RNA-Binding Protein IGF2BP1 in Cutaneous Squamous Cell Carcinoma

HHS Public Access Author manuscript Author Manuscript J Invest Dermatol. Author manuscript; available in PMC 2018 March 01. Published in final edited form as: J Invest Dermatol. 2017 March ; 137(3): 772–775. doi:10.1016/j.jid.2016.10.042. RNA-Binding Protein IGF2BP1 in Cutaneous Squamous Cell Carcinoma TaeWon Kim1,2,*, Thomas Havighurst3, KyungMann Kim3,4, Scott J Hebbring5, Zhan Ye5, Juliet Aylward1, Sunduz Keles3, Yaohui G. Xu1,*, and Vladimir S. Spiegelman1,6,* 1Department of Dermatology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin Author Manuscript 2Molecular and Environmental Toxicology Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 3Department of Biostatistics and Medical Informatics, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 4Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 5Center for Human Genetics, Marshfield Clinic Research Foundation, Marshfield, Wisconsin 6Department of Pediatrics, Pennsylvania State University, Hershey, Pennsylvania Author Manuscript Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, accounting for 20% of non-melanoma skin cancer. While most small cSCCs are readily curable by surgical approaches, surgery is not effective for patients with advanced and metastasized cSCC. Prospective study showed that the rate of nodal metastasis and diseasespecific death for cSCC are 4% and 1.5%, respectively (Brantsch et al., 2008). Thus, identification of additional therapeutic targets remains imperative for inoperable SCC. Author Manuscript Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a multifunctional RNAbinding protein and has been shown to affect multiple pro-survival, anti-apoptotic, and drug resistance pathways (Elcheva et al., 2008; Noubissi et al., 2010; Craig and Spiegelman, 2012). IGF2BP1 has an oncofetal pattern of expression and whereas 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 * Address correspondence to: Yaohui G. Xu, M.D., Ph.D. Department of Dermatology, University of Wisconsin School of Medicine and Public Health, 7th Floor, 1 South Park Street, Madison, WI, 53715, Ph: 608-287-2620, Fax: 608-287-2676, [email protected], or, Vladimir S. Spiegelman, MD, PhD, Department of Pediatrics, Division of Pediatric Hematology/ Oncology, Pennsylvania State University, College of Medicine, Milton S. Hershey Medical Center, PO Box 850, MC H085, C7830E, 500 University Drive, Hershey, Pennsylvania 17033-0850, Ph: 717-531-0003 Ext 287721, FAX: 717-531-4789, [email protected]. Conflict of Interest: The authors state no conflict of interest. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Kim et al. Page 2 Author Manuscript proliferation and inhibition of apoptosis (Noubissi et al., 2014). IGF2BP1 has also been shown to drive tumorigenesis in vivo (Tessier et al., 2004). IGF2BP1 was shown to regulate several proto-oncogenes and oncogenic signaling pathways that are activated in SCC (Noubissi et al., 2006; Bhatia and Spiegelman, 2005; Pierceall et al., 1991). In this report we analyzed the role of IGF2BP1 in the pathogenesis of cSCC. Author Manuscript In order to assess the levels of IGF2BP1 in human cSCC, we isolated RNA from fresh frozen cSCC tissues as well as 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 RT-PCR revealed that IGF2BP1 mRNA was significantly overexpressed in SCCs compared to their matching controls (Fig 1a, Supplementary Fig S1). 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 (Fig 1c, 1d). Immunoblot analysis confirmed that IGF2BP1 protein was over-expressed in SCC samples as compared to matching controls (Fig 1b). Author Manuscript To further test association of IGF2BP1 and clinical features, the following patient information was collected: age, sex, lesion size at time of biopsy, lesion size prior to Mohs surgery, defect size post Mohs surgery, keratoacanthoma type, cell differentiation (well, moderate, and poor), transplant vs. non-transplant, and stage (T0, T1, T2a, T2b and T3) based on Brigham Women Hospital (BWH) staging system (Jambusaria-Pahlajani et al., 2013). Some tumors may be under-staged 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 poor-differentiated cells expressed higher level of IGF2BP1 mRNA than moderately- and well-differentiated cells (p=0.008, F-test) (Fig S2a, Table S1). Also, models showed that increase in tumor staging is associated with increase in IGF2BP1 mRNA expression level (p=0.017, F-test) (Fig S2b, S2c, 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. Author Manuscript In order to further investigate involvement of IGF2BP1 in cSCC development we analyzed its role in proliferation and survival of SCC cells in vitro. Knockdown of IGF2BP1 by IGF2BP1-specific sh-RNA (sh-IGF2BP1) significantly reduced cell growth and induced apoptosis in human and mouse cSCC cell lines, SCC-13 and Ca3/7, respectively (Fig 2a, b). We further studied whether SCC patients have genetic variation in the gene IGF2BP1. There were 199 SCC cases and 9453 controls that were identified in Marshfield Clinic's Personalized Medicine Research Project (PMRP) with available genetic data (McCarty et al., J Invest Dermatol. Author manuscript; available in PMC 2018 March 01. Kim et al. Page 3 Author Manuscript Author Manuscript 2005). By focusing on the IGF2BP1 genetic region (+/- 5 kb), 128 common variants (minor allele frequencies >1%) could be identified. Association studies identified seven intronic single nucleotide polymorphisms (SNPs) that were nominally associated with SCC (p<0.05) (Table S2), 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 atSNP to quantify in silico the potential impact of these SNPs to transcription factor binding (Fig S3) (Zuo et al., 2015). This analysis suggested that these noncoding SNPs may impact 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 (Fig. S4), 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. Author Manuscript 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. Intriguingly, patients with SCC contain common SNPs in non-coding region 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 downregulation of IGF2BP1 may potentially become an effective approach for the management of cSCC in the future. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Acknowledgments This work was supported by Dermatology Foundation Career Development Award (to Y.G.X.); NIH grants AR063361, CA121851 (to V.S.S.); UL1TR000427, K22LM011938, U01HG006389, and R01GM114128 (to S.J.H.). The authors acknowledge the NCI 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, Drs. Andy Swanson and Eric Berg for recruiting patients, and Diane Bock, Mike Hetzer, and Tisha Kawahara for their administrative support. Author Manuscript References Bhatia N, Spiegelman VS. Activation of Wnt/β-catenin/Tcf signaling in mouse skin carcinogenesis. Mol Carcinog. 2005; 42:213–21. [PubMed: 15765534] Brantsch KD, 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; 9:713–20. [PubMed: 18617440] Craig EA, Spiegelman VS. 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Marshfield Clinic Personalized Medicine Research Project (PMRP): design, methods and recruitment for a large population-based biobank. Personalized Med. 2005; 2:49–79. Noubissi FK, 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; 441:898–901. [PubMed: 16778892] Noubissi FK, Nikiforov MA, Colburn N, Spiegelman VS. Transcriptional Regulation of CRD-BP by cmyc: Implications for c-myc Functions. Genes Cancer. 2010; 1:1074–82. [PubMed: 21779431] Noubissi FK, Kim T, Kawahara TN, Aughenbaugh WD, Berg E, Longley BJ, et al. Role of CRD-BP in the growth of human basal cell carcinoma cells. J Invest Dermatol. 2014; 134:1718–24. [PubMed: 24468749] Pierceall WE, Goldberg LH, Tainsky MA, Mukhopadhyay T, Ananthaswamy HN. Ras gene mutation and amplification in human nonmelanoma skin cancers. Mol Carcinog. 1991; 4:196–202. [PubMed: 2064725] Tessier CR, Doyle GA, Clark BA, Pitot HC, Ross J. Mammary tumor induction in transgenic mice expressing an RNA-binding protein. Cancer Res. 2004; 64:209–14. [PubMed: 14729626] Ye Z, Mayer J, Ivacic L, Zhou Z, He M, Schrodi SJ, et al. Phenome-wide association studies (PheWASs) for functional variants. Eur J Hum Genet. 2015; 23:523–9. [PubMed: 25074467] Zuo C, Shin S, Keles S. atSNP: transcription factor binding affinity testing for regulatory SNP detection. Bioinformatics. 2015; 31:3353–5. [PubMed: 26092860] Author Manuscript Abbreviations IGF2BP1 Insulin-like growth factor-2-mRNA-binding protein 1 cSCC cutaneous squamous cell carcinoma Author Manuscript J Invest Dermatol. Author manuscript; available in PMC 2018 March 01. Kim et al. Page 5 Author Manuscript Author Manuscript Figure 1. IGF2BP1 and its targets are overexpressed in SCC. a. Box plot of IGF2BP1 Log2 fold change corresponding to Fig S1. Mean fold change is 9.85 (23.30); median (dark line) is 11.00 (23.46) (p<0.0001, one-sample Student t-test). b. Immunoblot of randomly selected cSCC samples from Fig S1. c–d. Scatterplots between expression level of IGF2BP1 mRNA and its targets, β-TrCP1 and c-Myc. The Spearman rank correlation coefficient and p-value are presented on each plot. A LOWESS smoothing curve shows the relationship between the variables. Author Manuscript Author Manuscript J Invest Dermatol. Author manuscript; available in PMC 2018 March 01. Kim et al. Page 6 Author Manuscript Author Manuscript Author Manuscript Figure 2. Inhibition of IGF2BP1 suppressed SCC growth. a. Ca3/7 (mouse) and SCC-13 (human SCC) were cotransfected either with scrambled shRNA (shScram) and pTK-puro plasmid or with sh-IGF2BPl and pTK-puro plasmid. Cells were treated with puromycin (Ca3/7: 2.5 µg/mL, SCC-13: 0.5 µg/mL) until colonies become visible to the naked eye. Colonies were stained with crystal violet. Mean ± SD (n = 3, * p<0.05, one- sample Student t-test) are presented. b. Ca3/7 and SCC-13 cells were cotransfected either with shScram. and Amaxa-GFP plasmid or with sh-IGF2BPl and Amaxa-GFP plasmid. AnnexinV-positive and GFP-positive cells were counted using flow cytometry. Mean ± SD (n = 3, * p<0.05, one-sample Student t-test) are presented. c. Immunoblot analysis of SCC cell lines transfected with IGF2BPl-specific shRNA. Author Manuscript J Invest Dermatol. Author manuscript; available in PMC 2018 March 01.