Molecular Genetic and Epigenetic Characterization of Tumor Suppressor Rbl2/p130 in Human Breast Cancer

Abstract

Molecular Genetic and Epigenetic Characterization of Tumor Suppressor Rbl2/p130 in Human Breast Cancer Tumor suppressor retinoblastoma like protein-2 (Rbl2/p130) regulate G1/S transition through its binding with basal transcription factors (E2F4 and E2F5). Activities of Rbl2 protein are regulated through phosphorylation and acetylation in a cell cycle dependent manner. Deregulated expression of Rbl2 and E2F4/5 may lead to neoplastic alterations. The involvement of Rbl2 and E2F4/5 genes in the development and progression of breast cancer among local Pakistani population was investigated. Various genetic and epigenetic mechanisms were probed to explore their potential involvement in gene expressions and tumor growth formation. Rbl2/p130 gene was comprehensively screened for mutations in all exons through single strand conformational polymorphism (SSCP) analysis followed by direct sequencing of suspected samples. Total 200 breast cancer women with mean age of ~45 years were participated in this study. Out of which 126 were at stage I (SI), 40 at stage II (SII) and 34 at stage III (SIII) of breast cancer. Moreover, 138 patients were from invasive ductal carcinoma (IDC), 48 from Invasive lobular carcinoma (ILC) and 14 from ductal carcinoma in situ (DCIS). Among these, 79 mutations in blood and 87 mutations in tissues were detected. Out of these, 12 mutations were previously reported whereas the remaining were found novel. Rbl2 gene was found heavily mutated in exons 19-22 encompassing pocket and carboxyl terminal domain of protein. Among total 28 mutations in exon-21, an A>G transition in codon 1083 that codes for an acetylatable lysine present in the bipartite nuclear localization signals (NLS) 1081PSKRLR1086, was observed. This lysine was mutated to an arginine in 102 (51%) blood and 118 (59%) tissue samples, which suggests that impairment in acetylation status of Rbl2 proteins as well as alterations in nuclear localization might be a key event in tumor initiation and progression. Rbl2/p130 gene was found down regulated while E2F4 and E2F5 genes were upregulated in all study cohorts devised on the basis of age, disease severity and tissue origin. Deregulated expression of these genes was investigated in connection to xi their promoter methylation status using methylation specific PCR (MSP) of bisulphite converted genomic DNA. Rbl2 promoter was found hyper-methylated while promoters of E2F4/5 genes were hypo-methylated in patient‟s samples, suggesting a potential role of promoter methylation in breast carcinogenesis. The CpG positions (-1, +3, +15 and +75) in Rbl2 promoter were specifically found methylated, however positions (-8, +9, +21, +28, +47 and +52) were un-methylated. Rbl2 promoter methylation was found positively associated with various disease stages. Dynamic positioning of nucleosomes is pivotal in determining levels of gene expression especially on or around transcription start site (TSS) of a gene. Nucleosome position around TSS of Rbl2/p130 was determined using micrococcal nuclease (MNAse) digestion assay and ChIP-PCR. Region between -145 -to- +140 around TSS was scanned for 6 positions (P1=-137 -to- +69; P2=-90 -to- +69; P3=-33 -to- +140; RX=-137 -to- +7; F1=-54 -to- +99; F2=-11 -to- +140). About 66% breast tumors and 26.6% ANCT samples were positive for P1. The difference was found statistically significant (p < 0.0001) with an odd ratio (OD) of 9.143, suggesting that nucleosome formation in this region is ~9 times more probable in tumor samples, whereas P2, P3, RX, F1 and F2 were not that suitable for nucleosome positioning. These results indicate that nucleosomes are present slightly downstream of TSS under normal physiological circumstances, that upon breast carcinogenesis slides 55 bases upstream of the TSS, aligning position +1 at the center of nucleosome, hence hindering access to the transcriptional machinery. Breast tumor samples were found methylated at H3K4 to a lesser extent and these modifications were not found associated with Rbl2 proteins, although in precipitates where Rbl2 protein as enriched small fractions of K4 methylation were observed. Conversely, H3K9 tri-methylation as well as Suv39 H2 methyltransferase were found positively associated with Rbl2 proteins, which suggests a putative role of Rbl2 proteins in creating a pre-apoptotic environment in tumor tissues. Our results, highlights a cumulative impact of alterations in genetic and epigenetic mechanisms that culminate in neoplastic transformation. It is obvious from these findings that a cell cycle regulator like Rbl2 can act as a transducer molecule that provide platform for various other molecules to manipulate physiological environment of the cell. With this, we hypothesize that activities of Rbl2/p130 proteins are not limited to tumor suppressor only, rather it has the potential to regulate other physiological and biochemical activities of the cell.