RNF144A functions as a tumor suppressor in breast cancer through ubiquitin ligase activity-dependent regulation of stability and oncogenic functions of HSPA2

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Deregulation of E3 ubiquitin ligases is intimately implicated in breast cancer pathogenesis and progression, but the underlying mechanisms still remain elusive. Here we report that RING finger protein 144A (RNF144A), a poorly characterized member of the RING-in-between-RING family of E3 ubiquitin ligases, functions as a tumor suppressor in breast cancer. RNF144A was  downregulated in a subset of primary breast tumors and restoration of RNF144A suppressed breast cancer cell proliferation, colony formation, migration, invasion in vitro, tumor growth, and lung metastasis in vivo. In contrast, knockdown of RNF144A promoted malignant phenotypes of breast cancer cells. Quantitative proteomics and biochemical analysis revealed that RNF144A interacted with and targeted heat-shock protein family A member 2 (HSPA2), a putative oncoprotein that is frequently upregulated in human cancer and promotes tumor growth and progression, for ubiquitination and degradation. Notably, the ligase activity-defective mutants of RNF144A impaired its ability to induce ubiquitination and degradation of HSPA2, and to suppress breast cancer cell proliferation, migration, and invasion as compared with its wild-type counterpart. Moreover, RNF144A-mediated suppression of breast cancer cell proliferation, migration, and invasion was rescued by ectopic HSPA2 expression. Clinically, low RNF144A and high HSPA2 expression in breast cancer patients was correlated with aggressive clinicopathological characteristics and decreased overall and disease-free survival. Collectively, these findings reveal a previously unappreciated role for RNF144A in suppression of breast cancer growth and metastasis, and identify RNF144A as the first, to our knowledge, E3 ubiquitin ligase for HSPA2 in human cancer.

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  1. 1.

    Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

  2. 2.

    Chen C, Seth AK, Aplin AE. Genetic and expression aberrations of E3 ubiquitin ligases in human breast cancer. Mol Cancer Res. 2006;4:695–707.

  3. 3.

    Buetow L, Huang DT. Structural insights into the catalysis and regulation of E3 ubiquitin ligases. Nat Rev Mol Cell Biol. 2016;17:626–42.

  4. 4.

    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;11:629–43.

  5. 5.

    Rotin D, Kumar S. Physiological functions of the HECT family of ubiquitin ligases. Nat Rev Mol Cell Biol. 2009;10:398–409.

  6. 6.

    Walden H, Rittinger K. RBR ligase-mediated ubiquitin transfer: a tale with many twists and turns. Nat Struct Mol Biol. 2018;25:440–5.

  7. 7.

    Meszaros B, Kumar M, Gibson TJ, Uyar B, Dosztanyi Z. Degrons in cancer. Sci Signal 2017, 10:pii: eaak9982.

  8. 8.

    Marin I, Lucas JI, Gradilla AC, Ferrus A. Parkin and relatives: the RBR family of ubiquitin ligases. Physiol Genom. 2004;17:253–63.

  9. 9.

    Spratt DE, Walden H, Shaw GS. RBR E3 ubiquitin ligases: new structures, new insights, new questions. Biochem J. 2014;458:421–37.

  10. 10.

    Eisenhaber B, Chumak N, Eisenhaber F, Hauser MT. The ring between ring fingers (RBR) protein family. Genome Biol. 2007;8:209.

  11. 11.

    Ho SR, Lee YJ, Lin WC. Regulation of RNF144A E3 ubiquitin ligase activity by self-association through its transmembrane domain. J Biol Chem. 2015;290:23026–38.

  12. 12.

    Ho SR, Mahanic CS, Lee YJ, Lin WC. RNF144A, an E3 ubiquitin ligase for DNA-PKcs, promotes apoptosis during DNA damage. Proc Natl Acad Sci USA. 2014;111:E2646–2655.

  13. 13.

    Zhang Y, Yang YL, Zhang FL, Liao XH, Shao ZM, Li DQ. Epigenetic silencing of RNF144A expression in breast cancer cells through promoter hypermethylation and MBD4. Cancer Med. 2018;7:1317–25.

  14. 14.

    Zhang Y, Liao XH, Xie HY, Shao ZM, Li DQ. RBR-type E3 ubiquitin ligase RNF144A targets PARP1 for ubiquitin-dependent degradation and regulates PARP inhibitor sensitivity in breast cancer cells. Oncotarget. 2017;8:94505–18.

  15. 15.

    Scieglinska D, Krawczyk Z. Expression, function, and regulation of the testis-enriched heat shock HSPA2 gene in rodents and humans. Cell Stress Chaperon. 2015;20:221–35.

  16. 16.

    Eddy EM. Role of heat shock protein HSP70-2 in spermatogenesis. Rev Reprod. 1999;4:23–30.

  17. 17.

    Garg M, Kanojia D, Seth A, Kumar R, Gupta A, Surolia A, et al. Heat-shock protein 70-2 (HSP70-2) expression in bladder urothelial carcinoma is associated with tumour progression and promotes migration and invasion. Eur J Cancer. 2010;46:207–15.

  18. 18.

    Garg M, Kanojia D, Saini S, Suri S, Gupta A, Surolia A, et al. Germ cell-specific heat shock protein 70-2 is expressed in cervical carcinoma and is involved in the growth, migration, and invasion of cervical cells. Cancer. 2010;116:3785–96.

  19. 19.

    Jagadish N, Agarwal S, Gupta N, Fatima R, Devi S, Kumar V, et al. Heat shock protein 70-2 (HSP70-2) overexpression in breast cancer. J Exp Clin Cancer Res. 2016;35:150.

  20. 20.

    Jagadish N, Parashar D, Gupta N, Agarwal S, Suri V, Kumar R, et al. Heat shock protein 70-2 (HSP70-2) is a novel therapeutic target for colorectal cancer and is associated with tumor growth. BMC Cancer. 2016;16:561.

  21. 21.

    Gupta N, Jagadish N, Surolia A, Suri A. Heat shock protein 70-2 (HSP70-2) a novel cancer testis antigen that promotes growth of ovarian cancer. Am J Cancer Res. 2017;7:1252–69.

  22. 22.

    Rohde M, Daugaard M, Jensen MH, Helin K, Nylandsted J, Jaattela M. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms. Genes Dev. 2005;19:570–82.

  23. 23.

    Singh S, Suri A. Targeting the testis-specific heat-shock protein 70-2 (HSP70-2) reduces cellular growth, migration, and invasion in renal cell carcinoma cells. Tumour Biol. 2014;35:12695–706.

  24. 24.

    Zhang H, Chen W, Duan CJ, Zhang CF. Overexpression of HSPA2 is correlated with poor prognosis in esophageal squamous cell carcinoma. World J Surg Oncol. 2013;11:141.

  25. 25.

    Fu Y, Zhao H, Li XS, Kang HR, Ma JX, Yao FF, et al. Expression of HSPA2 in human hepatocellular carcinoma and its clinical significance. Tumour Biol. 2014;35:11283–7.

  26. 26.

    Zhang H, Gao H, Liu C, Kong Y, Wang C. Expression and clinical significance of HSPA2 in pancreatic ductal adenocarcinoma. Diagn Pathol. 2015;10:13.

  27. 27.

    Huang WJ, Xia LM, Zhu F, Huang B, Zhou C, Zhu HF, et al. Transcriptional upregulation of HSP70-2 by HIF-1 in cancer cells in response to hypoxia. Int J Cancer. 2009;124:298–305.

  28. 28.

    Sasaki T, Marcon E, McQuire T, Arai Y, Moens PB, Okada H. Bat3 deficiency accelerates the degradation of Hsp70-2/HspA2 during spermatogenesis. J Cell Biol. 2008;182:449–58.

  29. 29.

    Bromfield E, Aitken RJ, Nixon B. Novel characterization of the HSPA2-stabilizing protein BAG6 in human spermatozoa. Mol Hum Reprod. 2015;21:755–69.

  30. 30.

    Bromfield EG, Aitken RJ, McLaughlin EA, Nixon B. Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse. Mol Hum Reprod. 2017;23:91–105.

  31. 31.

    Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate breast cancer metastasis to lung. Nature. 2005;436:518–24.

  32. 32.

    Zhang FL, Cao JL, Xie HY, Sun R, Yang LF, Shao ZM, et al. Cancer-associated MORC2-mutant M276I regulates an hnRNPM-mediated CD44 splicing switch to promote invasion and metastasis in triple-negative breast cancer. Cancer Res. 2018;78:5780–92.

  33. 33.

    Sun R, Xie HY, Qian JX, Huang YN, Yang F, Zhang FL, et al. FBXO22 possesses both protumorigenic and antimetastatic roles in breast cancer progression. Cancer Res. 2018;78:5274–86.

  34. 34.

    Liang CC, Park AY, Guan JL. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007;2:329–33.

  35. 35.

    Liao XH, Zhang Y, Dong WJ, Shao ZM, Li DQ. Chromatin remodeling protein MORC2 promotes breast cancer invasion and metastasis through a PRD domain-mediated interaction with CTNND1. Oncotarget. 2017;8:97941–54.

  36. 36.

    Liu X, Zheng W, Wang W, Shen H, Liu L, Lou W, et al. A new panel of pancreatic cancer biomarkers discovered using a mass spectrometry-based pipeline. Br J Cancer. 2017;117:1846–54.

  37. 37.

    Liu J, Zhang C, Zhao Y, Yue X, Wu H, Huang S, et al. Parkin targets HIF-1alpha for ubiquitination and degradation to inhibit breast tumor progression. Nat Commun. 2017;8:1823.

  38. 38.

    Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012;486:346–52.

  39. 39.

    Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature. 2000;406:747–52.

  40. 40.

    Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nat Rev Clin Oncol. 2016;13:674–90.

  41. 41.

    Chambers AF, Groom AC, MacDonald IC. Dissemination and growth of cancer cells in metastatic sites. Nat Rev Cancer. 2002;2:563–72.

  42. 42.

    Zhou W, Slingerland JM. Links between oestrogen receptor activation and proteolysis: relevance to hormone-regulated cancer therapy. Nat Rev Cancer. 2014;14:26–38.

  43. 43.

    Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L. Treatment of HER2-positive breast cancer: current status and future perspectives. Nat Rev Clin Oncol. 2011;9:16–32.

  44. 44.

    Scieglinska D, Piglowski W, Chekan M, Mazurek A, Krawczyk Z. Differential expression of HSPA1 and HSPA2 proteins in human tissues; tissue microarray-based immunohistochemical study. Histochem Cell Biol. 2011;135:337–50.

  45. 45.

    Scieglinska D, Piglowski W, Mazurek A, Malusecka E, Zebracka J, Filipczak P, et al. The HspA2 protein localizes in nucleoli and centrosomes of heat shocked cancer cells. J Cell Biochem. 2008;104:2193–206.

  46. 46.

    Ghanta KS, Li DQ, Eswaran J, Kumar R. Gene profiling of MTA1 identifies novel gene targets and functions. PLoS ONE. 2011;6:e17135.

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We sincerely acknowledge the staff members of the Pathology Core Facility (Shanghai Cancer Center), the Proteomic Center (Institutes of Biomedical Sciences), the Animal Resource Center (State Key Laboratory of Oncogene and Related Gene), and members in the Li laboratory for their excellent technical assistance. The work in the Li laboratory is supported, in whole or in part, by the National Natural Science Foundation of China (No. 81372847, 81572584, and 81772805), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning (No. 2013-06), the Science and Technology Innovation Action Plan of Shanghai Municipal Science and Technology Commission (No. 16JC1405400), and start-up fund for new investigators from Fudan University.

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Correspondence to Da-Qiang Li or Zhi-Min Shao.

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