Mutational and Expressional Analysis of Base Excision Repair (BER) Pathway Genes in Breast Cancer Patients

Abstract

Mutational and Expressional Analysis of Base Excision Repair (BER) Pathway Genes in Breast Cancer Patients Base excision repair pathway plays an indispensable role in maintaining genomic integrity and its ability to mediate and repair carcinogen-induced DNA lesions is a key determinant of susceptibility to carcinogenesis. This study was designed for germline mutational screening and expressional analysis of base excision repair (BER) pathway genes at transcriptional and translational level in breast cancer patients along with healthy controls and in vitro characterization of these genes using breast cancer cell lines. Hence, this study was divided into three parts; in first part, three BER pathway genes (APEX1, OGG1 & XRCC1) were screened for germline mutations and their possible association with breast carcinogenesis. Second part comprised of expression analysis of BER genes and proliferation marker Ki-67 at mRNA and protein level to correlate observed deregulations with risk and progression of breast cancer. Third part of this study was an in-vitro analysis, to elucidate the role of BER pathway genes in breast carcinogenesis, using two breast cancer cell lines (MCF-7 and MDA-MB-MDA-231). Mutational analysis was carried out using PCR-SSCP followed by DNA sequencing in 925 individuals including 530 breast cancer patients and 395 cancer free healthy individuals. Sequence analysis of APEX1 revealed fourteen mutations, which included seven 5 ́UTR, one 3 ́UTR, two intronic and four missense mutations. Among identified mutations one 5 ́UTR (rs41561214), one 3 ́UTR (rs17112002) and one missense mutation (Ser129Arg) have already been reported while remaining elven mutations were novel. Six novel mutations (g.20923366T>G, g.20923435G>A, g.20923462G>A, g.20923516G>A, 20923539G>A, g.20923529C>T) were observed in 5 ́UTR region, two (g.20923585T>G, g.20923589T>G) in intron1 and three missense mutations (Glu101Lys, Ala121Pro, Ser123Trp) in exon 4. A significant association was observed between APEX1 mutations and increased breast cancer risk {(~9 fold (OR = 8.68, 95 % CI = 2.64 to 28.50) with g.20923435G>A (5’UTR) mutation, ~13 fold (OR= 12.63, 95 % CI = 3.01 to 53.01) with g.20923539G>A (5’UTR) mutation and ~5 fold increase with three missense mutations (Glu101Lys (OR = 4.82, 95 % CI = 1.97 to 11.80)), Ser123Trp (OR = 4.62, 95 % CI = 1.7 to 12.19), Ser129Arg (OR = 4.86, 95 % CI = 1.43 to 16.53) }. DNA sequencing for OGG1 revealed fifteen mutations, which included five intronic (g.9792260 insert_T; g.9793748G>A; g.9798336T>G; g.9798349T>A; g.9793680G>A, rs55846930), four splice site (g.9792109delT, g.9798307T>G, g.9798502T>G & g.9800972T>G), two 3’UTR (g.9798848G>A, g.9798896T>C), three missense (Val159Gly, Gly221Arg, Ser326Cys) and one non-sense (Trp375STOP) mutation. Among identified mutations, one intronic (g.9793680G>A, rs55846930) and two missense mutations (Gly221Arg, TMP_ESP_3_9796483 and Ser326Cys, rs1052133) has already been reported while remaining twelve mutations are novel. Significantly increased breast cancer risk was found associated with different mutations in OGG1 when compared with controls. Significantly (p<0.001) increased (~29 fold) breast cancer risk was found associated with a splice site variant g.9800972T>G (OR =28.85, 95 % CI = 3.87 to 207.7) and 3 ́UTR variant g.9798848G>A (OR =29.20, 95 % CI = 33.98 to 213.74). Among intronic mutations, highest (~15 fold) increase in breast cancer risk was found associated with g.9793680G>A variation (OR =14.65, 95 % CI =1.95 to 109.9; p< 0.009). Similar trend was observed in all missense mutations in breast cancer patients when compared with controls and ~14 fold increased risk was associated with Val159Gly (OR=13.68, 95 % CI = 1.82 to 102.9; p<0.01), ~17 fold with Gly221Arg (OR=16.85, 95 % CI =2.26 to 125.53; p<0.005) and ~18 fold with Ser326Cys (OR=18.45, 95 % CI =2.49 to 136.99; p<0.004) in breast cancer patients compared with controls. Whereas analysis of nonsense mutation showed that ~13 fold (OR =12.90, 95 % CI =1.71 to 97.28; p<0.01) increased breast cancer risk was associated with Trp375STOP in patients compared to controls. Mutational screening of XRCC1 revealed, twenty five mutations in different coding regions of XRCC1 including eighteen novel and seven already reported mutations. Among these, thirteen were missense mutations (Gly61Ala, Val72Gly, Asn183Ser, Arg194Trp, Arg280His, Ala283Asp, Asp356Asn, Asn510Ser, Arg559Gly, Arg560Gly, Tyr576Asn, Val629Gly & Val630Gly), eight synonymous mutations (Pro206Pro, Glu122Glu, Gln331Gln, Gln430Gln, Gln449Gln, Glu521Glu, Arg608Arg & Gln632Gln), three frameshift (Gly143fs*1, Ala182Argfs*29 & Ala587Serfs*9) and one nonsense mutation (Trp125*). Significantly higher frequency of one synonymous mutations Pro206Pro (p<0.003), seven missense mutations Gly61Ala (p<0.001), Val72Gly (p<0.0007), Arg194Trp (p<0.0001), Arg280His (p<0.03), Arg559Gly (p<0.03), Arg560Gly (p<0.009), Tyr576Asn (p<0.03) and two frameshift mutations Ala182Argfs*29 (p<0.01), Ala587Serfs*9 (p<0.008) was observed in breast cancer patients when compared with controls. Some of the observed mutations in BER genes were found significantly correlated (p<0.03) with family history of cancer, menopausal age and use of tobacco. For expressional analysis, BER genes (APEX1, OGG1 and XRCC1) and proliferation marker (Ki-67) were studied in tumor tissues and control samples of study cohort-2 and 3 (104 and 111 samples), at mRNA level using real-time PCR. Statistically significant down-regulation of XRCC1 (p<0.01) and OGG1 (p<0.04) was observed in breast tumor samples compared to control samples. Ki-67 (p<0.03) and APEX1 (p=0.32) were up-regulated in breast tumor samples compared with controls. Immunohistochemical analysis of APEX1, OGG1, XRCC1 and Ki-67 revealed that among 104 breast tumor samples, 81% samples showed OGG1 down-regulation, 79% showed APEX1 up-regulation, 73% showed XRCC1 down-regulation and 84% samples exhibited Ki-67 up-regulation. In mutational and expressional studies, APEX1, OGG1 and XRCC1 were observed as important factors responsible for onset and progression of breast cancer. Our data suggests that germline mutations in BER genes and down-regulation of BER pathway genes, such as OGG1 and XRCC1 combined with over-expression of APEX1 and Ki-67 (proliferation marker) may contribute to the initiation and progression of breast cancer in Pakistani population. In third part of this study in-vitro experiments were performed to analyse the role of selected BER pathway genes (APEX1, OGG1 and XRCC1) in cancer cell behaviour during tumorigenesis using two breast cancer cell lines (MCF-7 & MDA-MB-MDA- 231). APEX1 knockdown cells of both cell lines showed significantly decreased growth (p<0.001), invasion (p<0.05) and migration (p<0.01) abilities and increased adhesion (p<0.05) ability. Knockdown of OGG1 and XRCC1 caused significant increase in cancerous characteristics of both breast cancer cell lines showing decreased adhesion (p<0.05) but enhanced proliferation (p<0.05), invasion (p<0.05) and rapid migration (p<0.05), however this significance level of difference was slightly variable in both cancer cell lines. Results from these studies suggest that APEX1, OGG1 and XRCC1 may be intriguing potential targets for anticancer strategies and diagnostics.