High Expression of G9a Induces Cisplatin Resistance in Hepatocellular Carcinoma

Document Type : Original Article

Authors

1 Central Laboratory, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, Zhejiang Province, China

2 Department of Hepatobiliary and Pancreatic Surgery, Affiliated Jinhua Hospital, Zhejiang University School of Medicine, Jinhua, Zhejiang Province, China

Abstract

Objective: Chemotherapeutic drug resistance is the main obstacle that affects the efficacy of current therapies of
hepatocellular carcinoma (HCC), which needs to be addressed urgently. High expression of histone methyltransferase
G9a was reported to play a pivotal role in the progression of HCC. Regulatory mechanism of aberrant activation of G9a
in HCC and the association with subsequent cisplatin (DDP) resistance still remains ambiguous. This study strived to
investigate mechanism of G9a overexpression and its impact on cisplatin resistance in HCC cells.
Materials and Methods: In this experimental study, we investigated effects of different concentrations of cisplatin in
combination with BIX-01294 or PR-619 on viability and apoptosis of HuH7 and SNU387 cells via CCK-8 kit and flow
cytometric analysis, respectively. Colony formation capacity was applied to evaluate effect of cisplatin with or without
BIX-01294 on cell proliferation, and western blotting was used to verify expression level of the related proteins. Global
mRNA expression profile analysis was adopted to identify differentially expressed genes associated with overexpression
of G9a.
Results: We observed that overexpression of G9a admittedly promoted cisplatin resistance in HCC cells. Global
mRNA expression profile analysis after G9a inhibition showed that DNA repair and cell cycle progression were downregulated.
Moreover, we identified that deubiquitination enzymes (DUBs) stabilized high expression of G9a in HCC
through deubiquitination. Additionally, cisplatin could significantly inhibit proliferation of DUBs-deficient HCC cells, while
promoting their apoptosis.
Conclusion: Collectively, our data indicated that DUBs stabilize G9a through deubiquitination, thereby participating in
the cisplatin resistance of HCC cells. The elucidation of this mechanism contributes to propose a potential alternative
intervention strategy for the treatment of HCC patients harboring high G9a levels.

Keywords


  1. Vivarelli S, Salemi R, Candido S, Falzone L, Santagati M, Stefani S, et al. Gut microbiota and cancer: from pathogenesis to therapy. Cancers (Basel). 2019; 11(1): 38.
  2. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021; 71(3): 209-249.
  3. Cancer Genome Atlas Research Network. Electronic address: wheeler@bcm.edu; Cancer Genome Atlas Research Network. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell. 2017; 169(7): 1327-1341. e23.
  4. Shokoohian B, Negahdari B, Aboulkheyr EsH, Abedi-Valugerdi M, Baghaei K, Agarwal T, et al. Advanced therapeutic modalities in hepatocellular carcinoma: novel insights. J Cell Mol Med. 2021; 25(18): 8602-8614.
  5. Chen Z, Xie H, Hu M, Huang T, Hu Y, Sang N, et al. Recent progress in treatment of hepatocellular carcinoma. Am J Cancer Res. 2020; 10(9): 2993-3036.
  6. Petrowsky H, Fritsch R, Guckenberger M, De Oliveira ML, Dutkowski P, Clavien PA. Modern therapeutic approaches for the treatment of malignant liver tumours. Nat Rev Gastroenterol Hepatol. 2020; 17(12): 755-772.
  7. Heimbach JK, Kulik LM, Finn RS, Sirlin CB, Abecassis MM, Roberts LR, et al. AASLD guidelines for the treatment of hepatocellular carcinoma. Hepatology. 2018; 67(1): 358-380.
  8. Chino F, Stephens SJ, Choi SS, Marin D, Kim CY, Morse MA, et al. The role of external beam radiotherapy in the treatment of hepatocellular cancer. Cancer. 2018; 124(17): 3476-3489.
  9. Fang D, Guo Y, Zhu Z,Chen W. Silence of p15 expression by RNAi enhances cisplatin resistance in hepatocellular carcinoma cells. Bosn J Basic Med Sci. 2012; 12(1): 4-9.
  10. Ghosh S. Cisplatin: the first metal based anticancer drug. Bioorg Chem. 2019; 88: 102925.
  11. Zhang B, Cao K, Liu Z, Shan W, Wen Q, Wang R. Receptor interacting protein kinase 3 promotes cisplatin-induced necroptosis in apoptosis-resistant HepG2/DDP cells. Neoplasma. 2019; 66(5): 694-703.
  12. Margueron R, Reinberg D. Chromatin structure and the inheritance of epigenetic information. Nat Rev Genet. 2010; 11(4): 285-296.
  13. Momparler RL. Cancer epigenetics. Oncogene. 2003; 22(42): 6479-6483.
  14. Nagaraju GP, Dariya B, Kasa P, Peela S, El-Rayes BF. Epigenetics in hepatocellular carcinoma. Semin Cancer Biol. 2022; 86(Pt 3): 622-632.
  15. Shafabakhsh R, Arianfar F, Vosough M, Mirzaei HR, Mahjoubin- Tehran M, Khanbabaei H, et al. Autophagy and gastrointestinal cancers: the behind the scenes role of long non-coding RNAs in initiation, progression, and treatment resistance. Cancer Gene Ther. 2021; 28(12): 1229-1255.
  16. Yokoyama M, Chiba T, Zen Y, Oshima M, Kusakabe Y, Noguchi Y, et al. Histone lysine methyltransferase G9a is a novel epigenetic target for the treatment of hepatocellular carcinoma. Oncotarget. 2017; 8(13): 21315-21326.
  17. Albert M, Helin K. Histone methyltransferases in cancer. Semin Cell Dev Biol. 2010; 21(2): 209-220.
  18. Shinkai Y, Tachibana M. H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev. 2011; 25(8): 781-788.
  19. Casciello F, Windloch K, Gannon F, Lee JS. Functional Role of G9a histone methyltransferase in cancer. Front Immunol. 2015; 6: 487.
  20. Wei L, Chiu DK, Tsang FH, Law CT, Cheng CL, Au SL, et al. Histone methyltransferase G9a promotes liver cancer development by epigenetic silencing of tumor suppressor gene RARRES3. J Hepatol. 2017; 67(4): 758-769.
  21. Bai K, Cao Y, Huang C, Chen J, Zhang X, Jiang Y. Association of histone methyltransferase G9a and overall survival after liver resection of patients with hepatocellular carcinoma with a median observation of 40 months. Medicine (Baltimore). 2016; 95(2): e2493.
  22. Swatek KN, Komander D. Ubiquitin modifications. Cell Res. 2016; 26(4): 399-422.
  23. Wilkinson KD. Regulation of ubiquitin-dependent processes by deubiquitinating enzymes. FASEB J. 1997; 11(14): 1245-1256.
  24. Lv XY, Duan T, Li J. The multiple roles of deubiquitinases in liver cancer. Am J Cancer Res. 2020; 10(6): 1647-1657.
  25. Ciechomska IA, Marciniak MP, Jackl J,Kaminska B. Pre-treatment or post-treatment of human glioma cells with BIX01294, the inhibitor of histone methyltransferase G9a, sensitizes cells to temozolomide. Front Pharmacol. 2018; 9: 1271.
  26. Liu CW, Hua KT, Li KC, Kao HF, Hong RL, Ko JY, et al. Histone methyltransferase G9a drives chemotherapy resistance by regulating the glutamate-cysteine ligase catalytic subunit in head and neck squamous cell carcinoma. Mol Cancer Ther. 2017; 16(7): 1421-1434.
  27. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009; 25(9): 1105-1111.
  28. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNASeq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28(5): 511-515.
  29. Wang L, Feng Z, Wang X, Wang X, Zhang X. DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics. 2010; 26(1): 136-138.
  30. Nakatsuka T, Tateishi K, Kato H, Fujiwara H, Yamamoto K, Kudo Y, et al. Inhibition of histone methyltransferase G9a attenuates liver cancer initiation by sensitizing DNA-damaged hepatocytes to p53- induced apoptosis. Cell Death Dis. 2021; 12(1): 99.
  31. Guo Y, Zhao YR, Liu H, Xin Y, Yu JZ, Zang YJ, et al. EHMT2 promotes the pathogenesis of hepatocellular carcinoma by epigenetically silencing APC expression. Cell Biosci. 2021; 11(1): 152.
  32. Hu Y, Zheng Y, Dai M, Wang X, Wu J, Yu B, et al. G9a and histone deacetylases are crucial for Snail2-mediated E-cadherin repression and metastasis in hepatocellular carcinoma. Cancer Sci. 2019; 110(11): 3442-3452.
  33. Harrigan JA, Jacq X, Martin NM, Jackson SP. Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov. 2018; 17(1): 57-78.
  34. Zhu H, Yan F, Yuan T, Qian M, Zhou T, Dai X, et al. USP10 Promotes proliferation of hepatocellular carcinoma by deubiquitinating and Stabilizing YAP/TAZ. Cancer Res. 2020; 80(11): 2204-2216.
  35. Liao Y, Shao Z, Liu Y, Xia X, Deng Y, Yu C, et al. USP1-dependent RPS16 protein stability drives growth and metastasis of human hepatocellular carcinoma cells. J Exp Clin Cancer Res. 2021; 40(1): 201.
  36. Liu H, Chen W, Liang C, Chen BW, Zhi X, Zhang S, et al. WP1130 increases doxorubicin sensitivity in hepatocellular carcinoma cells through usp9x-dependent p53 degradation. Cancer Lett. 2015; 361(2): 218-225.
  37. Gao R, Buechel D, Kalathur RKR, Morini MF, Coto-Llerena M, Ercan C, et al. USP29-mediated HIF1alpha stabilization is associated with Sorafenib resistance of hepatocellular carcinoma cells by upregulating glycolysis. Oncogenesis. 2021; 10(7): 52.