Nathrath M, Burdach S, Bielack S, von Lüttichau I. Pathological fracture and prognosis of high-grade osteosarcoma of the extremities: an analysis of 2,847 Consecutive Cooperative Osteosarcoma Study Group (COSS) patients. J Clin Oncol 2020; 38(8): 823–833

[2] Duffaud F, Mir O, Boudou-Rouquette P, Piperno-Neumann S, Penel N, Bompas E, Delcambre C, Kalbacher E, Italiano A, Collard O, Chevreau C, Saada E, Isambert N, Delaye J, Schiffler C, Bouvier C, Vidal V, Chabaud S, Blay JY; French Sarcoma Group. Efficacy and safety of regorafenib in adult patients with metastatic osteosarcoma: a non-comparative, randomised, double-blind, placebo-controlled, phase 2 study. Lancet Oncol 2019; 20(1): 120–133

[3] Zhao J, Dean DC, Hornicek FJ, Yu X, Duan Z. Emerging next-generation sequencing-based discoveries for targeted osteosarcoma therapy. Cancer Lett 2020; 474: 158–167

[4] Anwar MA, El-Baba C, Elnaggar MH, Elkholy YO, Mottawea M, Johar D, Al Shehabi TS, Kobeissy F, Moussalem C, Massaad E, Omeis I, Darwiche N, Eid AH. Novel therapeutic strategies for spinal osteosarcomas. Semin Cancer Biol 2020; 64: 83–92

[5] Heymann D. Metastatic osteosarcoma challenged by regorafenib. Lancet Oncol 2019; 20(1): 12–14

[6] Italiano A, Mir O, Mathoulin-Pelissier S, Penel N, Piperno-Neumann S, Bompas E, Chevreau C, Duffaud F, Entz-Werlé N, Saada E, Ray-Coquard I, Lervat C, Gaspar N, Marec-Berard P, Pacquement H, Wright J, Toulmonde M, Bessede A, Crombe A, Kind M, Bellera C, Blay JY. Cabozantinib in patients with advanced Ewing sarcoma or osteosarcoma (CABONE): a multicentre, single-arm, phase 2 trial. Lancet Oncol 2020; 21(3): 446–455

[7] Lamb CA, Yoshimori T, Tooze SA. The autophagosome: origins unknown, biogenesis complex. Nat Rev Mol Cell Biol 2013; 14(12): 759–774

[8] Kim KH, Lee MS. Autophagy—a key player in cellular and body metabolism. Nat Rev Endocrinol 2014; 10(6): 322–337

[9] Heras-Sandoval D, Pérez-Rojas JM, Hernández-Damián J, Pedraza-Chaverri J. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal 2014; 26(12): 2694–2701

[10] Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ 2011; 18(4): 571–580

[11] Janku F, McConkey DJ, Hong DS, Kurzrock R. Autophagy as a target for anticancer therapy. Nat Rev Clin Oncol 2011; 8(9): 528–539

[12] Cicchini M, Karantza V, Xia B. Molecular pathways: autophagy in cancer—a matter of timing and context. Clin Cancer Res 2015; 21(3): 498–504

[13] White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer 2012; 12(6): 401–410

[14] Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer 2017; 17(9): 528–542

[15] Galluzzi L, Bravo-San Pedro JM, Demaria S, Formenti SC, Kroemer G. Activating autophagy to potentiate immunogenic chemotherapy and radiation therapy. Nat Rev Clin Oncol 2017; 14(4): 247–258

[16] Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer 2007; 7(12): 961–967

[17] Li X, He S, Ma B. Autophagy and autophagy-related proteins in cancer. Mol Cancer 2020; 19(1): 12

[18] Bursch W. The autophagosomal-lysosomal compartment in programmed cell death. Cell Death Differ 2001; 8(6): 569–581

[19] Chen S, Rehman SK, Zhang W, Wen A, Yao L, Zhang J. Autophagy is a therapeutic target in anticancer drug resistance. Biochim Biophys Acta 2010; 1806(2): 220–229

[20] Sui X, Chen R, Wang Z, Huang Z, Kong N, Zhang M, Han W, Lou F, Yang J, Zhang Q, Wang X, He C, Pan H. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment. Cell Death Dis 2013; 4(10): e838

[21] Dalby KN, Tekedereli I, Lopez-Berestein G, Ozpolat B. Targeting the prodeath and prosurvival functions of autophagy as novel therapeutic strategies in cancer. Autophagy 2010; 6(3): 322–329

[22] Kim M, Jung JY, Choi S, Lee H, Morales LD, Koh JT, Kim SH, Choi YD, Choi C, Slaga TJ, Kim WJ, Kim DJ. GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy. Autophagy 2017; 13(1): 149–168

[23] Huang J, Liu K, Yu Y, Xie M, Kang R, Vernon P, Cao L, Tang D, Ni J. Targeting HMGB1-mediated autophagy as a novel therapeutic strategy for osteosarcoma. Autophagy 2012; 8(2): 275–277

[24] Horie R, Nakamura O, Yamagami Y, Mori M, Nishimura H, Fukuoka N, Yamamoto T. Apoptosis and antitumor effects induced by the combination of an mTOR inhibitor and an autophagy inhibitor in human osteosarcoma MG63 cells. Int J Oncol 2016; 48(1): 37–44

[25] Liu K, Ren T, Huang Y, Sun K, Bao X, Wang S, Zheng B, Guo W. Apatinib promotes autophagy and apoptosis through VEGFR2/STAT3/BCL-2 signaling in osteosarcoma. Cell Death Dis 2017; 8(8): e3015

[26] Lin C, He H, Liu H, Li R, Chen Y, Qi Y, Jiang Q, Chen L, Zhang P, Zhang H, Li H, Zhang W, Sun Y, Xu J. Tumour-associated macrophages-derived CXCL8 determines immune evasion through autonomous PD-L1 expression in gastric cancer. Gut 2019; 68(10): 1764–1773

[27] Wang W, Wang X, Fujioka H, Hoppel C, Whone AL, Caldwell MA, Cullen PJ, Liu J, Zhu X. Parkinson’s disease-associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes. Nat Med 2016; 22(1): 54–63

[28] Ren T, Zheng B, Huang Y, Wang S, Bao X, Liu K, Guo W. Osteosarcoma cell intrinsic PD-L2 signals promote invasion and metastasis via the RhoA-ROCK-LIMK2 and autophagy pathways. Cell Death Dis 2019; 10(4): 261

[29] Morrow JJ, Bayles I, Funnell APW, Miller TE, Saiakhova A, Lizardo MM, Bartels CF, Kapteijn MY, Hung S, Mendoza A, Dhillon G, Chee DR, Myers JT, Allen F, Gambarotti M, Righi A, DiFeo A, Rubin BP, Huang AY, Meltzer PS, Helman LJ, Picci P, Versteeg HH, Stamatoyannopoulos JA, Khanna C, Scacheri PC. Positively selected enhancer elements endow osteosarcoma cells with metastatic competence. Nat Med 2018; 24(2): 176–185

[30] Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods 2012; 9(4): 357–359

[31] Feng J, Liu T, Qin B, Zhang Y, Liu XS. Identifying ChIP-seq enrichment using MACS. Nat Protoc 2012; 7(9): 1728–1740

[32] Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010; 26(6): 841–842

[33] Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, Cheng JX, Murre C, Singh H, Glass CK. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell 2010; 38(4): 576–589

[34] Patil SL, Palat A, Pan Y, Rajapakshe K, Mirchandani R, Bondesson M, Yustein JT, Coarfa C, Gunaratne PH. MicroRNA-509-3p inhibits cellular migration, invasion, and proliferation, and sensitizes osteosarcoma to cisplatin. Sci Rep 2019; 9(1): 19089

[35] Schott CR, Ludwig L, Mutsaers AJ, Foster RA, Wood GA. The autophagy inhibitor spautin-1, either alone or combined with doxorubicin, decreases cell survival and colony formation in canine appendicular osteosarcoma cells. PLoS One 2018; 13(10): e0206427

[36] Mori M, Hitora T, Nakamura O, Yamagami Y, Horie R, Nishimura H, Yamamoto T. Hsp90 inhibitor induces autophagy and apoptosis in osteosarcoma cells. Int J Oncol 2015; 46(1): 47–54

[37] Salminen A, Kaarniranta K, Kauppinen A, Ojala J, Haapasalo A, Soininen H, Hiltunen M. Impaired autophagy and APP processing in Alzheimer’s disease: the potential role of Beclin 1 interactome. Prog Neurobiol 2013; 106-107: 33–54

[38] Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A, Rosen J, Eskelinen EL, Mizushima N, Ohsumi Y, Cattoretti G, Levine B. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest 2003; 112(12): 1809–1820

[39] Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, Levine B. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999; 402(6762): 672–676

[40] Guo Y, Huang C, Li G, Chen T, Li J, Huang Z. Paxilitaxel induces apoptosis accompanied by protective autophagy in osteosarcoma cells through hypoxia-inducible factor-1α pathway. Mol Med Rep 2015; 12(3): 3681–3687

[41] Tao H, Chen F, Liu H, Hu Y, Wang Y, Li H. Wnt/β-catenin signaling pathway activation reverses gemcitabine resistance by attenuating Beclin1-mediated autophagy in the MG63 human osteosarcoma cell line. Mol Med Rep 2017; 16(2): 1701–1706

[42] Dong M, Wan XB, Yuan ZY, Wei L, Fan XJ, Wang TT, Lv YC, Li X, Chen ZH, Chen J, Lin Q, Wen JY, Ma XK, Liu Q, Wu XY. Low expression of Beclin 1 and elevated expression of HIF-1α refine distant metastasis risk and predict poor prognosis of ER-positive, HER2-negative breast cancer. Med Oncol 2013; 30(1): 355

[43] Chen YB, Hou JH, Feng XY, Chen S, Zhou ZW, Zhang XS, Cai MY. Decreased expression of Beclin 1 correlates with a metastatic phenotypic feature and adverse prognosis of gastric carcinomas. J Surg Oncol 2012; 105(6): 542–547

[44] Zhao H, Yang M, Zhao J, Wang J, Zhang Y, Zhang Q. High expression of LC3B is associated with progression and poor outcome in triple-negative breast cancer. Med Oncol 2013; 30(1): 475

[45] Scholz BA, Sumida N, de Lima CDM, Chachoua I, Martino M, Tzelepis I, Nikoshkov A, Zhao H, Mehmood R, Sifakis EG, Bhartiya D, Göndör A, Ohlsson R. WNT signaling and AHCTF1 promote oncogenic MYC expression through super-enhancer-mediated gene gating. Nat Genet 2019; 51(12): 1723–1731

[46] Bahr C, von Paleske L, Uslu VV, Remeseiro S, Takayama N, Ng SW, Murison A, Langenfeld K, Petretich M, Scognamiglio R, Zeisberger P, Benk AS, Amit I, Zandstra PW, Lupien M, Dick JE, Trumpp A, Spitz F. A Myc enhancer cluster regulates normal and leukaemic haematopoietic stem cell hierarchies. Nature 2018; 553(7689): 515–520

[47] Chipumuro E, Marco E, Christensen CL, Kwiatkowski N, Zhang T, Hatheway CM, Abraham BJ, Sharma B, Yeung C, Altabef A, Perez-Atayde A, Wong KK, Yuan GC, Gray NS, Young RA, George RE. CDK7 inhibition suppresses super-enhancer-linked oncogenic transcription in MYCN-driven cancer. Cell 2014; 159(5): 1126–1139

[48] Ying Y, Wang Y, Huang X, Sun Y, Zhang J, Li M, Zeng J, Wang M, Xiao W, Zhong L, Xu B, Li L, Tao Q, Wang X, Shu XS. Oncogenic HOXB8 is driven by MYC-regulated super-enhancer and potentiates colorectal cancer invasiveness via BACH1. Oncogene 2020; 39(5): 1004–1017

[49] Jiang Y, Jiang YY, Xie JJ, Mayakonda A, Hazawa M, Chen L, Xiao JF, Li CQ, Huang ML, Ding LW, Sun QY, Xu L, Kanojia D, Jeitany M, Deng JW, Liao LD, Soukiasian HJ, Berman BP, Hao JJ, Xu LY, Li EM, Wang MR, Bi XG, Lin DC, Koeffler HP. Co-activation of super-enhancer-driven CCAT1 by TP63 and SOX2 promotes squamous cancer progression. Nat Commun 2018; 9(1): 3619

[50] Borck PC, Guo LW, Plutzky J. BET epigenetic reader proteins in cardiovascular transcriptional programs. Circ Res 2020; 126(9): 1190–1208

[51] Chapuy B, McKeown MR, Lin CY, Monti S, Roemer MG, Qi J, Rahl PB, Sun HH, Yeda KT, Doench JG, Reichert E, Kung AL, Rodig SJ, Young RA, Shipp MA, Bradner JE. Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell 2013; 24(6): 777–790

[52] Chen D, Zhao Z, Huang Z, Chen DC, Zhu XX, Wang YZ, Yan YW, Tang S, Madhavan S, Ni W, Huang ZP, Li W, Ji W, Shen H, Lin S, Jiang YZ. Super enhancer inhibitors suppress MYC driven transcriptional amplification and tumor progression in osteosarcoma. Bone Res 2018; 6(1): 11

RIGHTS & PERMISSIONS

Higher Education Press