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RNA-Binding Protein IGF2BP1 in Cutaneous Squamous Cell Carcinoma

2016; Elsevier BV; Volume: 137; Issue: 3 Linguagem: Inglês

10.1016/j.jid.2016.10.042

1523-1747

TaeWon Kim, Thomas C. Havighurst, KyungMann Kim, Scott J. Hebbring, Zhan Ye, Juliet L. Aylward, Sündüz Keleş, Yaohui G. Xu, Vladimir S. Spiegelman,

Cutaneous Melanoma Detection and Management

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, accounting for 20% of nonmelanoma skin cancer. Although most small cSCCs are readily curable by surgical approaches, surgery is not effective for patients with advanced and metastasized cSCC. A prospective study showed that the rate of nodal metastasis and disease-specific death for cSCC are 4% and 1.5%, respectively (Brantsch et al., 2008Brantsch K.D. Meisner C. Schonfisch B. Trilling B. Wehner-Caroli J. Rocken M. et al.Analysis of risk factors determining prognosis of cutaneous squamous-cell carcinoma: a prognostic study.Lancet Oncol. 2008; 9: 713-720Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). Thus, identification of additional therapeutic targets remains imperative for inoperable SCC. IGF2BP1 is a multifunctional RNA-binding protein that has been shown to affect multiple prosurvival, anti-apoptotic, and drug resistance pathways (Craig and Spiegelman, 2012Craig E.A. Spiegelman V.S. Inhibition of coding region determinant binding protein sensitizes melanoma cells to chemotherapeutic agents.Pigment Cell Melanoma Res. 2012; 25: 83-87Crossref PubMed Scopus (18) Google Scholar, Elcheva et al., 2008Elcheva I. Tarapore R.S. Bhatia N. Spiegelman V.S. Overexpression of mRNA-binding protein CRD-BP in malignant melanomas.Oncogene. 2008; 27: 5069-5074Crossref PubMed Scopus (44) Google Scholar, Noubissi et al., 2010Noubissi F.K. Nikiforov M.A. Colburn N. Spiegelman V.S. Transcriptional regulation of CRD-BP by c-myc: Implications for c-myc Functions.Genes Cancer. 2010; 1: 1074-1082Crossref PubMed Google Scholar). IGF2BP1 has an oncofetal pattern of expression and although it is important for normal embryonic development, in normal tissues it is expressed at very low levels, and its down-regulation in vitro and in vivo has no significant physiological effect. Conversely, we have found that ectopic expression of IGF2BP1 in normal keratinocytes leads to increased proliferation and inhibition of apoptosis (Noubissi et al., 2014Noubissi F.K. Kim T. Kawahara T.N. Aughenbaugh W.D. Berg E. Longley B.J. et al.Role of CRD-BP in the growth of human basal cell carcinoma cells.J Invest Dermatol. 2014; 134: 1718-1724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). IGF2BP1 has also been shown to drive tumorigenesis in vivo (Tessier et al., 2004Tessier C.R. Doyle G.A. Clark B.A. Pitot H.C. Ross J. Mammary tumor induction in transgenic mice expressing an RNA-binding protein.Cancer Res. 2004; 64: 209-214Crossref PubMed Scopus (92) Google Scholar). IGF2BP1 was shown to regulate several proto-oncogenes and oncogenic signaling pathways that are activated in SCC (Bhatia and Spiegelman, 2005Bhatia N. Spiegelman V.S. Activation of Wnt/β-catenin/Tcf signaling in mouse skin carcinogenesis.Mol Carcinog. 2005; 42: 213-221Crossref PubMed Scopus (44) Google Scholar, Noubissi et al., 2006Noubissi F.K. Elcheva I. Bhatia N. Shakoori A. Ougolkov A. Liu J. et al.CRD-BP mediates stabilization of betaTrCP1 and c-myc mRNA in response to beta-catenin signalling.Nature. 2006; 441: 898-901Crossref PubMed Scopus (186) Google Scholar, Pierceall et al., 1991Pierceall W.E. Goldberg L.H. Tainsky M.A. Mukhopadhyay T. Ananthaswamy H.N. Ras gene mutation and amplification in human nonmelanoma skin cancers.Mol Carcinog. 1991; 4: 196-202Crossref PubMed Scopus (252) Google Scholar). In this report we analyzed the role of IGF2BP1 in the pathogenesis of cSCC. To assess the levels of IGF2BP1 in human cSCC, we isolated RNA from fresh frozen cSCC tissues and matching control skin from 68 patients who presented for Mohs micrographic surgery. All human studies have been approved by our institutional review board, and patients have given their written informed consent. Quantitative reverse transcriptase–PCR showed that IGF2BP1 mRNA was significantly overexpressed in SCCs compared with their matching controls (Figure 1a, and see Supplementary Figure S1 online). We also measured mRNA levels of two IGF2BP1-binding targets, β-TrCP1 and c-Myc, which play roles in cell cycle progression, growth and apoptosis. Both were overexpressed in most patients along with IGF2BP1 overexpression, and statistical analysis confirmed a positive correlation between expression levels of IGF2BP1 and both β-TrCP1 and c-Myc (Figure 1c and d). Immunoblot analysis confirmed that IGF2BP1 protein was overexpressed in SCC samples compared with matching controls (Figure 1b). To further test the association of IGF2BP1 and clinical features, the following patient information was collected: age, sex, lesion size at time of biopsy, lesion size before Mohs surgery, defect size after Mohs surgery, keratoacanthoma type, cell differentiation (well, moderate, and poor), transplant versus non-transplant recipient, and stage (T0, T1, T2a, T2b, and T3) based on Brigham and Women's Hospital staging system (Jambusaria-Pahlajani et al., 2013Jambusaria-Pahlajani A. Kanetsky P.A. Karia P.S. Hwang W.T. Gelfand J.M. Whalen F.M. et al.Evaluation of AJCC tumor staging for cutaneous squamous cell carcinoma and a proposed alternative tumor staging system.JAMA Dermatol. 2013; 149: 402-410Crossref PubMed Scopus (228) Google Scholar). Some tumors may be understaged because our study does not allow a complete assessment of tumor depth histologically. We also attempted to collect clinical outcome data (local recurrence, regional metastasis, distant metastasis, or death) with a follow-up duration of 1–4 years; only four patients had adverse outcomes related to SCC. We performed univariate regression models of log2 fold change on these various parameters. We found that poorly differentiated cells expressed higher levels of IGF2BP1 mRNA than moderately and well-differentiated cells (P = 0.008, F test) (see Supplementary Figure S2a and Supplementary Table S1 online). Also, models showed that increase in tumor staging is associated with increase in IGF2BP1 mRNA expression level (P = 0.017, F test) (see Supplementary Figure S2b and c and Supplementary Table S1). Statistically significant association was not found between IGF2BP1 expression and the other listed parameters, including clinical outcomes. These data collectively show that IGF2BP1 and its targets are overexpressed in human cSCC. The levels of IGF2BP1 expression inversely correlate with cell differentiation and directly correlate with tumor staging in human cSCC. To further investigate involvement of IGF2BP1 in cSCC development, we analyzed its role in the proliferation and survival of SCC cells in vitro. Knockdown of IGF2BP1 by IGF2BP1-specific short hairpin RNA (sh-IGF2BP1) significantly reduced cell growth and induced apoptosis in human and mouse cSCC cell lines, SCC-13 and Ca3/7, respectively (Figure 2a and b). We further studied whether SCC patients have genetic variation in the gene IGF2BP1. There were 199 SCC cases and 9,453 control subjects who were identified in the Marshfield Clinic's Personalized Medicine Research Project with available genetic data (McCarty et al., 2005McCarty C.A. Wilke R.A. Giampetro P.F. Wesbrook S.D. Caldwell M.D. Marshfield Clinic Personalized Medicine Research Project (PMRP): design, methods and recruitment for a large population-based biobank.Personalized Med. 2005; 2: 49-79Crossref PubMed Google Scholar). By focusing on the IGF2BP1 genetic region (± 5 kilo base pairs), we could identify 128 common variants (minor allele frequencies > 1%). Association studies identified seven intronic single nucleotide polymorphisms (SNPs) that were nominally associated with SCC (P < 0.05) (see Supplementary Table S2 online), although these were not significant after Bonferroni adjustment. The top SNP was rs4265867, and the second most significant variant was rs150781440, bearing little linkage disequilibrium with rs4265867 (r < 0.01). We used Affinity Test for regulatory SNP detection bioinformatics tool (atSNP) to quantify in silico the potential impact of these SNPs to transcription factor binding (see Supplementary Figure S3 online) (Zuo et al., 2015Zuo C. Shin S. Keles S. atSNP: transcription factor binding affinity testing for regulatory SNP detection.Bioinformatics. 2015; 31: 3353-3355Crossref PubMed Scopus (47) Google Scholar). This analysis suggested that these noncoding SNPs may affect IGF2BP1 regulation by disrupting (MAZ, EN1) or enhancing (FOXG1, NFYA, NFY, SP1) transcription factor binding. We have found that expression level of IGF2BP1 is consistently higher in both normal skin and SCCs of patients heterozygous for rs74443707 (see Supplementary Figure S4 online); however, to reach statistical significance a larger cohort of patients may be needed. Although the case-control association analysis did not pass a conservative Bonferroni threshold (P < 0.00039, assuming α < 0.05 and 128 independent tests) and replication studies are required, these results suggest that common variants in IGF2BP1 may contribute to SCC risk. We have found that mRNA expression level of IGF2BP1 is positively correlated with higher SCC staging and poor cell differentiation. We have also shown that inhibition of IGF2BP1 function suppresses growth and induces apoptosis in SCC. Thus, we propose that overexpression of IGF2BP1 promotes cell proliferation and escape of apoptosis, leading to progression of SCC. Patients with SCC contain common SNPs in noncoding regions of IGF2BP1 that are predicted to alter the binding of several transcription factors and might potentially contribute to dysregulation of IGF2BP1 expression in SCC. This study uncovers a potential role of IGF2BP1 in SCC pathogenesis. Therapeutic down-regulation of IGF2BP1 may potentially become an effective approach for the management of cSCC in the future. The authors state no conflict of interest. This work was supported by Dermatology Foundation Career Development Award (to YGX); National Institutes of Health grants AR063361 and CA191550 (to VSS) and UL1TR000427, K22LM011938, U01HG006389, and R01GM114128 (to SJH). The authors acknowledge the National Cancer Institute Cancer Center Support Grant P30 CA014520 to the University of Wisconsin Carbone Cancer Center. The authors particularly thank patients who agreed to participate in the study; Andy Swanson and Eric Berg for recruiting patients; and Diane Bock, Mike Hetzer, and Tisha Kawahara for their administrative support. Download .pdf (.87 MB) Help with pdf files Supplementary Figures S1–S4 and Supplementary Tables S1 and S2