Abstract 2407: Investigation of Novel Rad51c-ATXN7 fusion gene in colorectal tumors

Abstract

Abstract The Fanconi Anemia (FA) pathway is a major mechanism of homologous recombination DNA repair in response to genotoxic insults. Formation of FANCD2 foci has been reported as a predictor of resistance to cisplatin and mitomycin C (MMC) treatment in cancer cells. Defects in Rad51C (FANCO) have been documented as the cause of Fanconi Anemia (FA) complementation group O (FANCO) disorder. A fusion gene formed between Rad51c (exon 1-7) and ATXN7 (ataxin7, involved in neurocerebral ataxia) exons 6-13 have been demonstrated by next generation sequencing in MCF-7 breast cancer cells. Given our observation of higher prevalence of somatic functional FA deficiency in colorectal tumors based on FA triple staining immunofluorescence (FATSI) method that we developed to assess FANCD2 foci in tumors, we evaluated the presence of the fusion gene Rad51C-ATXN7 in the FANCD2 foci negative and foci positive tumors using RT-PCR, immunoprecipitation and immunoblot analysis. RNA and DNA isolated from frozen colon tumor tissues with TRIzol reagent. RT-PCR primers spanned the region between Rad51c exon5 and ATXN7exon 8 to amplify fusion gene in colorectal tumors. To identify the fusion break point in RNA from colorectal tumors, we RT-PCR amplified six tumors and their products were Topo TA cloned. We identified three previously unknown isoforms of fusion mRNAs between Rad51c and ATXN7 among the 40 Topo TA clone sequences analyzed. We named the mRNA fused between Rad51c from exons 1-7 and ATXN7 exons 6-13 long form. From RNA sequence analysis by translation tool, we identified that the long form extends for only five amino acids (aa) after the fusion junction and results in a premature stop codon without producing a fusion protein. We also identified two short variants (1 & 2) in 40 clones. Sequencing confirmed the short variant form 1 between Rad51c (exon 1-6) and ATXN7 (exon 6-13). Immunoprecipitation and western blot analysis further confirmed a 110 KDa protein to be the short variant 1 in colorectal tumor cells. Sequence analysis of the short variant 2 showed that this variant formed between Rad51c (exon 1-5) and ATXN7 (exon 6-13), resulting in a fusion chimera of unknown protein with additional 37 aa and stop codon post fusion junction at C-terminus, but with an N-terminus resembling Rad51c. Thus only the variant 1 is able to produce a Rad51c-ATXN7 fusion protein. We further investigated the presence of the fusion gene according to FA functionality (FancD2 foci positive versus negative) by RT-PCR. Among 30 colorectal tumors evaluated 16 tumors were FANCD2 foci negative and 14 were positive. The Rad51c-ATXN7 short variant-1 was present in 6 of the 16 FANCD2 foci negative colorectal tumors as compared to 9 of the 14 FANCD2 foci positive tumors (p=0.153, t-test). In conclusion, a novel fusion protein was identified at relatively higher frequency in colorectal tumors. Further studies are required to investigate its function in either malignant transformation, tumor progression or in response to genotoxic insults. Citation Format: Arjun Kalvala, Li Gao, Brittany Barnwell, Greg A Otterson, Miguel A Villalona-Calero, Wenrui Duan. Investigation of Novel Rad51c-ATXN7 fusion gene in colorectal tumors. [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 2407. doi:10.1158/1538-7445.AM2014-2407