Stathmin 1 Is a Potential Novel Oncogene in Melanoma

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 data from one of three independent experiments are shown. (c) Conserved miRNA-binding sites on the 30-UTR of STMN1 mRNA (modified from http://www.targetscan.org/cgi-bin/targetscan/vert_50/view_gene.cgi?taxid=9606&gs=STMN1 &showcnc=0&shownc=0). (d) miR-193b and its predicted seed-binding site in the 30-UTR of STMN1. Vector psiCHECK-STMN1 contains wild- type STMN1 30-UTR (top), while vector psiCHECK-STMN1 M contains a deletion at the seed-binding site (bottom). The underlined eight nucleotides indicate the miR-193b seed region. The primer sequences used to construct report vectors and procedures were described in Supplementary methods. (e) Relative luciferase activity is shown for reporter constructs psiCHECK-STMN1 or psiCHECK-STMN1 M in cells transfected with miR-193b or negative control. Malme-3M cells were seeded at 75 000 cells per well in a 12-well plate the day before transfection. The cells were cotransfected with 5 nmol/l miRNA precursor (either miR-193b or negative control) and 100 ng reporter vectors using Lipofectamine 2000 (Invitrogen). Renilla luciferase activity was measured 24 h after transfection using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). Data were normalized to firefly luciferase. Data shown are the mean±s.e.m. from three independent experiments, each performed in triplicate. *Po0.05 were calculated using the independent samples t-test (IBM SPSS, Armonk, NY, USA).

& 2013 Macmillan Publishers Limited Oncogene (2013) 1330 – 1337 Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1332 relatively low levels of endogenous STMN1 among tested cell miRNA-binding sites predicted by TargetScan. miR-193b/a, two lines. As expected, decreasing miR-193b expression resulted in miRNAs that contain the same miRNA ‘seed,’ are predicted to bind STMN1 levels increasing by 1.7, 1.4 and 1.3 folds, respectively to nucleotides 58–64 of the STMN1 30-UTR, while miR-101 is (Figure 1b). These results suggest that miR-193b is involved in the predicted to bind to nucleotides 284–290 (Figure 1c). Of these regulation of STMN1. three miRNAs, miR-193b is the only one that appears signiﬁcantly Early experimental data showed that most metazoan miRNA dysregulated in melanoma.12 target sites have perfect Watson–Crick base pairing with the To determine whether STMN1 is a direct or indirect target of miRNA ‘seed’, a 50 region of the miRNA centered on nucleotides miR-193b, we constructed two luciferase reporter plasmids: one 2–7.31 TargetScan (www.targetscan.org) is a predictive algorithm wild-type vector cloned with a full length 30-UTR of STMN1 and the developed to predict miRNA targets based on this concept other mutant vector cloned with the 30-UTR containing a deletion of stringent miRNA seed binding and evolutionary conservation at the miR-193b seed-binding site (Figure 1d). A B70% decrease of target site.32,33 The STMN1 30-UTR harbors two conserved in luciferase activity was observed when the wild-type vector was

Nevus Primary Melanoma Metastatic Melanoma

* 800 60 r = -0.43 600 40 400 miR-193b STMN1 20 200 Microarray Signal

Staining Intensity (%) 0 0 0 20 40 60 Nevi STMN1 Staining Intensity (%)

Primary Melanoma Metastatic Melanoma

100 *** * *** 80

40 Log-rank test p=0.96

STMN1 Staining Intensity (%) 20

Nevi

Primary Melanoma Metastatic Melanoma Figure 2. Increased expression of STMN1 in melanoma, and the inverse correlation between STMN1 and miR-193b expression levels. Ethics approval for use of the tissue was obtained from the Faculty of Health Sciences Ethics Board at Queen’s University. (a) Representative images of STMN1 immunostaining in melanocytic tissues. Immunohistochemistry was performed on a TMA slide using STMN1 antibody (1:250, Santa Cruz Biotechnology) according to a standard protocol in the Department of Pathology and Molecular Medicine at the Kingston General Hospital. The stained slide was scanned by an Aperio ScanScope (Aperio Technologies). (b) STMN1 staining intensities in eight nevi, six primary melanoma and eight metastatic melanoma. The intensity was assessed by Aperio’s ImageScope software as percentage of total number of positive pixels/ total number of pixels. The black bar indicates mean value for each group. (c) Pearson’s correlation between miR- 193b expression and STMN1 staining intensity in 22 melanocytic tissues. (d) The expression of STMN1 in a second independent melanocytic TMA. This TMA includes 27 nevi, 53 primary melanoma and 40 metastatic melanoma. *Po0.05 and ***Po0.001 were calculated using the independent samples t-test. (e) Kaplan–Meier analysis comparing survival between patients with median and above (X6.7) vs below median (o6.7) of STMN expression. Fifty-three primary melanoma patients from the second cohort with average 5.6 years of follow-up were included in this analysis. P value was calculated from the log-rank test. The analysis was performed using SAS version 9.2 (SAS Inc., Cary, NC, USA).

Oncogene (2013) 1330 – 1337 & 2013 Macmillan Publishers Limited Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1333 Table 1. Summary of genes involved in migration/cell cycle progression identiﬁed by IPA after STMN1 knockdown

Symbol Entrez gene name Fold Function change

STMN1 Stathmin 1 À 10.87 Migration/cell cycle progression CCNE2 Cyclin E2 À 3.55 Cell cycle progression CYR61 Cysteine-rich, angiogenic inducer, 61 À 2.63 Migration/cell cycle progression EXO1 Exonuclease 1 À 2.59 Cell cycle progression TGM2 Transglutaminase 2 (C polypeptide, protein-glutamine-gamma- À 2.32 Migration glutamyltransferase) ITGA9 Integrin, alpha 9 À 2.17 Migration RAD54B RAD54 homolog B (S. cerevisiae) À 2.17 Cell cycle progression GANI1 Guanine nucleotide-binding protein (G protein), alpha inhibiting activity À 2.13 Cell cycle progression polypeptide 1 PPM1A Protein phosphatase, Mg2 þ /Mn2 þ dependent, 1A À 2.12 Cell cycle progression BBS4 Bardet-Biedl syndrome 4 À 2.09 Cell cycle progression NEK2 NIMA (never in mitosis gene a)-related kinase 2 À 1.95 Cell cycle progression RASGRF1 Ras protein-speciﬁc guanine nucleotide-releasing factor 1 À 1.94 Migration CREM cAMP responsive element modulator À 1.93 Cell cycle progression JUN Jun proto-oncogene À 1.86 Cell cycle progression EZH2 Enhancer of zeste homolog 2 (Drosophila) À 1.86 Migration/cell cycle progression TUSC2 Tumor suppressor candidate 2 À 1.80 Cell cycle progression CHL1 Cell adhesion molecule with homology to L1CAM (close homolog of L1) À 1.78 Migration VHL von Hippel–Lindau tumor suppressor À 1.76 Cell cycle progression GFI1B Growth factor independent 1B transcription repressor À 1.74 Cell cycle progression CENPH Centromere protein H À 1.71 Cell cycle progression UVRAG UV radiation-resistance-associated gene À 1.65 Cell cycle progression KIF3B Kinesin family member 3B À 1.59 Cell cycle progression MYLK Myosin light chain kinase À 1.54 Migration DIAPH3 Diaphanous homolog 3 (Drosophila) À 1.52 Cell cycle progression DDR2 Discoidin domain receptor tyrosine kinase 2 À 1.50 Migration/cell cycle progression IFNB1 Interferon, beta 1, ﬁbroblast 15.49 Cell cycle progression CCL3L1/ Chemokine (C–C motif) ligand 3-like 1 4.37 Migration CCL3L3 TNF Tumor necrosis factor 3.85 Migration/cell cycle progression CYP1B1 Cytochrome P450, family 1, subfamily B, polypeptide 1 2.24 Cell cycle progression UBE2B Ubiquitin-conjugating enzyme E2B 1.85 Cell cycle progression GATA6 GATA-binding protein 6 1.60 Cell cycle progression

cotransfected with miR-193b relative to the negative control. The Po0.05) between STMN1 staining intensity and miR-193b repressive effect of miR-193b was relieved when cells were expression (Figure 2c). Despite the small sample size, our data cotransfected with mutant vector (Figure 1e). These results suggest demonstrate that STMN1 is upregulated during the progression of that miR-193b post-transcriptionally regulates STMN1 expression melanoma, and suggest the elevated level of STMN1 may be in through direct interaction with the predicted seed-binding site. part associated with the downregulation of miR-193b. To validate the above results, a large independent TMA was constructed. The second TMA was composed of 27 nevi, 53 STMN1 is upregulated in Melanoma primary melanoma and 40 metastatic melanoma, none of which We examined STMN1 expression directly in a series of melanoma were used in the first TMA. STMN1 levels were significantly tumor samples. A tissue microarray (TMA) was assembled, increased in primary melanoma (10.5%) and metastatic melanoma including eight nevi, six primary melanoma and eight (18.4%) compared with nevi (0.4%) (Po0.001) in the second metastatic melanoma samples. The rationale for using nevi as a cohort (Figure 2d). The difference in expression levels between benign comparator has been discussed previously.17 Following primary and metastatic melanoma also reached significance immunohistochemical staining with a mouse mononclonal (Po0.05). Our data confirm that STMN1 expression is significantly antibody against STMN1, the TMA slide was scanned to upregulated in malignant melanoma compared with nevi. generate digital images (Figure 2a). The staining intensities were We found no significant association between STMN1 expression assessed using the Aperio ImageScope (Aperio Technologies, and patient survival. Kaplan–Meier analysis demonstrated that Vista, CA, USA). The average STMN1 staining intensity was overall survival was not different in 53 primary melanoma patients increased from 1.3% in nevi to 12.8% in primary melanoma to with high (median and above) compared with low (below median) 26.5% in metastatic melanoma (Figure 2b). STMN1 expression (P ¼ 0.96) (Figure 2e). Additionally, STMN1 We subsequently compared STMN1 expression to miR-193b expression was not significantly correlated with Breslow depth expression in the same 22 samples used to assemble the TMA (P ¼ 0.78), mitotic count (P ¼ 0.95) or patient age (P ¼ 0.92) using described above (Figure 2b). miR-193b expression levels were Spearman’s ranking test. Interestingly, the average expression of obtained from previous microarray studies.12,17 There was a STMN1 was twice as high in males (mean ¼ 13.1) compared to significant inverse correlation (Pearson’s coefficient r ¼À0.43, females (mean ¼ 6.5) (P ¼ 0.0022, Wilcoxon Rank-sum test),

& 2013 Macmillan Publishers Limited Oncogene (2013) 1330 – 1337 Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1334 although it is not clear that this is linked to poorer prognosis of cohort, males had 4.3 times the rate of death of females, while the melanoma in males compared with females. The Cox proportional rate of death increased by 48% per decade of patient age. hazards model confirmed that STMN1 expression did not correlate Surprisingly, Breslow depth, the commonly used prognostic with the risk of patient death (HR ¼ 0.998, 95% confidence marker in melanoma, was only slightly correlated with increased interval: 0.967–1.031, P ¼ 0.92) (Supplementary Table 1). In this patient risk and did not reach significance (HR ¼ 1.1, 95%

1.20 *** ***

1.00 Malme-3M A375 0.80 Control siRNA + – + – 0.60 STMN1 siRNA – + – + STMN1 Relative 0.40

Gamma tubulin 0.20 BrdU Incorporation Rate 0.00 Malme-3M, Malme3M, A375, A375, control STMN1 control STMN1 siRNA siRNA siRNA siRNA

300 *** ***

250

200

150 Malme-3M, control siRNA Malme-3M, STMN1 siRNA 100

50 Migrated Cells per Field 0 Malme-3M, Malme-3M, A375, A375, control STMN1 control STMN1 siRNA siRNA siRNA siRNA A375, control siRNA A375, STMN1 siRNA

*** 1.60

Malme-3M 1.40 1.20 WPI control + – 1.00 STMN1-HA – + 0.80 STMN1

Relative 0.60 Gamma tubulin 0.40 0.20 BrdU Incorporation Rate 0.00 Malme-3M, Malme-3M, WPI control STMN1-HA

*** 160 140 120 100 80 60 40 20 Migrated Cells per Field 0 Malme-3M, WPI control Malme-3M, STMN1-HA Malme-3M, Malme-3M, WPI control STMN1-HA

Malme-3M A375 Negative Control+––– – – – + miR-193b –––+ –––+ Control siRNA – – + – – – + – STMN1 siRNA –––+ ––– + p21

p27 STMN1

Gamma tubulin

Oncogene (2013) 1330 – 1337 & 2013 Macmillan Publishers Limited Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1335 confidence interval: 0.943–1.283, P ¼ 0.23). The reliability, sensitiv- The microtubule destabilization mechanism of STMN1 has been ity and power of the estimates from our study could be limited well studied. STMN1 can either sequester tubulin dimers through because of the relatively small sample size. Nevertheless, its C-terminal region or promote microtubule catastrophe through important genes in melanoma pathogenesis are not necessarily its N-terminal region, depending on cellular pH.37 STMN1 contains ideal prognostic markers. For example, studies have shown that four phosphorylation sites: Ser 16, Ser 25, Ser 38 and Ser 63. BRAF and NRAS mutation status, cytoplasmic phospho-AKT In response to extracellular signals or during mitosis,38 expression and PTEN expression levels do not correlate with phosphorylation at one or more of the serine sites can weaken overall survival of melanoma patients.34–36 Further studies are the tubulin-binding ability of STMN1 and increase free tubulin required to understand the link, if any, between STMN1 expression concentrations for microtubule assembly.39 At the leading edge of and patient survival. migrating cells, Rac1 can activate PAK1, which phosphorylates STMN1 at Ser 16 and promotes localized microtubule growth.40 During mitosis, cyclin-dependent kinases, CDK1 and CDK2, can The roles of STMN1 as an oncogene in melanoma phosphorylate STMN1 at Ser 25 and Ser 38. STMN1 is a key factor in controlling microtubule dynamics, which However, the pathways downstream of STMN1-mediated is critical to cellular processes such as cell motility and cell division microtubule destabilization are poorly understood. Therefore, we during mitosis.23 To investigate the biological effect of STMN1 in performed a gene expression analysis to globally screen genes melanoma, we decided first to reduce expression of STMN1 using and pathways affected by STMN1 knockdown. By comparing the small-interfering RNA (siRNA) in an in vitro cell line model. Malme- transcriptome of Malme-3M cells transfected with STMN1 siRNA to 3M and A375 cells, two melanoma cell lines, were transfected with cells transfected with control siRNA, we identified 272 differen- either negative control or STMN1 siRNA. As shown in Figure 3a, tially expressed genes, including 170 downregulated and 102 STMN1 levels were greatly decreased in cells transfected with upregulated genes (Supplementary Table 2). Not surprisingly, STMN1 siRNA. Using the BrdU incorporation assay from Roche STMN1 was one of the most downregulated genes, showing (Laval, Que´bec, Canada), we detected a significantly decreased a 10.87-fold decrease in expression. Through pathway analysis proliferation rate in cells treated with STMN1 siRNA relative to cells of the set of differentially expressed genes, 25 downregulated treated with negative control (Figure 3b). We next examined cell and 6 upregulated genes were categorized into functions motility using a transwell migration assay. Reduction of STMN1 affecting cell cycle progression and/or migration (Table 1). expression notably reduced the migration potential of melanoma Integrin, actin, tight junction, Rac and Cdc42 signaling pathways cells by B85% in Malme-3M and B50% in A375 cells (Figure 3c). were among the top canonical pathways associated with down- Therefore, reducing STMN1 expression significantly hampered the regulated genes (Supplementary Table 3), suggesting that proliferation and migration potential of melanoma cells. To test interconnected cytoskeletal networks are directly affected whether ectopic overexpression of STMN1 would achieve the by STMN1 knockdown. Downregulated genes such as Cyr61, opposite effects, we subsequently expressed STMN1 in Malme-3M TGM2, CHL1, MYLK or DDR2 could promote migration depending cells using a hemagglutinin (HA)-tagged STMN1 construct in a on the specific cellular context.41–45 INFB1 and TNF, two of the lentiviral system. The western blot analysis confirmed increased most upregulated genes, could inhibit cell proliferation through expression of STMN1 following infection, showing the HA-tagged induction of p21Cip1/Waf1,46,47 while downregulated genes such as STMN1 protein migrating slightly slower than the endogenous CCNE2, EZH2, JUN and GFI1B are known to promote G1/S transition protein (Figure 3d). As anticipated, ectopic expression of STMN1 by regulating p21Cip1/Waf1 and p27Kip.48–51 Consequently, we significantly increased the cell proliferation rate in Malme-3M cells found p21Cip1/Waf1 and p27Kip levels were elevated on western by B30% (Figure 3e) and migration by B60% (Figure 3f). Previous blots following STMN1 knockdown (Figure 3g). Our exploratory studies have shown miR-193b could repress cell proliferation and study suggested that cyclin-dependent kinase inhibitors could be migration.12,18,21 Ectopic expression of STMN1 could partially important downstream effectors of STMN1 signaling. Further relieve the repressive effects of miR-193b on proliferation and investigation is needed to examine the relationship between migration (Supplementary Figure S1), indicating miR-193b func- STMN1 and those differentially expressed genes, and whether tions at least in part through suppressing the expression of STMN1. STMN1 could form protein complexes that directly affect cell These results confirmed that STMN1 promotes cell proliferation proliferation and migration. and migration, and suggests overexpression of STMN1 could have In summary, our study suggests STMN1 behaves as an an oncogenic role during progression of melanoma. oncogene in melanoma, in keeping with data from other cancers.

Figure 3. Functional studies of STMN1 in melanoma. (a) STMN1 level was assessed by western blotting in Malme-3M and A375 cells transfected with control siRNA or STMN1 siRNA. Cells were transfected with 10 nmol/l oligos and were harvested 72 h after transfection. STMN1 and negative control siRNA are DsiRNA oligos purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA). (b) Knocking down STMN1 reduced cell proliferation. Cell proliferation rate was measured using the Cell Proliferation ELISA, BrdU (chemiluminescence) by Roche Applied Biosciences. Transfected Malme-3M and A375 cells, as described in (a), were seeded at 3500 cells per well into 96-well black plate 72 h post- transfection, and incubated with BrdU substrate. After 16 h, cells were ﬁxed, labeled with anti-BrdU antibody and assessed using a microplate luminometer. Representative data from one of three independent experiments are shown. The data are mean±s.e.m. measured in septuplicates. (c) Knocking down STMN1 repressed cell migration. In all, 4 Â 104 Malme-3M cells and 3 Â 104 A375 cells were plated into the top chamber of 24-well insert (pore size 8 mm; BD Biosciences, Bedford, MA, USA) with RPMI medium after transfection. 10% fetal bovine serum was added in RPMI medium used as a chemoattractant in the lower chamber. Incubated for 16 h, cells that did not migrate through the pores were removed by a cotton swab. Cells on the lower side of membrane were ﬁxed with 10% neutral buffered formalin, stained with 1% crystal violet and counted. Representative data from one of three independent experiments were shown as mean±s.e.m. (d) Ectopic expression of STMN1. Malme-3M cells were infected with lentivirus, stably expressing either WPI lentiviral vector (WPI control) or WPI lentiviral vector with HA-tagged STMN1 (STMN1-HA). The detailed lentiviral vector construction was described in Supplementary methods. The expression of STMN1 was examined by western blotting. (e, f) Proliferation and migration effects in Malme-3M cells overexpressing STMN1. The procedures were described in (b) and (c). ***Po0.001 were calculated using the independent samples t-test. (g) p21Cip1/Waf1 and p27Kip levels in Malme-3M and A375 cells transfected with miRNA precursors (negative control or miR-193b) or with siRNA oligos (control or STMN1). The procedure was performed as described in Figures 1a and 3a. p21Cip1/Waf1 (2947 P) was purchased from Cell Signaling and p27Kip (sc-528) was purchased from Santa Cruz.

& 2013 Macmillan Publishers Limited Oncogene (2013) 1330 – 1337 Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1336 We demonstrated that miR-193b directly regulated STMN1 22 Curmi PA, Gavet O, Charbaut E, Ozon S, Lachkar-Colmerauer S, Manceau V et al. expression, and that upregulation of STMN1 may be at least in Stathmin and its phosphoprotein family: general properties, biochemical and part due to reduced expression of miR-193b in malignant functional interaction with tubulin. Cell Struct Funct 1999; 24: 345–357. melanoma. Inhibition of STMN1 significantly repressed cell 23 Belletti B, Nicoloso MS, Schiappacassi M, Berton S, Lovat F, Wolf K et al. Stathmin proliferation and migration, making it a potential therapeutic activity influences sarcoma cell shape, motility, and metastatic potential. Mol Biol target in melanoma. Cell 2008; 19: 2003–2013. 24 Bieche I, Lachkar S, Becette V, Cifuentes-Diaz C, Sobel A, Lidereau R et al. Over- expression of the stathmin gene in a subset of human breast cancer. Br J Cancer 1998; 78: 701–709. CONFLICT OF INTEREST 25 Chen G, Wang H, Gharib TG, Huang CC, Thomas DG, Shedden KA et al. The authors declare no conflict of interest. Overexpression of oncoprotein 18 correlates with poor differentiation in lung adenocarcinomas. Mol Cell Proteomics 2003; 2: 107–116. 26 Ghosh R, Gu G, Tillman E, Yuan J, Wang Y, Fazli L et al. Increased expression and differential phosphorylation of stathmin may promote prostate cancer progres- ACKNOWLEDGEMENTS sion. Prostate 2007; 67: 1038–1052. This work is supported by the Canadian Institutes of Health Research (CIHR) Grant 27 Hsieh SY, Huang SF, Yu MC, Yeh TS, Chen TC, Lin YJ et al. Stathmin1 (VAT; HEF). JC is funded in part by the Queen’s University Terry Fox Foundation overexpression associated with polyploidy, tumor-cell invasion, early Training Program in Transdisciplinary Cancer Research in partnership with CIHR. recurrence, and poor prognosis in human hepatoma. Mol Carcinog 2010; 49: 476–487. 28 Jeon TY, Han ME, Lee YW, Lee YS, Kim GH, Song GA et al. Overexpression of stathmin1 in the diffuse type of gastric cancer and its roles in proliferation and REFERENCES migration of gastric cancer cells. Br J Cancer 2010; 102: 710–718. 1 Gray-Schopfer V, Wellbrock C, Marais R. Melanoma biology and new targeted 29 Singer S, Malz M, Herpel E, Warth A, Bissinger M, Keith M et al. Coordinated therapy. Nature 2007; 445: 851–857. expression of stathmin family members by far upstream sequence element- 2 Rothhammer T, Bosserhoff AK. Epigenetic events in malignant melanoma. binding protein-1 increases motility in non-small cell lung cancer. Cancer Res Pigment Cell Res 2007; 20: 92–111. 2009; 69: 2234–2243. 3 Miller AJ, Mihm Jr MC. Melanoma. N Engl J Med 2006; 355: 51–65. 30 Zheng P, Liu YX, Chen L, Liu XH, Xiao ZQ, Zhao L et al. Stathmin, a new 4 Cohen C, Zavala-Pompa A, Sequeira JH, Shoji M, Sexton DG, Cotsonis G et al. target of PRL-3 identified by proteomic methods, plays a key role in Mitogen-actived protein kinase activation is an early event in melanoma pro- progression and metastasis of colorectal cancer. J Proteome Res 2010; 9: gression. Clin Cancer Res 2002; 8: 3728–3733. 4897–4905. 5 Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 31 Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell 2009; 136: 116: 281–297. 215–233. 6 Baek D, Villen J, Shin C, Camargo FD, Gygi SP, Bartel DP. The impact of microRNAs 32 Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB. Prediction of on protein output. Nature 2008; 455: 64–71. mammalian microRNA targets. Cell 2003; 115: 787–798. 7 Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N. 33 Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adeno- Widespread changes in protein synthesis induced by microRNAs. Nature 2008; sines, indicates that thousands of human genes are microRNA targets. Cell 2005; 455: 58–63. 120:15–20. 8 Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are con- 34 Akslen LA, Angelini S, Straume O, Bachmann IM, Molven A, Hemminki K et al. served targets of microRNAs. Genome Res 2009; 19: 92–105. BRAF and NRAS mutations are frequent in nodular melanoma but are not asso- 9 Sayed D, Abdellatif M. MicroRNAs in development and disease. Physiol Rev 2011; ciated with tumor cell proliferation or patient survival. J Invest Dermatol 2005; 91: 827–887. 125: 312–317. 10 Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat Rev 35 Edlundh-Rose E, Egyhazi S, Omholt K, Mansson-Brahme E, Platz A, Hansson J et al. Cancer 2006; 6: 259–269. NRAS and BRAF mutations in melanoma tumours in relation to clinical 11 Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer 2006; characteristics: a study based on mutation screening by pyrosequencing. 6: 857–866. Melanoma Res 2006; 16: 471–478. 12 Chen J, Feilotter HE, Pare GC, Zhang X, Pemberton JG, Garady C et al. MicroRNA- 36 Slipicevic A, Holm R, Nguyen MT, Bohler PJ, Davidson B, Florenes VA. Expression of 193b represses cell proliferation and regulates cyclin D1 in melanoma. Am J activated Akt and PTEN in malignant melanomas: relationship with clinical out- Pathol 2010; 176: 2520–2529. come. Am J Clin Pathol 2005; 124: 528–536. 13 Mueller DW, Rehli M, Bosserhoff AK. miRNA expression profiling in melanocytes 37 Howell B, Larsson N, Gullberg M, Cassimeris L. Dissociation of the tubulin- and melanoma cell lines reveals miRNAs associated with formation and pro- sequestering and microtubule catastrophe-promoting activities of oncoprotein gression of malignant melanoma. J Invest Dermatol 2009; 129: 1740–1751. 18/stathmin. Mol Biol Cell 1999; 10: 105–118. 14 Segura MF, Hanniford D, Menendez S, Reavie L, Zou X, Alvarez-Diaz S et al. 38 Andersen SS, Ashford AJ, Tournebize R, Gavet O, Sobel A, Hyman AA et al. Mitotic Aberrant miR-182 expression promotes melanoma metastasis by repressing chromatin regulates phosphorylation of Stathmin/Op18. Nature 1997; 389: FOXO3 and microphthalmia-associated transcription factor. Proc Natl Acad Sci USA 640–643. 2009; 106: 1814–1819. 39 Cassimeris L. The oncoprotein 18/stathmin family of microtubule destabilizers. 15 Boyle GM, Woods SL, Bonazzi VF, Stark MS, Hacker E, Aoude LG et al. Melanoma Curr Opin Cell Biol 2002; 14: 18–24. cell invasiveness is regulated by miR-211 suppression of the BRN2 transcription 40 Niethammer P, Bastiaens P, Karsenti E. Stathmin-tubulin interaction gradients in factor. Pigment Cell Melanoma Res 2011; 24: 525–537. motile and mitotic cells. Science 2004; 303: 1862–1866. 16 Levy C, Khaled M, Iliopoulos D, Janas MM, Schubert S, Pinner S et al. Intronic miR- 41 Buhusi M, Midkiff BR, Gates AM, Richter M, Schachner M, Maness PF. Close 211 assumes the tumor suppressive function of its host gene in melanoma. Mol homolog of L1 is an enhancer of integrin-mediated cell migration. J Biol Chem Cell 2010; 40: 841–849. 2003; 278: 25024–25031. 17 Chen J, Zhang X, Lentz C, Abi-Daoud M, Pare GC, Yang X et al. miR-193b Regulates 42 Kamm KE, Stull JT. Dedicated myosin light chain kinases with diverse cellular Mcl-1 in Melanoma. Am J Pathol 2011; 179: 2162–2168. functions. J Biol Chem 2001; 276: 4527–4530. 18 Li XF, Yan PJ, Shao ZM. Downregulation of miR-193b contributes to enhance 43 Li Z, Xu X, Bai L, Chen W, Lin Y. Epidermal growth factor receptor-mediated urokinase-type plasminogen activator (uPA) expression and tumor progression tissue transglutaminase overexpression couples acquired tumor necrosis and invasion in human breast cancer. Oncogene 2009; 28: 3937–48. factor-related apoptosis-inducing ligand resistance and migration through 19 Leivonen SK, Makela R, Ostling P, Kohonen P, Haapa-Paananen S, Kleivi K et al. c-FLIP and MMP-9 proteins in lung cancer cells. J Biol Chem 2011; 286: Protein lysate microarray analysis to identify microRNAs regulating estrogen 21164–21172. receptor signaling in breast cancer cell lines. Oncogene 2009; 28: 3926–36. 44 Olaso E, Labrador JP, Wang L, Ikeda K, Eng FJ, Klein R et al. Discoidin domain 20 Rauhala HE, Jalava SE, Isotalo J, Bracken H, Lehmusvaara S, Tammela TL et al. miR- receptor 2 regulates fibroblast proliferation and migration through the extra- 193b is an epigenetically regulated putative tumor suppressor in prostate cancer. cellular matrix in association with transcriptional activation of matrix metallo- Int J Cancer 2010; 127: 1363–1372. proteinase-2. J Biol Chem 2002; 277: 3606–3613. 21 Xu C, Liu S, Fu H, Li S, Tie Y, Zhu J et al. MicroRNA-193b regulates proliferation, 45 Sun ZJ, Wang Y, Cai Z, Chen PP, Tong XJ, Xie D. Involvement of Cyr61 in growth, migration and invasion in human hepatocellular carcinoma cells. Eur J Cancer migration, and metastasis of prostate cancer cells. Br J Cancer 2008; 99: 2010; 46: 2828–2836. 1656–1667.

Oncogene (2013) 1330 – 1337 & 2013 Macmillan Publishers Limited Stathmin 1 is a potential novel oncogene in melanoma J Chen et al 1337 46 Kumar PS, Shiras A, Das G, Jagtap JC, Prasad V, Shastry P. Differential expression 49 Lauper N, Beck AR, Cariou S, Richman L, Hofmann K, Reith W et al. Cyclin E2: a and role of p21cip/waf1 and p27kip1 in TNF-alpha-induced inhibition of pro- novel CDK2 partner in the late G1 and S phases of the mammalian cell cycle. liferation in human glioma cells. Mol Cancer 2007; 6:42. Oncogene 1998; 17: 2637–2643. 47 Wellen J, Walter J, Jangouk P, Hartung HP, Dihne M. Neural precursor 50 Tong B, Grimes HL, Yang TY, Bear SE, Qin Z, Du K et al. The Gﬁ-1B proto-onco- cells as a novel target for interferon-beta. Neuropharmacology 2009; 56: protein represses p21WAF1 and inhibits myeloid cell differentiation. Mol Cell Biol 386–398. 1998; 18: 2462–2473. 48 Fan T, Jiang S, Chung N, Alikhan A, Ni C, Lee CC et al. EZH2-dependent sup- 51 Wang CH, Tsao YP, Chen HJ, Chen HL, Wang HW, Chen SL. Transcriptional pression of a cellular senescence phenotype in melanoma cells by inhibition of repression of p21((Waf1/Cip1/Sdi1)) gene by c-jun through Sp1 site. Biochem p21/CDKN1A expression. Mol Cancer Res 2011; 9: 418–429. Biophys Res Commun 2000; 270: 303–310.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

& 2013 Macmillan Publishers Limited Oncogene (2013) 1330 – 1337