Downregulation of Smurf2, a tumor-suppressive ubiquitin ligase, in triple-negative breast cancers: Involvement of the RB-microRNA axis

BMC Cancer

Hiroaki Kiyokawa²  Yiran Zhou²  Alfred W Rademaker³  Ashley B Flowers¹  Limin Sun²  Xin Gu¹  Xianpeng Liu² 

[1]Department of Pathology, Louisiana State University Health Science Center, Shreveport, LA, USA

[2]Department of Molecular Pharmacology & Biological Chemistry, Northwestern University, Chicago, IL 60611, USA

[3]Robert H Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL 60611, USA

关键词: Tumor suppressor;    Retinoblastoma;    MicroRNA;    Ubiquitination;    Triple-negative breast cancer;   

Others  :  859100
DOI  :  10.1186/1471-2407-14-57

 received in 2013-08-20, accepted in 2014-01-27,  发布年份 2014

【 摘 要 】

Background

The HECT family ubiquitin ligase Smurf2 regulates cell polarity, migration, division, differentiation and death, by targeting diverse substrates that are critical for receptor signaling, cytoskeleton, chromatin remodeling and transcription. Recent studies suggest that Smurf2 functions as a tumor suppressor in mice. However, no inactivating mutation of SMURF2 has been reported in human, and information about Smurf2 expression in human cancer remains limited or complicated. Here we demonstrate that Smurf2 expression is downregulated in human breast cancer tissues, especially of the triple-negative subtype, and address the mechanism of Smurf2 downregulation in triple-negative breast cancer cells.

Methods

Human breast cancer tissues (47 samples expressing estrogen receptor (ER) and 43 samples with triple-negative status) were examined by immunohistochemistry for the expression of Smurf2. Ten widely-studied human breast cancer cell lines were examined for the expression of Smurf2. Furthermore, microRNA-mediated regulation of Smurf2 was investigated in triple-negative cancer cell lines.

Results

Immunohistochemical analysis showed that benign mammary epithelial cells expressed high levels of Smurf2, so did cells in ductal carcinomas in situ. In contrast, invasive ductal carcinomas showed focal or diffuse decrease in Smurf2 expression, which was observed more frequently in triple-negative tumors than in ER-positive tumors. Consistently, human triple-negative breast cancer cell lines such as BT549, MDA-MB-436, DU-4475 and MDA-MB-468 cells showed significantly lower expression of Smurf2 protein, compared to ER + or HER2+ cell lines. Studies using quantitative PCR and specific microRNA inhibitors indicated that increased expression of miR-15a, miR-15b, miR-16 and miR-128 was involved in Smurf2 downregulation in those triple-negative cancer cell lines, which have mutations in the retinoblastoma (RB) gene. Forced expression of RB increased levels of Smurf2 protein with concomitant decreases in the expression of the microRNAs.

Conclusions

This study provides evidence of posttranscriptional downregulation of Smurf2 in triple-negative breast cancers, and demonstrates that the loss of RB function is involved in microRNA-mediated interference with Smurf2 translation. The new link from RB inactivation to Smurf2 downregulation is likely to play a role in malignant phenotypes of triple-negative breast cancer cells.

【 授权许可】

   
2014 Liu et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Lehmann BD, Bauer JA, Chen X, Sanders ME, Chakravarthy AB, Shyr Y, Pietenpol JA: Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest 2011, 121:2750-2767.
• [2]Morris GJ, Naidu S, Topham AK, Guiles F, Xu Y, McCue P, Schwartz GF, Park PK, Rosenberg AL, Brill K, et al.: Differences in breast carcinoma characteristics in newly diagnosed African-American and Caucasian patients: a single-institution compilation compared with the National Cancer Institute’s Surveillance, Epidemiology, and End Results database. Cancer 2007, 110:876-884.
• [3]Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka CA, Lickley LA, Rawlinson E, Sun P, Narod SA: Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Canc Res 2007, 13:4429-4434.
• [4]David D, Nair SA, Pillai MR: Smurf E3 ubiquitin ligases at the cross roads of oncogenesis and tumor suppression. Biochim Biophys Acta 1835, 2013:119-128.
• [5]Fukuchi M, Fukai Y, Masuda N, Miyazaki T, Nakajima M, Sohda M, Manda R, Tsukada K, Kato H, Kuwano H: High-level expression of the Smad ubiquitin ligase Smurf2 correlates with poor prognosis in patients with esophageal squamous cell carcinoma. Canc Res 2002, 62:7162-7165.
• [6]Ray D, Ahsan A, Helman A, Chen G, Hegde A, Gurjar SR, Zhao L, Kiyokawa H, Beer DG, Lawrence TS, et al.: Regulation of EGFR protein stability by the HECT-type ubiquitin ligase SMURF2. Neoplasia 2011, 13:570-578.
• [7]Blank M, Tang Y, Yamashita M, Burkett SS, Cheng SY, Zhang YE: A tumor suppressor function of Smurf2 associated with controlling chromatin landscape and genome stability through RNF20. Nat Med 2012, 18:227-234.
• [8]Hellwinkel OJ, Asong LE, Rogmann JP, Sultmann H, Wagner C, Schlomm T, Eichelberg C: Transcription alterations of members of the ubiquitin-proteasome network in prostate carcinoma. Prostate Canc PD 2011, 14:38-45.
• [9]Ramkumar C, Kong Y, Cui H, Hao S, Jones SN, Gerstein RM, Zhang H: Smurf2 regulates the senescence response and suppresses tumorigenesis in mice. Canc Res 2012, 72:2714-2719.
• [10]Osmundson EC, Ray D, Moore FE, Gao Q, Thomsen GH, Kiyokawa H: The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint. J Cell Biol 2008, 183:267-277.
• [11]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001, 25:402-408.
• [12]Singh R, Mo YY: Role of microRNAs in breast cancer. Canc Biol Ther 2013, 14:201-212.
• [13]Liu H: MicroRNAs in breast cancer initiation and progression. Cell Mol Life Sci 2012, 69(21):3587-3599.
• [14]Zhu Y, Yu F, Jiao Y, Feng J, Tang W, Yao H, Gong C, Chen J, Su F, Zhang Y, et al.: Reduced miR-128 in breast tumor-initiating cells induces chemotherapeutic resistance via Bmi-1 and ABCC5. Clin Canc Res 2011, 17:7105-7115.
• [15]Polytarchou C, Iliopoulos D, Struhl K: An integrated transcriptional regulatory circuit that reinforces the breast cancer stem cell state. Proc Natl Acad Sci USA 2012, 109:14470-14475.
• [16]Ofir M, Hacohen D, Ginsberg D: MiR-15 and miR-16 are direct transcriptional targets of E2F1 that limit E2F-induced proliferation by targeting cyclin E. Mol Canc Res 2011, 9:440-447.
• [17]Subhawong AP, Subhawong T, Nassar H, Kouprina N, Begum S, Vang R, Westra WH, Argani P: Most basal-like breast carcinomas demonstrate the same Rb-/p16+ immunophenotype as the HPV-related poorly differentiated squamous cell carcinomas which they resemble morphologically. Am J Surg Pathol 2009, 33:163-175.
• [18]Dick FA, Rubin SM: Molecular mechanisms underlying RB protein function. Nat Rev Mol Cell Bio 2013, 14:297-306.
• [19]Bandi N, Zbinden S, Gugger M, Arnold M, Kocher V, Hasan L, Kappeler A, Brunner T, Vassella E: miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Canc Res 2009, 69:5553-5559.
• [20]Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, Barbosa-Morais NL, Teschendorff AE, Green AR, Ellis IO, et al.: MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol 2007, 8:R214. BioMed Central Full Text
• [21]Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H, Taylor M, Harris AL, Ragoussis J: microRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Canc Res 2011, 71:5635-5645.
• [22]Qian P, Banerjee A, Wu ZS, Zhang X, Wang H, Pandey V, Zhang WJ, Lv XF, Tan S, Lobie PE, et al.: Loss of SNAIL regulated miR-128-2 on chromosome 3p22.3 targets multiple stem cell factors to promote transformation of mammary epithelial cells. Canc Res 2012, 72:6036-6050.
• [23]Donzelli S, Fontemaggi G, Fazi F, Di Agostino S, Padula F, Biagioni F, Muti P, Strano S, Blandino G: MicroRNA-128-2 targets the transcriptional repressor E2F5 enhancing mutant p53 gain of function. Cell Death Differ 2012, 19:1038-1048.
• [24]Kavsak P, Rasmussen RK, Causing CG, Bonni S, Zhu H, Thomsen GH, Wrana JL: Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 2000, 6:1365-1375.
• [25]Bonni S, Wang HR, Causing CG, Kavsak P, Stroschein SL, Luo K, Wrana JL: TGF-beta induces assembly of a Smad2-Smurf2 ubiquitin ligase complex that targets SnoN for degradation. Nat Cell Biol 2001, 3:587-595.
• [26]Massague J: TGFbeta signalling in context. Nat Rev Mol Cell Biol 2012, 13:616-630.
• [27]Moses H, Barcellos-Hoff MH: TGF-beta biology in mammary development and breast cancer. CSH Perspect Biol 2011, 3:a003277.
• [28]Nie J, Xie P, Liu L, Xing G, Chang Z, Yin Y, Tian C, He F, Zhang L: Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by stabilizing the E3 ligase MDM2. J Biol Chem 2010, 285:22818-22830.
• [29]Kong Y, Cui H, Zhang H: Smurf2-mediated ubiquitination and degradation of Id1 regulates p16 expression during senescence. Aging Cell 2011, 10:1038-1046.
• [30]Gupta GP, Perk J, Acharyya S, De Candia P, Mittal V, Todorova-Manova K, Gerald WL, Brogi E, Benezra R, Massague J: ID genes mediate tumor reinitiation during breast cancer lung metastasis. Proc Natl Acad Sci USA 2007, 104:19506-19511.
• [31]Ohtani N, Zebedee Z, Huot TJ, Stinson JA, Sugimoto M, Ohashi Y, Sharrocks AD, Peters G, Hara E: Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence. Nature 2001, 409:1067-1070.
• [32]Fukunaga E, Inoue Y, Komiya S, Horiguchi K, Goto K, Saitoh M, Miyazawa K, Koinuma D, Hanyu A, Imamura T: Smurf2 induces ubiquitin-dependent degradation of Smurf1 to prevent migration of breast cancer cells. J Biol Chem 2008, 283:35660-35667.
• [33]Jin C, Yang YA, Anver MR, Morris N, Wang X, Zhang YE: Smad ubiquitination regulatory factor 2 promotes metastasis of breast cancer cells by enhancing migration and invasiveness. Canc Res 2009, 69:735-740.