Identification Of Genomic Disruptions And Rearrangements In Esophageal Squamous Cell Carcinoma

Genetic disruptions and gene rearrangements are the most common genetic alterations in human malignant tumors. So far, a few functional genetic disruptions and gene rearrangements have been identified in several solid tumors, but no such evidence was found in esophageal carcinoma. The purpose of the present study was to clarify the genetic alterations in esophageal squamous cell carcinoma (ESCC).Six ESCC samples were collected for SNP micro-array and a few disruptions were found including MTAP, CDKN2B-AS1, NRP2, ANXA13and PPP3CA. Frequency of MTAP, NRP2, ODZ2and CADM2disruptions were identified by real-time PCR and dual-color fluorescence in situ hybridization (FISH). Alterations of MTAP and NRP2expression in tumors were detected by western blotting. RACE technique is applied to detect the possible fusion transcripts. Cell proliferation assays and transwell assays were carried out to investigate the effect of MTAP alteration on malignant phenotype of ESCC cells.Real-time PCR results showed that MTAP disruptions were present in22.5%(9/40) of ESCCs and the frequency of ODZ2, CADM2and NRP2disruptions was found to be12.5%,8.3%and16.7%. Three fusion transcripts were detected in one ESCC sample which were1-5exons of MTAP fused with13,14and19exons of CDKN2B-AS1respectively. In another sample, NRP2and ANXA13formed the fusion transcript which was the first exon of ANXA13fused with the1-14exons of NRP2. PPP3CA and LPHN3self-rearrangements were identified in the other two ESCC samples.Further analysis of earlier59ESCC SNP micro-array confirmed high frequency deletion and disruption of MTAP. Down-regulation of MTAP was detected in5out of the9ESCC samples with MTAP disruptions by western blotting. Functional studies indicated that silencing MTAP by small interfering RNA induced epithelial-mesenchymal transition (EMT), accompanied by a decrease of E-cadherin and an increase of N-cadherin and Slug. Also, MTAP knockdown promoted cell migration and invasion in ESCC KYSE150and KYSE170cells. Our results indicate that MTAP disruptions and reduced expression exist in ESCCs and that MTAP plays a part in mediating cell migration, invasion and EMT of ESCC cells.In conclusion, two new fusion transcripts:MTAP-CDKN2B-AS1and NRP2-ANXA13are identified for the first time. Functional study show that MTAP knocking down induces EMT in esophageal cancer cells. Our work provides important clues in exploring the significance of MTAP and NRP2in esophageal squamous carcinoma.