Abstract 3802: EWSR1: Identification and functional characterization of a novel target gene locus in Lynch syndrome
Authors: Piscuoglio Salvatore , Kovac Michal , Kishore Shivendra , Mele Valentina , Zavolan Mihaela , Müller Hansjakob , Terracciano Luigi M. , Heinimann Karl 
Published: June 2012

Abstract
Background: Lynch syndrome (also Hereditary Non-Polyposis Colon Cancer, HNPCC) represents the most common, autosomal dominantly inherited cancer predisposition worldwide and accounts for 3-5% of the total colorectal cancer (CRC) burden. It is caused by germline mutations in DNA mismatch repair (MMR) genes (mainly MLH1 and MSH2). MMR deficiency results in microsatellite instability (MSI), i.e. genome-wide accumulation of somatic alterations at repetitive DNA sequence motifs, mostly non-coding microsatellite markers. MSI, used as a diagnostic tool to identify HNPCC-related CRCs, can also affect repeat tracts within or immediately adjacent to the coding sequence of so-called target genes (like TGFbRII, BAX, etc) which are thought to fuel carcinogenesis in HNPCC. In search for novel target gene loci we identified a large poly-T tract, (T)16, in the 3’ untranslated region (3’UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene.
Aims: To determine i) type and frequency of instability at the EWSR1 (T)16 in 78 HNPCC and 85 sporadic colorectal cancers and ii) its possible effect on EWS expression
Materials and methods: We determined the length of the EWSR1 3’UTR tract motif, (T)16, by PCR amplification and fragment analysis of 78 CRCs from HNPCC patients with identified germline mutation (MLH1: n=50; MSH2: n=28), 85 sporadic microsatellite-stable CRCs as well as 5 CRC cell lines (2 MMR proficient, 3 MMR deficient). EWS protein expression was assessed by immunohistochemistry (IHC) on a tissue microarray and immunoreactivity scored semi-quantitatively. Statistical comparisons were performed using Chi-square or Student's t test where appropriated (two-tailed p values, considering p&lt;0.05 to be statistically significant).
Results: Novel alleles at the EWSR1 3’UTR (T)16 tract were observed in all (n=78) HNPCC but none (0/85) of the sporadic CRCs. The type (insertion/deletion) and frequency of somatic alterations were: ins1-2 (2.5%), del1-3 (22.6%), del4 (34.4%), del5 (28.54%) and del&gt;6 (11.92%). Similar observations were made for the MMR-deficient cell lines (HCT116/MLH1: del3/del4; LoVo/MSH2: del4) whereas the MMR-proficient ones were stable. RNA secondary structure prediction suggested gross structural alterations for deletions &gt;4 Ts. IHC showed significant downregulation of EWS expression in sporadic CRCs (p&lt;0.005), but no difference in HNPCC CRCs (p=0.7). Since the (T)16 tract may represent a binding site for AU-rich element binding proteins it could have an effect on EWSR1 mRNA stability/translation (currently being tested).
Conclusion: The (T)16 tract in the 3’UTR of the EWSR1 gene represents a novel target gene locus in Lynch syndrome allowing for highly sensitive and specific identification of MMR-deficient CRCs. Moreover, IHC analysis suggests a regulatory role of this locus on EWS protein expression in HNPCC colorectal cancers.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3802. doi:10.1158/1538-7445.AM2011-3802