miR-24-2 regulates genes in survival pathway and demonstrates potential in reducing cellular viability in combination with docetaxel.

MicroRNAs the small (18-22 in length) noncoding RNA molecules are negative regulators of gene expression, modulating biological processes of cell differentiation, survival and death. The latter two phenomena are critical in tumour biology. We provide here the results of human genome wide target prediction of one such microRNA, hsa-miR-24-2, shown to target genes essential for initiating cellular stability and cell survival. The protein-protein interaction study showed important nodes which could affect cell cycle progression and differential oncogenesis. An analysis of hsa-miR-24-2 in sporadic breast tumours showed a negative correlation with metastasis and increasing nodes. The conclusion drawn of hsa-miR-24-2 targeting the genes of cell survival correlated with the methylation profile and resultant transcription factor binding site gain or loss in support of absence of cell survival. In order to accentuate the potential of hsa-miR-24-2 to reduce cellular viability under experimental conditions, in vitro studies in the presence and absence of anti-cancer drugs, such as docetaxel resulted in a significant decrease in cellular viability even at a 200-fold reduced dose of the drug in combination with hsa-miR-24-2.

MiR-101 induces senescence and prevents apoptosis in the background of DNA damage in MCF7 cells.

Moderately increased DNA damage due to the exogenous miR-101 (4 fold) over-expression in MCF7 cells was substantiated by an increase in the number of γ-H2AX foci, correlating with a simple-to-do Halo-assay. miR-101 induced mild/moderate DNA damage favoured senescence rather than apoptosis. An experimental support emanated from the induced mild/moderate DNA damage with 1 µM/5 µM etoposide in MCF7 cells, which resulted in an endogenous miR-101 over-expression (10/4 fold, respectively), followed by senescence. On the other hand, the severe DNA damage induced with 10 µM etoposide, resulted in a low (<1 fold) endogenous expression of miR-101 and an elevated percentage of apoptotic cells. Using bioinformatics tools along with in-vitro and in-vivo validations, miR-101 was found to target and downregulate the mRNA expression of UBE2N and SMARCA4, involved in DNA damage repair (DDR) pathways. Recovery of the expression of the two novel targets in anti-miR-101 transfection validated the results. We conclude that a threshold range of over-expressed miR-101, capable of inducing mild/moderate DNA damage, is sensed by cells to become senescent. The observation derives further support from in-silico protein-protein network analysis where the two novel targets showed their involvement in senescence pathway.

miR-24-2 controls H2AFX expression regardless of gene copy number alteration and induces apoptosis by targeting antiapoptotic gene BCL-2: a potential for therapeutic intervention.

INTRODUCTION: New levels of gene regulation with microRNA (miR) and gene copy number alterations (CNAs) have been identified as playing a role in various cancers. We have previously reported that sporadic breast cancer tissues exhibit significant alteration in H2AX gene copy number. However, how CNA affects gene expression and what is the role of miR, miR-24-2, known to regulate H2AX expression, in the background of the change in copy number, are not known. Further, many miRs, including miR-24-2, are implicated as playing a role in cell proliferation and apoptosis, but their specific target genes and the pathways contributing to them remain unexplored. METHODS: Changes in gene copy number and mRNA/miR expression were estimated using real-time polymerase chain reaction assays in two mammalian cell lines, MCF-7 and HeLa, and in a set of sporadic breast cancer tissues. In silico analysis was performed to find the putative target for miR-24-2. MCF-7 cells were transfected with precursor miR-24-2 oligonucleotides, and the gene expression levels of BRCA1, BRCA2, ATM, MDM2, TP53, CHEK2, CYT-C, BCL-2, H2AFX and P21 were examined using TaqMan gene expression assays. Apoptosis was measured by flow cytometric detection using annexin V dye. A luciferase assay was performed to confirm BCL-2 as a valid cellular target of miR-24-2. RESULTS: It was observed that H2AX gene expression was negatively correlated with miR-24-2 expression and not in accordance with the gene copy number status, both in cell lines and in sporadic breast tumor tissues. Further, the cells overexpressing miR-24-2 were observed to be hypersensitive to DNA damaging drugs, undergoing apoptotic cell death, suggesting the potentiating effect of mir-24-2-mediated apoptotic induction in human cancer cell lines treated with anticancer drugs. BCL-2 was identified as a novel cellular target of miR-24-2. CONCLUSIONS: mir-24-2 is capable of inducing apoptosis by modulating different apoptotic pathways and targeting BCL-2, an antiapoptotic gene. The study suggests that miR-24-2 is more effective in controlling H2AX gene expression, regardless of the change in gene copy number. Further, the study indicates that combination therapy with miR-24-2 along with an anticancer drug such as cisplatin could provide a new avenue in cancer therapy for patients with tumors otherwise resistant to drugs.

Investigation of DNA damage response and apoptotic gene methylation pattern in sporadic breast tumors using high throughput quantitative DNA methylation analysis technology.

BACKGROUND: Sporadic breast cancer like many other cancers is proposed to be a manifestation of abnormal genetic and epigenetic changes. For the past decade our laboratory has identified genes involved in DNA damage response (DDR), apoptosis and immunosurveillance pathways to influence sporadic breast cancer risk in north Indian population. Further to enhance our knowledge at the epigenetic level, we performed DNA methylation study involving 17 gene promoter regions belonging to DNA damage response (DDR) and death receptor apoptotic pathway in 162 paired normal and cancerous breast tissues from 81 sporadic breast cancer patients, using a high throughput quantitative DNA methylation analysis technology. RESULTS: The study identified five genes with statistically significant difference between normal and tumor tissues. Hypermethylation of DR5 (P=0.001), DCR1 (P=0.00001), DCR2 (P=0.0000000005) and BRCA2 (P=0.007) and hypomethylation of DR4 (P=0.011) in sporadic breast tumor tissues suggested a weak/aberrant activation of the DDR/apoptotic pathway in breast tumorigenesis. Negative correlation was observed between methylation status and transcript expression levels for TRAIL, DR4, CASP8, ATM, CHEK2, BRCA1 and BRCA2 CpG sites. Categorization of the gene methylation with respect to the clinicopathological parameters showed an increase in aberrant methylation pattern in advanced tumors. These uncharacteristic methylation patterns corresponded with decreased death receptor apoptosis (P=0.047) and DNA damage repair potential (P=0.004) in advanced tumors. The observation of BRCA2 -26 G/A 5'UTR polymorphism concomitant with the presence of methylation in the promoter region was novel and emerged as a strong candidate for susceptibility to sporadic breast tumors. CONCLUSION: Our study indicates that methylation of DDR-apoptotic gene promoters in sporadic breast cancer is not a random phenomenon. Progressive epigenetic alterations in advancing tumors result in aberrant DDR-apoptotic pathway thereby promoting tumor development. We propose, since pathological epigenetic changes of the DDR-apoptotic genes are reversible modifications, these could further be targeted for therapeutic interventions.

Role of H2AX in DNA damage response and human cancers.

H2AX, the evolutionarily conserved variant of histone H2A, has been identified as one of the key histones to undergo various post-translational modifications in response to DNA double-strand breaks (DSBs). By virtue of these modifications, that include acetylation, phosphorylation and ubiquitination, H2AX marks the damaged DNA double helix, facilitating local recruitment and retention of DNA repair and chromatin remodeling factors to restore genomic integrity. These modifications are essential for effective DSB repair, so is their removal for cell, to recover from checkpoint arrest. Because of these vital roles during DSB signaling and also its activation during early cancer stages, H2AX is emerging as an intriguing gene in tumor biology, supported further by frequent deletion of the region harboring this gene. This review focuses on the insights gained from recent studies on dynamic regulation of H2AX in DSB repair. Also, posing future challenges in the area of chromatin reorganization and retention of epigenetic signature post-DSB-repair with implication of its haploinsufficiency in human cancers.

Copy number alterations of the H2AFX gene in sporadic breast cancer patients.

In addition to being a structural component of chromatin, histone H2AX also has an important role in preserving genetic integrity. The histone H2AFX gene maps to the chromosome region 11q23.2 approximately 11q23.3 that is deleted in most human cancers. Mouse model studies also have clearly shown its involvement in tumorigenesis in a dosage-dependent manner. Therefore, in this study, DNA from 65 paired sporadic breast cancer tissues was systematically screened for gene mutations and changes in gene copy numbers. Although whole H2AFX gene scans showed an absence of mutation in the studied samples, the H2AFX gene copy number was altered in 37% of tumor samples. Furthermore, a twofold reduction in gene copy number in the MCF7 cell line strongly suggests the involvement of H2AFX alteration in breast carcinogenesis. Analysis of clinicopathologic association revealed a convincing correlation with positive estrogen/progesterone receptor status. To our knowledge, this is the first report of a change in H2AFX gene copy number in human cancer.

Homologous recombination-mediated double-strand break repair in mouse testicular extracts and comparison with different germ cell stages.

Homologous recombination (HR) is established as a significant contributor to double-strand break (DSB) repair in mammalian somatic cells; however, its role in mammalian germ cells has not been characterized, although being conservative in nature it is anticipated to be the major pathway in germ cells. The germ cell system has inherent limitations by which intact cell approaches are not feasible. The present study, therefore, investigates HR-mediated DSB repair in mouse germ cell extracts by using an in vitro plasmid recombination assay based on functional rescue of a neomycin (neo) gene. A significantly high-fold increase in neo+ (Kan(R)) colonies following incubation of two plasmid substrates (neo delta1 and neo delta2) with testicular extracts demonstrated the extracts' ability to catalyze intermolecular recombination. A significant enhancement in recombinants upon linearization of one of the plasmids suggested the existence of an HR-mediated DSB repair activity. Comparison of the activity at sequential developmental stages, spermatogonia, spermatocytes and spermatids revealed its presence at all the stages; spermatocyte being the most proficient stage. Further, restriction analysis of recombinant plasmids indicated the predominance of gene conversion in enriched spermatocytes (mostly pachytenes), in contrast to gonial and spermatid extracts that showed higher reciprocal exchange. In conclusion, this study demonstrates HR repair activity at all stages of male germ cells, suggesting an important role of HR-mediated DSB repair during mammalian spermatogenesis. Further, the observed preference of gene conversion over reciprocal exchange at spermatocyte stage correlates with the close association of gene conversion with the meiotic recombination program.