Epigenetic changes and PTCH1, SHh and IHh protein expression in serous ovarian carcinomas

Serozni karcinomi jajnika najčešći su oblik epitelnih tumora jajnika. Uglavnom se dijagnosticiraju u uznapredovalom stadiju bolesti i imaju visoku stopu smrtnosti. Kod raka jajnika primijećena je poremećena aktivacija više signalnih puteva među kojima je i signalni put Hedgehog (Hh). Uloga signalnog puta Hh u raku jajnika, kao i u pojedinim histološkim podtipovima raka jajnika, nije dovoljno istražena. U ovoj disertaciji istražili smo ulogu proteina PTCH1, SHh i IHh, kao i promjene u metilacijskom statusu promotora istoimenih gena, u seroznim karcinomima jajnika. Istraživanje je provedeno na uzorcima seroznih karcinoma jajnika niskog (LGSC) i visokog (HGSC) stupnja malignosti, dok su uzorci zdravih jajnika i jajovoda služili kao kontrole. U istraživanju su korištene i stanične linije HGSC-a, OVCAR5, OVCAR8 i OVSHAO, i kontrolna stanična linija normalnih epitelnih stanica jajovoda, FNE1. Ekspresija proteina PTCH1, SHh i IHh u tkivnim uzorcima analizirana je metodom imunohistokemije, dok je u staničnim linijama ispitana uz pomoć imunofluorescencije i Western blota. Metilacijski obrazac DNA u promotorima gena PTCH1, SHh i IHh analizirali smo koristeći PCR ovisan o metilaciji (MSP). Ekspresija proteina PTCH1, SHh i IHh bila je značajno veća u karcinomima HGSC i LGSC u odnosu na kontrolna tkiva, kao i u karcinomskim u odnosu na kontrolnu staničnu liniju. U tumorskom tkivu i tumorskim stanicama u kulturi bila je izražena nuklearna ekspresija proteina PTCH1 što navodi na zaključak da bi navedeni protein mogao igrati aktivnu, tumor-promotorsku ulogu u jezgri malignih stanica seroznih karcinoma jajnika. U staničnim linijama detektirani su fragmenti proteina PTCH1 različite molekularne mase što upućuje na mogućnost proteolitičkog cijepanja ovog proteina. Autokrina signalizacija ovisna o SHh-ligandu utvrđena je u karcinomima HGSC i LGSC, dok su autokrina i parakrina signalizacija ovisna o IHh-ligandu uočene u karcinomima HGSC. Metilacija DNA u promotorima gena PTCH1, SHh i IHh nije bila povezana s razinom ekspresije ovih proteina što navodi na zaključak da ovaj mehanizam nije uključen u patogenezu seroznih karcinoma jajnika. Dobiveni rezultati upućuju na aktivnu ulogu proteina PTCH1, SHh i IHh u patogenezi seroznih karcinoma jajnika te doprinose boljem razumijevanju molekularne patogeneze ovih karcinoma.Serous ovarian carcinomas are the most common type of epithelial ovarian cancers. They are mostly diagnosed in the advanced stage of the disease and have a high mortality rate. In ovarian cancer, aberrant activation of several signaling pathways, including the Hh signaling pathway, has been observed. The role of Hh signaling pathway in ovarian cancer, as well as in certain histological subtypes of ovarian cancer, has not been sufficiently investigated. In this dissertation, we investigated the role of PTCH1, SHh and IHh proteins, as well as changes in the promoter methylation status of the corresponding genes, in serous ovarian carcinomas. Low- (LGSC) and high-grade (HGSC) serous ovarian carcinoma tissue samples were used in this study, while normal ovarian and fallopian tube tissue samples served as controls. HGSC cell lines, OVCAR5, OVCAR8 and OVSHAO, and control cell line, normal fallopian tube non-ciliated epithelium cell line FNE1, were also used in the study. PTCH1, SHh and IHh protein expression levels were analyzed using immunohistochemistry in tissue samples, and by immunofluorescence and Western blot in cell lines. DNA methylation pattern of PTCH1, SHh and IHh genes were analyzed by methylation-specific PCR (MSP). PTCH1, SHh and IHh protein expression levels were significantly higher in HGSCs and LGSCs compared to control tissues, as well as in cancer cell lines compared to control cell line. Nuclear expression of the PTCH1 protein in tumor tissue and cultured tumor cells suggests that this protein could play an active, tumor-promoter role in the nuclei of serous ovarian cancer cells. PTCH1 protein fragments of different molecular weight were detected in the cell lines, indicating possible proteolytic cleavage of this protein. Autocrine SHh signaling was found in HGSCs and LGSCs, while autocrine and paracrine IHh signaling were observed in HGSCs. DNA methylation of PTCH1, SHh, and IHh gene promoters were not in line with the expression levels of these proteins, leading to the conclusion that this mechanism is not involved in the pathogenesis of serous ovarian carcinoma. Our results indicate that PTCH1, SHh and IHh proteins play an active role in pathogenesis of serous ovarian carcinoma, and contribute to a better understanding of the molecular pathogenesis of these cancers