Abstract 1557: Overexpressions of ALEX1 gene play a negative role in human colorectal tumorigenesis

Abstract Background: Arm protein lost in epithelial cancers, on chromosome X (ALEX) is a novel subgroup within the armadillo family which has several ARM repeat domain. The role and the expression profile of ALEX1 gene in colorectal tumor are not well examined. Our studies have revealed overexpression of ALEX1 suppressed colony formation ability of stable human colorectal carcinoma cell lines and clinical significance of ALEX1 expression in colorectal cancer patients. Materials and methods: Human colon cancer cell lines (HCT116, SW480) and breast cancer cell line (MCF-7) were used in this study. Human ALEX1 gene was amplified by PCR and inserted into the XhoI site of pCAGIPuro plasmid. Colony formation assay and Soft agar colony formation assay were performed to examine the effect of overexpression of the ALEX1 on cancer cell proliferation. Genomic DNA was purified by the phenol chloroform extraction and bisulfite genomic sequencing was carried out with the EpiTect bisulfite kit. We examined the expression level of ALEX1 mRNA in matched tissue pairs of normal colorectal mucosa and colorectal tumor tissue by Quantitative real-time RT-PCR Tumor specimens along with adjacent normal tissues were obtained from 49 patients with primary colorectal cancer undergoing complete surgical resection of tumors and lymph nodes. The pathological stages of the patients were as follows: stage I, 9 patients; stage II, 16 patients and stage III, 24 patients by pTNM classification. The post-surgical observation period was from 277 to 3631 days (1625.0 ± 1033.0 days). Results: Overexpression of ALEX1 in colorectal carcinoma cells was capable of impairing colony formation and suppressed the anchorage-dependent and -independent colony formation of human colorectal carcinoma cell lines by the study of stable clones of HCT116 cells expressing ALEX1 protein. Bisulfite genomic sequencing revealed that the promoter region of ALEX1 gene was highly methylated in both HCT116 and I SW480 cells in comparison to those in PANC-1 and MCF-7 cells which express endogenous ALEX1 mRNA, indicating the capability of promoter methylation to silence ALEX1 gene in HCT116 and SW480 cells. In 34 cases out of 49 (69%) colorectal tumor tissues, greater than a 50% reduction of the ALEX1 mRNA level was observed in comparison to adjacent normal mucosa tissues. ALEX1 mRNA was significantly reduced in colorectal tumor tissues than those in normal mucosa (P=0.01459, Mann-Whitney U-test). The 17 cases with higher ALEX1 gene expression (Tumor/normal value ≧0.20) significantly revealed a better disease-free survival rate than the other 32 cases (< 0.20; P = 0.045, log-rank test), which showed significant correlations between ALEX1 expression and better prognosis. Conclusions: Our findings may support that ALEX1 functions as a tumor suppressor in colorectal cancer progression. Examination of ALEX1 expression might be helpful for predicting the prognosis of patients with curative resected colorectal cancer. Citation Format: Akihiko Takeda. Overexpressions of ALEX1 gene play a negative role in human colorectal tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1557. doi:10.1158/1538-7445.AM2014-1557