ABERRANT EXPRESSION OF COL14A1, CELRS3, and CTHRC1 IN BREAST CANCER СELLS.

BACKGROUND: Collagens, which are the major components of the extracellular matrix involved in the regulation of tumor microenvironment, could be differentially expressed in breast cancer (BC) with different transcriptome profiling. AIM: To analyze the transcript level expression of COL1A1, COL5A1, COL10A1, COL11A1, COL12A1, COL14A1, CTHRC1, and CELRS3 genes and the clinical relevance of their differential expression in BC. MATERIALS AND METHODS: The transcript level expression of the genes was analyzed using the quantitative real-time PCR (qPCR) in tumor tissue of 60 BC patients. RESULTS: Overexpression of COL1A1, COL5A1, COL10A1, COL11A1, COL12A1, CTHRC, and CELRS3 anddown-regulated expression of COL14A1 were observed. COL14A1 down-regulation was associated with aggressive, basal, and Her-2/neu BC subtypes (p = 0.031). Overexpression of CELSR3 was found to be associated with the older age of the patients (> 55 years, p = 0.049). Further analysis with the TCGA BC data set has shown a concordance in the differential expression of the above genes. Furthermore, overexpression of CTHRC1 was associated with poor overall survival (OS), particularly with poor prognosis (p = 0.00042) for the luminal BC subtype. On the other hand, CELSR3 overexpression was associated with mucinous tumors and poor prognosis in post-menopausal women. In silicotarget prediction identified several BC-associated miRNAs and members of miR-154, -515, and -10 families to perform a likely regulatory role in the above ECM genes. CONCLUSION: The present study shows that the expression of COL14A1 and CTHRC1 may serve as potential biological markers for the detection of basal BC and the prognosis of survival for patients with the luminal subtype of BC.

Study of Gene Expression Profiles of Breast Cancers in Indian Women.

Breast cancer is the most common cancer among women globally. In India, the incidence of breast cancer has increased significantly during the last two decades with a higher proportion of the disease at a young age compared to the west. To understand the molecular processes underlying breast cancer in Indian women, we analysed gene expression profiles of 29 tumours and 9 controls using microarray. In the present study, we obtained 2413 differentially expressed genes, consisting of overexpressed genes such as COL10A1, COL11A1, MMP1, MMP13, MMP11, GJB2, and CST1 and underexpressed genes such as PLIN1, FABP4, LIPE, AQP7, LEP, ADH1A, ADH1B, and CIDEC. The deregulated pathways include cell cycle, focal adhesion and metastasis, DNA replication, PPAR signaling, and lipid metabolism. Using PAM50 classifier, we demonstrated the existence of molecular subtypes in Indian women. In addition, qPCR validation of expression of metalloproteinase genes, MMP1, MMP3, MMP11, MMP13, MMP14, ADAMTS1, and ADAMTS5 showed concordance with that of the microarray data; wherein we found a significant association of ADAMTS5 down-regulation with older age (≥55 years) of patients. Together, this study reports gene expression profiles of breast tumours from the Indian subcontinent, throwing light on the pathways and genes associated with the breast tumourigenesis in Indian women.

Clinical significance of promoter hypermethylation of RASSF1A, RARbeta2, BRCA1 and HOXA5 in breast cancers of Indian patients.

Promoter hypermethylation of genes is implicated in the pathogenesis of many cancers, including breast cancer. Herein, we analyzed the promoter methylation status of a panel of critical growth regulatory genes, RASSF1A, RARbeta2, BRCA1 and HOXA5, in 54 breast cancers and 5 distant normal breast tissues of Indian patients. The methylation data were correlated with clinicopathological characteristics and hormone receptor status to determine the impact of methylation in breast carcinogenesis. Promoter hypermethylation of RASSF1A was observed in 39/54 (72%), HOXA5 in 36/54 (67%), BRCA1 in 15/54 (28%) and RARbeta2 in 8/54 (15%) breast cancers. Our most significant findings were the association of RASSF1A methylation with nodal metastasis (p=0.05); and RARbeta2 methylation with age (all tumors in patients in the older age group were methylated, p=0.04). Further, the interactions between DNA methylation and hormone receptor biology in breast cancer cells are beginning to be clearly understood. In this context the association of HOXA5 methylation with loss of ERalpha (p=0.009) is noteworthy.

A sensitive test for the detection of specific DSB repair defects in primary cells from breast cancer specimens.

Increasing evidence indicates that breast cancer pathogenesis is linked with DNA double-strand break (DSB) repair dysfunction. This conclusion is based on advances in the study of functions of breast cancer susceptibility genes such as BRCA1 and BRCA2, on the identification of breast cancer-associated changes regarding the genetics, expression, and localization of multiple DSB repair factors, and on observations indicating enhanced radiation-induced chromosomal damage in cells from predisposed individuals and sporadic breast cancer patients. In this pilot study, we describe a sensitive method for the analysis of DSB repair functions in mammary carcinomas. Using this method we firstly document alterations in pathway-specific DSB repair activities in primary cells originating from familial as well as sporadic breast cancer. In particular, we identified increases in the mutagenic nonhomologous end joining and single-strand annealing mechanisms in sporadic breast cancers with wild-type BRCA1 and BRCA2, and, thus, similar phenotypes to tumors with mutant alleles of BRCA1 and BRCA2. This suggests that detection of error-prone DSB repair activities may be useful to extend the limits of genotypic characterization of high-risk susceptibility genes. This method may, therefore, serve as a marker for breast cancer risk assessment and, even more importantly, for the prediction of responsiveness to targeted therapies such as to inhibitors of poly(ADP-ribose)polymerase (PARP1).

Frequent loss of Dab2 protein and infrequent promoter hypermethylation in breast cancer.

Disabled-2 (Dab2), a putative tumor suppressor protein, is lost in 80-90% ovarian tumors and ovarian/breast cancer cell lines. The clinical significance of Dab2 protein in breast cancer remains yet unknown. Immunohistochemical analysis of Dab2 protein showed no detectable expression in 67/91 (74%) breast tumors, while all 10 normal tissues showed presence of Dab2 protein. We hypothesized that epigenetic silencing of Dab2 may account for loss of protein in breast cancer. Methylation of Dab2 exon 1, a putative promoter, was analyzed in six breast cancer cell lines and in 54 primary breast tumors by methylation specific PCR. Methylation was observed in MDA-MB-231 and MDA-MB-157 cells and in 6 of 54 (11%) primary breast tumors that also showed loss of Dab2 protein. Expression of Dab2 transcripts was detected in all cell lines except MDA-MB-157. However, none of these six cell lines showed detectable levels of Dab2 protein by western blotting, while non-malignant mammary epithelial cell line MCF 10A showed Dab2 protein expression. To our knowledge this is the first report showing low frequency of Dab2 (putative) promoter methylation (11%) in primary breast tumors. Frequent loss of Dab2 protein (74%) suggest that hypermethylation of Dab2 promoter may only be one of the mechanisms accounting for its loss in breast cancer. Further, in silico analysis of Dab2 3'-UTR revealed existence of miRNA complimentary to this region of the gene, suggesting microRNA mediated targeting of Dab2 mRNA might account for loss of the protein in breast cancer.