Abstract 814: Identification of PCDHB3 as a potential tumor suppressor gene in colorectal cancer by exome sequencing.

Abstract

Abstract Colorectal cancer (CRC) is one of the most common cancers in worldwide. Previous studies have identified hundreds of recurrent mutations in CRC. To gain a comprehensive view of the genetic alterations underlying colorectal tumorigenesis, we sequenced the exome in colorectal cancer from 10 patients without chemo-/radio- therapy histories. In total, we identified 378 novel somatic alterations, in which 341 missense, 41 small indels, and 7 splice-site mutations, 9 genes with more than two novel alterations were discovered in the list. Sequenom MassARRAY validation reached a positive rate of 85%. Two PCDHB family members which totally mutated in 4 sites attracted our attentions. We focused on PCDHB3, which had three missense mutations in 3 individuals and has never been investigated before. We re-sequenced another 104 cases of CRC genomic DNA through PCR arrays focused on the PCDHB3 exon and reached a final mutation rate of 7.02%. A semi-quantitative RT-PCR assay was performed to compare the PCDHB3 mRNA levels among 98 pairs of CRC tissues. A statistical significant decrease of PCDHB3 level was observed in tumor tissues compared with normal intestinal epithelium mucosa (P<0.001). IHC results showed that PCDHB3 expressed less and weaker in tumor. Furthermore, reduced PCDHB3 expression is associated with poor clinical outcome (overall survival: P=0.028; progression free survival: P=0.004) in advanced CRC patients with stage III and IV. It indicates that tumor tissues with higher and stronger expression of PCDHB3 are less aggressive in inducing metastasis and leads to longer survival of patients. In vitro studies, over-expressed PCDHB3 in DLD1, a CRC cell line, showed a decreased number of cells capable of passing through the filter in a transwell migration assay, whereas the SW480 cell line with decreased PCDHB3 passed through more compared to its control. In the MTT assays, HT29/DLD1 cells with higher PCDHB3 levels showed a lower ratio of proliferative performance compared to their negative controls; and SW480 with less PCDHB3 had an advantage over its control. In vivo assays of nude mice confirmed that with higher expression of wild-type PCDHB3, tumor developed significantly smaller than the control group, as well as the mutant-type group. It comes to a conclusion that PCDHB3 may function as a potential tumor suppressor gene in CRC, and we made a hypothesis that there are two mechanisms underlying PCDHB3-related CRC tumorigenesis: by mutations affecting amino acid sequence or by down-regulated expression of amino acid. In summary, our study provides a catalog of genetic alterations in colorectal cancer from 10 patients. The discovery of PCDHB3 as a potential tumor suppressor gene and the elucidation of mutated genes involved in pathways known to be relevant with colorectal cancer has broadened our understanding of molecular basis in colorectal tumorigenesis. Citation Format: Wen Ye, Jiangxue Wu, Ranyi Liu, Wenlin Huang. Identification of PCDHB3 as a potential tumor suppressor gene in colorectal cancer by exome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 814. doi:10.1158/1538-7445.AM2013-814