Abstract 3866: Potential oncogenic function of Rad51C splice variant in colorectal tumors

Rad51c is a tumor suppressor gene and known for its function in homologous recombination and DNA repair in early and late stages of HR (Homologous Recombination). Studies from breast and ovarian cancer patients reveled a bi-allelic homozygous germline missense mutation in the codon 773 and 258 of Rad51C gene. At the cellular level the effects include chromosomal instability (increased chromosomal breakage) associated with hypersensitivity to DNA damaging agents, and defective DNA damage repair. Mutations, which include base insertions and missense, have been reported in the Rad51C gene. Alternatively spliced transcriptomes have been shown to play a different or antagonistic biological role compared to the full length wild type mRNA. Some of the important diseases caused by cis acting or trans acting protein splicing factors include cystic fibrosis, dementia, premature aging and cancer. In addition, promoter methylation has been associated with gene silencing, while DNA methylation at both intronic and exonic regions are shown to correlate with isoform-specific transcription by alternative splicing or by utilizing alternate promoters. DNA methylation at the cytosine residues (5-methylcytosine) of the CpG di-nucleotides is carried out by DNA methyltransferases (DNMT) and is generally considered to be a repressive epigenetic modification. By RT-PCR, we identified splice variants of Rad51C gene that include variant-1 (without exon-7), variant-2 (without exon-6, 7) and variant-3 (without exon-7, 8) in colorectal tumors. Of the 38 colorectal tumors, 18 contained variant 1, 12 contained variant 2, 14 contained variant 3, and eight had no expression of any of the variants. Bisulfate DNA sequencing and Methylation Specific PCR (MS-PCR) showed promoter methylation of Rad51C in tumor cells. 5-Azacytidine treatment of LS-174T cells caused a 14 fold increase in variant 1, a 4.8 fold increase for variant 3 and 3.4 fold for variant 2 compared to no treatment. Real time PCR analysis of 9 pair-matched colorectal tumors and non-tumors showed that variant 1 was overexpressed in tumors comparing to matched non-tumors. Expression of Rad51C variants in these tumors was associated with microsatellite stability and with maintenance of functionality of the fanconi anemia repair pathway. In vitro transient overexpression of Rad51C variant-1 in colorectal tumor cells over the wild type Rad51C caused a 1.8 fold increase in the cell proliferation as analyzed by BrdU immunofluorescence staining and FACS. Given that the Rad51C splice variant-1 is overexpressed in colorectal tumors and has a role in promoting cell proliferation, further investigation regarding its potential role in oncogenesis in colorectal tumor cells is warranted. Citation Format: Arjun Kalvala, Li Gao, Kathleen Dotts, Fernando Ochoa Cortes, Brittany Barnwell, Greg Otterson, Miguel Villalona Calero, Wenrui Duan. Potential oncogenic function of Rad51C splice variant in colorectal tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3866. doi:10.1158/1538-7445.AM2015-3866