Kondratov O. Inactivation of potential tumor suppressor genes ITGA9, GLCE, LRRC3B and WNT7A in breast and renal carcinomas

The thesis work is devoted to study of the potential tumor suppressor genes - LRRC3B and WNT7A in clear cell renal cell carcinomas (RCC's), ITGA9 and GLCE in breast carcinomas. In previous work we have found that ITGA9, LRRC3B and WNT7A associated loci are subjected to genetic/epigenetic alterations in set of RCC's using NotI-microarray analysis. The NotI-microarray technology allows to search for genetic (deletion, amplification) and epigenetic (DNA methylation) alterations of genes/loci simultaneously, due to the fact that NotI sites are frequently associated with promoter regions of genes. Integrin alpha9 (ITGA9) is one of the less studied integrin subunits that facilitates accelerated cell migration and regulates diverse biological functions such as angiogenesis, lymphangiogenesis, cancer cell proliferation and migration. In this work, integrin alpha9 expression and its epigenetic regulation in primary breast tumors and breast cancer cell line MCF7 were studied. In breast cancer, ITGA9 expression was downregulated or lost in 44% of tumors while another 45% of tumors showed normal or increased ITGA9 expression level (11% of tumors demonstrated controversial results in multiplex and quantitative RT-PCR). Methylation of ITGA9 CpG-island located in the first intron of the gene was shown in 90% of the breast tumors with the decreased ITGA9 expression while no methylation at 5'-untranslated region of ITGA9 was observed. 5-aza-2'- deoxycytidine treatment restored integrin alpha9 expression in ITGA9-negative MCF7 breast carcinoma cells, Trichostatin A treatment did not influenced it but a combined treatment of the cells with 5-aza-2'- deoxycytidine /Trichostatin A doubled the ITGA9 activation. The obtained results suggest CpG methylation as a major mechanism of integrin alpha9 inactivation in breast cancer. D-glucuronyl C5-epimerase (GLCE) is a potential tumor-suppressor gene involved in heparan sulfate biosynthesis. GLCE expression is significantly decreased in breast tumors; however, the underlying molecular mechanisms remain unclear. This study examined the possible epigenetic mechanisms for GLCE inactivation in breast cancer. Very little methylation of the GLCE promoter region was detected in breast tumors in vivo and in breast cancer cells (MСF7 and T47D) in vitro and GLCE expression in breast cancer cells was not altered by 5-aza-2'- deoxycytidine treatment, suggesting that promoter methylation is not involved in regulating GLCE expression. And histone deacetylation was shown as potential mechanism of GLCE repression in breast cancer cell lines. The leucine rich repeat containing 3B (LRRC3B) gene is a putative tumor suppressor located on human chromosome 3 in the 3p24 region. LRRC3B is frequently altered in colon and gastric cancers and also in leukaemias. In this study we investigated the promoter region methylation as a possible mechanism of LRRC3B gene inactivation in clear cell renal cell carcinomas. We found that the LRRC3B gene promoter was methylated in 43% of clear cell renal carcinoma samples. However, no correlation between DNA methylation and LRRC3B expression was found. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. WNT7A (wingless-type MMTV integration site family, member 7A) is a known tumor suppressor gene of non-small cell lung carcinomas (NSCLC) and is frequently inactivated due to CpG-island hypermethylation in human cancers. The members of WNT family are involved in cell signaling and play crucial roles in cancer development. In the present work hypermethylation of the WNT7A gene was detected in 66% (29/44) of analyzed clear cell RCC's using methyl-specific PCR (MSP). Moreover, bisulfite sequencing confirmed intensive hypermethylation of the 5'-CpG island of the WNT7A gene. Methylation analysis revealed a statistically significant correlation exists between the WNT7A hypermethylation status and some of the clinical-pathological characteristics. The WNT7A gene is more frequently methylated in tumors at advanced stages (III-IV) and high nuclear grades (3-4) than in tumors at early stages (I-II) and low nuclear grades (1-2) of clear cell RCC's. To investigate the genetic alterations of the WNT7A gene locus, the microsatellite markers analysis of this region on chromosome 3p25 was also performed. The WNT7A gene is located between markers D3S2385, D3S2403 and D3S1252. A loss of heterozy- gosity at least with one marker was found in 85% (23/27) of informative cases. Moreover, for the first time we have shown a loss of the microsatellite markers D3S2385 and D3S2403 in cancer. Additionally, tumor suppressor properties of the WNT7A gene in RCC cell lines were investigated. For this purpose, the WNT7A gene was re-expressed in RCC cell lines A498 and KRC/Y. This led to a significant reduction in colony number in both cell lines. Thus it has been performed analysis of epigenetic and genetic alterations in potential tumor suppressor genes ITGA9, GLCE for breast cancer and LRRC3B, WNT7A for renal cancer. It was shown that the genetic and/or epigenetic influenced on expression of ITGA9, GLCE and WNT7A. Additionally, tumor suppressor functions were revealed for LRRC3B and WNT7A in renal cancer.