The thesis is dedicated to the discovery of genetic, epigenetic and gene expression changes in various types of prostate tumors.
Prostate cancer is the most frequently diagnosed male cancer in Western countries, taking the third place in morbidity and the second place in mortality in Ukraine in 2014 year.
Investigation of genetic and epigenetic changes, as well as gene expression in different types of prostate tumors, will enable not only to expand existing knowledge of the mechanisms of development and progression of prostate cancer. It will also help to identify potential prostate cancer-associated genes, which may include both proto-oncogenes and tumor suppressor genes that could help with stratification of various types of prostate tumors.
The study of genetic and epigenetic changes was performed by hybridization on NotI-microarrays in 33 clinical specimens of prostate tumors, among which there were 15 prostate adenoma and 18 adenocarcinoma samples. Four samples of conventionally normal prostate gland tissue were used as controls. Genetic/epigenetic alterations were found in the 88 genes / loci of the human chromosome three from the 180 ones.
After statistical analysis we obtained a difference in the deletion / methylation frequencies between the prostate adenoma and the various stages of prostate adenocarcinoma samples for 14 genes / loci (LOC440944 / SETD5, OSBPL10 / ZNF860, CLCN2, PRSS42 / MYL3, VHL, BBX, LMCD1, CMTM6, FAM19A4, CAND2, KY , GATA2, ALDH1L1, MAP4).
Validation of genetic and epigenetic changes found on microarrays was performed for the selected genes / loci by quantitative polymerase chain reaction and bisulfite sequencing, respectively. For qPCR we selected BHLHE40, BCL6, ITGA9 genes. Investigation was performed on 11 samples of prostate adenocarcinoma. It was found that the expression levels of all three genes were decreased by an average of 3 times in the vast majority of samples of adenocarcinoma compared to the CNT.
To verify the possible methylation detected by the Not-I hybridization we selected FGF12, GATA2, LMCD1, TESSP2 genes. Experiment was conducted on 12 samples of the prostate adenocarcinoma. Methylation of the NotI site of FGF12 gene was found in six out of 11 studied clones (55%) and was associated with methylation of the sequenced region. Other 11 sequenced samples showed from 40 to 80% of NotI site methylation. A high degree of methylation of samples (30-70%) was also found for GATA2 and LMCD1 genes. While for TESSP2, which was used as a control, almost no methylation (less than 10%) was found.
Next, we investigated levels of expression of 143 cancer-associated genes in the human prostate adenocarcinoma cell lines. Fifty differentially expressed genes were identified between the LNCaP and DU145 and PC3 cell lines, which are potentially associated with the invasive and metastatic type of prostate tumors. Among these genes we selected GLCE gene for detailed research due to the literature data. After transfection of the human prostate cancer cell lines LNCaP and PC3 with vector containing GLCE we have shown that expression of GLCE protein mostly effects on expression of the angiogenesis, invasiveness and metastasis genes in LNCaP and PC3 cell lines.
Due to the complexity of the signaling processes in cancer cells our data obtained on the cell ines requires testing on clinical samples. Therefore, seven genes (EFNA5, TAGLN, EPDR1, FOS, PLAU, TGFB1, IL1B) were selected for further validation. After analysis of the relative expression levels, a statistically significant difference was found between the groups of prostate adenoma, adenocarcinoma and conventionally normal tissues for FOS, EFNA5, IL1B and TGFB1 genes. For EFNA5, FOS and IL1B genes, the relative expression level in the adenocarcinoma group was elevated in comparison with the prostate adenoma group. For the TGFB1 gene, a decrease in the relative expression level in the prostate adenoma group was observed compared to the conventionally normal tissues group. In addition, for the PLAU gene, a significant reduction of the relative expression level in the adenocarcinoma group was shown compared with conventionally normal tissues.
Detected genes with genetic / epigenetic and altered expression levels could be used for further studies on the creation of biomarker panels for the detection and stratification of various types of prostate tumors.
