Studies on Genetic Characterization and Deregulation of Homologous Recombinant Repair (HRR) Pathway Genes in Thyroid Cancer

Abstract

XRCC2, XRCC3 and RAD51 are the main molecules of the homologous recombinant repair (HRR) pathway related to thyroid cancer. Polymorphisms in these genes have been reported frequently in literature and are known to show diverse patterns in different populations. The present study was designed to screen these genes in thyroid cancer patients and controls at the DNA, mRNA and protein levels. A total of 456 pathologically confirmed thyroid cancer patients and 400 healthy controls were recruited. ARMS-PCR was used for genetic analysis followed by sequencing. In this study, various reported polymorphisms were analysed in HRR pathway genes at the germline level in thyroid cancer patients. Significant association of these SNPs were observed with age of diagnosis, gender, smoking, staging, histological subtype and treatment strategies of cancer. Significant association of these functional, promoter based and non-coding polymorphisms in HRR pathway genes and associations of these SNPs with important risk factors highlights their possible role in thyroid carcinogenesis. Further haplotype analysis revealed that most of the haplotypes in XRCC2, XRCC3 and RAD51 are linked with a significant increase in thyroid cancer risk. While some of the haplotypes were associated with a significant reduced thyroid cancer risk. RAD51 SNP, rs1801321 were observed consistent in reduced risk of thyroid cancer in association with risk factors analysed in this study. HRR pathway genes were further investigated at mRNA and protein expression levels. Thyroid cancer samples (n=102) along with equal numbers of un-involved tissues as controls were used for expression analysis. Quantitative real time PCR was used for determination of mRNA expression levels and immunohistochemistry was performed to analyse the protein expression of these genes. Expression analysis of XRCC2, XRCC3, RAD51 and proliferation marker Ki67 at mRNA level revealed significant deregulations. Significant downregulation (p<0.01) of XRCC2 and up-regulations of XRCC3 (p<0.01), RAD51 (p<0.001) and proliferation marker Ki67 (p<0.001) were

observed in the expression profile of HRR molecules. These were significantly correlated (negatively and positively) with an up-regulated expression profile of the tumor proliferation marker, Ki67. XRCC3 and RAD51 expression was up-regulated in higher stages and aggressive tumor stages. Immunohistochemical analysis of HRR molecules revealed that among 102 tumor samples, 87% samples showed down- regulation of XRCC2 (p<0.0001), 75% samples showed up-regulation of XRCC3 (p<0.001), 76% samples showed up-regulation of RAD51 (p<0.0001) and 82% samples showed up-regulation (p<0.0001) of proliferation marker, Ki67. These IHC analysis results support our qPCR findings. Polymorphisms in HRR genes and abnormal expression at transcription and translation levels besides defective DNA damage may suggest that HRR pathway genes are correlated with thyroid tumorigenesis and aggressive proliferative behaviour of thyroid cancer in the Pakistani population. Furthermore, the role of XRCC2 and XRCC3 genes were explored in a thyroid cancer cell line (8505C) cells using in-vitro experiments. Effects of genetic modifications were observed for XRCC2 and XRCC3 in thyroid cancer cells by CRISPR-Cas9. These two genes were expressed using lentivirus having GFP, Cas9 and guide RNA. Gene knockouts for these genes were observed after single cell isolation. A gene cleavage assay showed successful genetic modifications and altered behaviour of these genes in thyroid cancer cells. Therefore, potential knockout of these genes gave an insight into molecular mechanism of thyroid cancer. XRCC3 gene function was further explored in the thyroid cancer cell line. Gene knockdown was produced by siRNA technology and successful knockdowns were confirmed using western blots. XRCC3 knockdown cells of anaplastic thyroid cancer (8505C) cell line showed decreased cell growth (p<0.01) as well as decreased cell proliferation after cell colony forming assay (p<0.001) when compared to controls. Decreased invasion (p<0.001), increased adhesion ability (p<0.001) and decreased migration abilities (i.e. wound closure for XRCC3 with siRNA knockdown was 86%) of the cells migrated for wound closure after 24 hours compared to ~98% cell migration of control cells. XRCC3 siRNA knockdown showed significant genetic alterations after treatment with topoisomerase inhibitor drugs (i.e. camptothecin, phleomycin and etoposide). Effect of these drugs on XRCC3 knockdowns were observed by colony PCR and metaphase analysis. Therefore overexpression of XRCC3 caused significant increase in cancerous characteristics of thyroid cancer which is in accordance with observed results at the mRNA and protein

levels of expression. For a better insight into these genes in thyroid carcinogenesis, further work is needed to explore the interesting domains playing important role in carcinogenesis. Study of different factors affecting the thyroid cancer will not only assist in understanding thyroid cancer disease progression but will also help to establish future personalized cancer risk prediction.