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Stathmin 1 Is a Potential Novel Oncogene in Melanoma

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Stathmin 1 Is a Potential Novel Oncogene in Melanoma Oncogene (2013) 32, 1330–1337 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc SHORT COMMUNICATION Stathmin 1 is a potential novel oncogene in melanoma J Chen, M Abi-Daoud, A Wang , X Yang, X Zhang, HE Feilotter and VA Tron In previous studies, we demonstrated that miR-193b expression is reduced in melanoma relative to benign nevi, and also that miR- 193b represses cyclin D1 and Mcl-1 expression. We suggested that stathmin 1 (STMN1) might be a target of miR-193b. STMN1 normally regulates microtubule dynamics either by sequestering free tubulin heterodimers or by promoting microtubule catastrophe. Increased expression of STMN1 has been observed in a variety of human malignancies, but its association with melanoma is unknown. We now report that STMN1 is upregulated during the progression of melanoma relative to benign nevi, and that STMN1 is directly regulated by miR-193b. Using an experimental cell culture approach, overexpression of miR-193b using synthetic microRNAs repressed STMN1 expression, whereas inhibition of miR-193b with anti-miR oligos increased STMN1 expression in melanoma cells. The use of a luciferase reporter assay confirmed that miR-193b directly regulates STMN1 by targeting the 30-untranslated region of STMN1 mRNA. We further demonstrated that STMN1 is overexpressed in malignant melanoma compared with nevi in two independent melanoma cohorts, and that its level is inversely correlated with miR-193b expression. However, STMN1 expression was not significantly associated with patient survival, Breslow depth, mitotic count or patient age. STMN1 knockdown by small-interfering RNA in melanoma cells drastically repressed cell proliferation and migration potential, whereas ectopic expression of STMN1 using lentivirus increased cell proliferation and migration rates. Subsequent gene expression analysis indicated that interconnected cytoskeletal networks are directly affected following STMN1 knockdown. In addition, we identified deregulated genes associated with proliferation and migration, and revealed that p21Cip1/Waf1 and p27Kip could be downstream effectors of STMN1 signaling. Taken together, our study suggests that downregulation of miR-193b may contribute to increased STMN1 expression in melanoma, which consequently promotes migration and proliferation of tumor cells. Oncogene (2013) 32, 1330–1337; doi:10.1038/onc.2012.141; published online 4 June 2012 Keywords: STMN1; miR-193b; melanoma; oncogene INTRODUCTION The dysregulation of specific miRNAs and their potential targets Melanoma is the most dangerous form of skin cancer, and the has been examined in melanoma, and begins to provide a clearer incidence of melanoma has increased steadily over the past picture of cellular processes that are impacted by changes in two decades.1 Melanoma arises from melanocytes, and the miRNA expression. For example, miR-182 is reported to be progression of the disease has been associated with genetic and upregulated in metastatic melanoma relative to nevi, and in vitro epigenetic changes,2 some of which are now identified. For overexpression of miR-182 in melanoma cells promotes example, cyclin-dependent kinase inhibitor 2A (CDKN2A), a tumor migration and survival by directly repressing microphthalmia- 14 suppressor gene, is often mutated in familial melanoma.3 BRAF associated transcription factor-M and FOXO3. In contrast, miR- mutations are presented in B50% of melanoma cases while 211, often downregulated or absent in melanoma, normally another B15% of tumors contain NRAS mutations, leading to a decreases migration and invasion capacity of cells by 15,16 constitutively activated MAPK pathway.3,4 suppressing IGF2R, TGFBR2, NFAT5 and POU3F2. In our Recent studies have begun to unveil the important roles of previous studies, miR-193b was identified as a significantly 12,17 microRNA (miRNA) dysregulation during the progression of downregulated molecule in malignant melanoma. We melanoma. miRNAs are B22 nt single-stranded non-coding RNAs demonstrated that miR-193b directly regulates cyclin D1 and that post-transcriptionally regulate gene expression through Mcl-1. This tumor suppressor potential of miR-193b has been also binding to the 30-untranslated region (UTR) of target mRNAs, reported in breast cancer, prostate cancer and hepatocellular 18–21 subsequently leading to mRNA destabilization and translation carcinoma. inhibition.5 More than 1000 miRNAs have been identified in The direct relationship between miRNAs and their target mRNAs the human genome (http://www.mirbase.org/cgi-bin/mirna_ provides a useful paradigm for studying disease progression. By summary.pl?org=hsa). A single miRNA can affect the expression examining downstream changes following disruption of expres- of hundreds of genes,6,7 and 460% of human protein-coding sion of a particular miRNA, key target genes can be identified and genes are now predicted to be regulated by miRNAs.8 miRNAs are their role in the process under study can be further investigated. involved in regulating virtually every aspect of cellular function, Using this approach, we report that stathmin 1 (STMN1) is a direct and are implicated in human diseases.9 Mounting evidence target of miR-193b, and further, that this protein is overexpressed indicates that miRNAs can function as oncogenes or tumor in melanoma. Reduction of STMN1 levels decreases the melanoma suppressor genes.10 Aberrant expression of miRNAs has been cell’s ability to proliferate and migrate, whereas ectopic expression reported in most human malignancies,11 including melanoma.12,13 of STMN1 promotes the opposite effects. Department of Pathology and Molecular Medicine, Queen’s University, Kingston, Ontario, Canada. Correspondence: Dr VA Tron, Department of Pathology and Molecular Medicine, Queen’s University, 88 Stuart Street, Richardson Laboratory, Room 202, Kingston, Ontario, Canada K7L 3N6. E-mail: [email protected] Received 30 October 2011; revised and accepted 27 February 2012; published online 4 June 2012 Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1331 RESULTS AND DISCUSSION cancer and colorectal cancer.23–30 However, its association with miR-193b directly regulates STMN1 melanoma is unknown. Because of our earlier studies suggesting that miR-193b was To confirm the role of miR-193b in regulating STMN1, human downregulated in melanoma relative to benign nevi, we had melanoma cell lines were transfected with synthetic miR-193b or undertaken gene expression profiling in melanoma cell lines to negative control oligonucleotides. We have previously demon- identify potential targets of miR-193b.12 From the list of genes strated increased levels of miR-193b in melanoma cells transfected showing reduced mRNA expression following ectopic expression with synthetic miR-193b oligos using northern blotting and real- of miR-193b, we selected candidates of interest, based on their time PCR.12,17 Ectopic expression of miR-193b was associated with known biological functions. One such gene of interest was STMN1. notably decreased STMN1 levels in all five melanoma cell lines STMN1 is an important protein that regulates microtubule tested by western blotting (Figure 1a). We were interested to dynamics through either sequestering free tubulin heterodimers know whether reducing endogenous miR-193b levels would or promoting rapid microtubule depolymerization and shrinkage achieve the opposite effect and increase STMN1 protein levels. (catastrophe).22 Overexpression of STMN1 has been observed in a To answer this question, chemically modified, single-stranded variety of human malignancies, including breast cancer, prostate oligonucleotides were used to inhibit endogenous miR-193 in cancer, sarcoma, non-small cell lung cancer, hepatoma, gastric Malme-3M, A375 and SK-MEL-2 cells, the cell lines expressing a b A375 Malme-3M MeWo SK-MEL-2 SK-MEL-28 Malme-3M A375 SK-MEL-2 Negative Control + – + – + – + – + – Anti-miR control + – + – + – miR-193b ––++++ – – – + Anti-miR-193b – + – + – + STMN1 STMN1 Densitometry 1.01.7 1.01.4 1.0 1.3 Gamma tubulin Gamma tubulin cd Human STMN1 3’UTR miR-193b/a miR-101 0 100 200 300 400 500 600 700 800 898 Conserved sites for miRNA families conserved among vertebrates e * 140 120 100 80 60 40 (Renilla/Firefly) 20 Relative Luciferase Activity 0 miR-193b miR-193b negative control negative control psiCHECK-STMN1 + psiCHECK-STMN1 + psiCHECK-STMN1 M + psiCHECK-STMN1 M + Figure 1. miR-193b represses STMN1 expression and directly targets STMN1. (a) Western blot analysis of STMN1 expression in A375, Malme- 3M, MeWo, SK-MEL-2 and SK-MEL-28 cells transfected with miR-193b or negative control. Seeded in 100-mm dishes at 6 Â 105 cells per plate the day before transfection, cells were transfected with 5 nmol/l miRNA precursor (negative control or miR-193b) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and were harvested 72 h after transfection. The STMN1 antibody (sc-48362) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and gamma tubulin from Sigma-Aldrich (Oakville, Ontario, Canada). (b) Increased STMN1 expression in Malme-3M cells transfected with anti-miR-193b. The procedure was as described in (a), except that Malme-3M cells were transfected with 100 nmol/l miRNA inhibitor (anti-miR control or anti-miR-193b). Gamma tubulin was used as the loading control. Densitometry was performed using Quantity One software (Bio-Rad, Mississauga, Ontario, Canada). Both miRNA precursors and anti-miR inhibitors were purchased from Ambion (Austin, TX, USA). Representative
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