Ekspresija proteina SFRP1, SFRP3, DVL1, DVL2 i DVL3 signalnog puta Wnt u tkivu seroznoga karcinoma jajnika

doctoral thesis

• Title (english): Expression of Wnt signaling pathway proteins SFRP1, SFRP3, DVL1, DVL2 and DVL3 in serous ovarian cancer
• Author: Vedran Kardum https://orcid.org/0000-0002-7397-344X
• Mentor: Ljiljana Šerman (mentor)
• Committee member: Slavko Orešković (predsjednik povjerenstva)
• Committee member: Anita Škrtić (član povjerenstva)
• Committee member: Sonja Levanat (član povjerenstva)
• Granter: University of Zagreb; School of Medicine; Zagreb
• Defense date and country: 2021-01-20, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Basic Medical Sciences; Cytology, Histology and Embryology
• Universal decimal classification (UDC): 612 - Physiology

Abstract

Uvod: Maligni epitelni tumori jajnika najsmrtonosniji su ginekološki tumori koji se, u najvećem broju slučajeva, dijagnosticiraju u uznapredovalom stadiju bolesti s lošom prognoznom preživljenja. Smrtnost od raka jajnika u razvijenom svijetu je u laganom padu, međutim taj pad je znatno sporiji od pada njegove incidencije. Još uvijek ne postoji učinkovita metoda primarnoga probira bolesnica, kao ni osjetljiva klinička ili laboratorijska dijagnostika ranoga stadija bolesti. U ovoj disertaciji obrađivane su komponente signalnog puta Wnt; SFRP1, SFRP3, DVL1, DVL2 i DVL3. Aberantna aktivacija ovog signalnog puta dokazana je u brojnim tumorima. -----
Materijal i metode: Provedeno je retrospektivno istraživanje na arhiviranim uzorcima seroznih karcinoma jajnika (52) prikupljenih u KB Merkur između 2000. i 2012. godine. Ekspresija proteina u tumorskom tkivu uspoređivana je s ekspresijom proteina u urednom tkivu jajnika (9) i/ili jajovoda (5) prikupljenih prilikom operativnih zahvata zbog neke druge nemaligne ginekološke bolesti. Provedena je imunohistokemijska analiza ekspresije proteina SFRP1, SFRP3, DVL1, DVL2 i DVL3 signalnog puta Wnt, te je određena njihova međusobna korelacija i korelacija s kliničkim parametrima bolesti. DNA je izolirana iz zdravog i tumorskog tkiva, nakon čega je analizirana metilacija DNA u promotorima gena SFRP1 uz pomoć PCR-a ovisnog o metilaciji. Ekspresija proteina DVL1, DVL2 i DVL3 analizirana je i na staničnim linijama seroznog karcinoma jajnika visokog stupnja malignosti i na normalnim imortaliziranim epitelnim stanicama jajovoda uz pomoć Western blot-a. -----
Rezultati: Disertacijom smo dokazali statistički značajno manju ekspresiju proteina SFRP1 i SFRP3 u tumorskom tkivu. Uzrok smanjene ekspresije proteina SFRP1 u HGSC skupini tumora je metilacija promotora gena SFRP1. Ekspresija proteina DVL1 i DVL3 statistički je značajno veća u LGSC skupini tumora nego u kontrolnim zdravim uzorcima jajnika i jajovoda, dok je ekspresija proteina DVL3 statistički značajno veća u HGSC skupini tumora nego u kontrolnom zdravom tkivu jajnika. Ekspresija proteina DVL2 i DVL3 je bila viša u staničnim linijama karcinoma jajnika u usporedbi sa staničnom linijom FNE1 kontrole. mRNA za DVL1, DVL2 i DVL3 je značajno veća u OVASHO staničnoj liniji. -----
Zaključak: Dokazali smo da je ekspresija proteina SFRP1 smanjena u većini HGSC tumora te da je smanjena ekspresija ovog proteina posljedica metilacije promotora gena SFRP1. Protein SFRP1 i SFRP3 dokazano djeluju kao tumor supresori u seroznom karcinomu jajnika visokog i niskog stupnja malignosti. Naši rezultati također upućuju i na aktivnu ulogu proteina iz obitelji Dishevelled u seroznim karcinomima jajnika.

Abstract (english)

Introduction: Ovarian cancer is the most lethal gynaecologic malignancy usually diagnosed in the advanced stage with a poor prognosis. The mortality rate is slightly declining but much slower than its incidence. We still do not have an effective primary screening method nor is there a sensitive clinical or laboratory test for detecting ovarian cancer in its early stage. In this study, we explored the components of the Wnt signalling pathway, SFRP1, SFRP3, DVL1, DVL2 and DVL3. Their aberrant activation has been proven in many other tumors. -----
Materials and methods: We performed a retrospective study on archived samples of serous ovarian cancer (52) collected at the University hospital Merkur between 2000 and 2012. Protein expression in tumor specimen was compared with protein expression in normal ovarian tissue (9) and/or fallopian tube (5) collected during surgery for other non-malignant gynaecological diseases. We explored SFRP1, SFRP3, DVL1, DVL2 and DVL3 protein expression using immunohistochemistry, their mutual correlation and correlation with clinical parameters of the disease. DNA was isolated from healthy and tumor tissue and SFRP1 gene methylation was analysed by methylation-specific PCR. The expression of DVL1, DVL2 and DVL3 proteins was analysed both on serous ovarian carcinoma cell lines as well as on TERT-immortalised epithelial fallopian tube using Western blot. -----
Results: We statistically demonstrated lower protein expression of SFRP1 and SFRP3 in tumor tissue. Loss of SFRP1 protein expression was caused by the SFRP1 promoter hypermethilation in a subset of HGSC. DVL1 and DVL3 protein expression was statistically higher in LGSC compared to normal ovaries and fallopian tubes, while DVL3 was statistically higher in HGSC compared to normal ovaries. DVL2 and DVL3 protein expression was higher in HGSC cell lines when compared with normal control cell line FNE1. DVL1, DVL2 and DVL3 mRNA expression was significantly increased only in OVASHO cell line. -----
Conclusion: We have shown that the SFRP1 protein was silenced in most HGSC tumors and the silencing was due to methylation of SFRP1 gene promoter. SFRP1 and SFRP3 protein have been shown to act as tumor suppressors in both, high and low grade, serous ovarian carcinoma. Our data show an active involvement of Dishevelled family proteins in serous ovarian carcinomas.

• Keywords: Žena; Jajove cijevi; Razbarušeni proteini; Metilacija; Karcinom; epitel jajnika; Novotvorine jajnika; SFRP1 protein; čovjek; Membranski proteini; Stanična linija; DNK; RNA; Glasnik
• Keywords (english): Female; Fallopian Tubes; Dishevelled Proteins; Methylation; Carcinoma; Ovarian Epithelial; Ovarian Neoplasms; SFRP1 protein; human; Membrane Proteins; Cell Line; DNA; RNA; Messenger
• Language: croatian
• URN:NBN: urn:nbn:hr:105:561754
• Study programme: Title: Biomedicine and Health Sciences; Study programme type: university; Study level: postgraduate; Academic / professional title: doktor/doktorica znanosti, područje biomedicine i zdravstvo (doktor/doktorica znanosti, područje biomedicine i zdravstvo)
• Type of resource: Text
• Extent: 100 str.
• File origin: Born digital
• Access conditions: Open access
• Created on: 2021-03-25 10:50:54