Date of Award

January 2018

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Elena S. Ratner

Abstract

TRANSFORMING GROWTH FACTOR beta (TGFb) DOWNREGULATES HOMOLOGOUS RECOMBINATION REPAIR GENES AND INCREASES POLY-(ADP-RIBOSE) POLYMERASE (PARP) INHIBITOR SENSITIVITY IN BREAST CANCER-ASSOCIATED GENE 2 (BRCA2)WILD-TYPE EPITHELIAL OVARIAN CANCER CELLS.

Mehida Alexandre, Z Ping Lin, Ruth Hanna, Elena S. Ratner. Department of Obstetrics and Gynecology, Division of Gynecologic Oncology. Yale University School of Medicine, New Haven, CT

Transforming growth factor (TGF) is a ubiquitous cytokine named for its ability to transform normal fibroblasts in culture. Recently, TGF has been linked to genomic instability in breast cancer and to increased susceptibility to Poly (ADP-Ribose) Polymerase (PARP) inhibition. Hereditary epithelial ovarian cancer (EOC) with deleterious BRCA mutations only accounts for 15% of all EOC but is heavily susceptible to synthetic lethality with PARP inhibition. With this in mind, we aim to render BRCA WT EOC susceptible to PARP inhibition by inducing “BRCAness” with TGF and to understand the mechanisms via which TGF causes genomic instability in late stage ovarian cancer.

BRCA2-mutated PEO1 and BRCA2 WT PEO4 EOC cells were derived from the same patient at first and second relapse, respectively, following platinum-based chemotherapy. Western blot analysis was performed to determine expression of mesenchymal markers and to measure expression of homologous recombination (HR) repair genes: BRCA1, BRCA2, and Rad51. Flow cytometry and luciferase assay were performed to measure HR activity and Non Homologous End Joining (NHEJ) activity, respectively with and without siRNA-mediated SMAD2 inhibition. Scratch wound assays were conducted to determine cells’ migratory ability. Clonogenic assay was done to determine cell survival in response to increasing concentrations of PARP inhibitor with or without addition of TGF.

TGF increases expression of mesenchymal markers and p-AKT in BRCA2-mutated EOC. TGF changes the morphology of BRCA2 WT EOC, making them fibroblast-like. TGF downregulates HR repair genes and decreases NHEJ activity as well as increases sensitivity of BRCA2 WT EOC to olaparib. siRNA silencing of SMAD2 inhibits the effect of TGF on HR activity.

The model of BRCA2-mutated PEO1 and WT PEO4 represents the progression of ovarian cancer from chemo-sensitive to resistant, respectively. TGF signaling promotes survival through EMT induction in BRCA2 mutated ovarian cancer. TGF/smad pathway activation creates genomic instability in chemoresistant BRCA2 WT which when exploited can lead to synthetic lethality with PARP inhibition. Identifying molecules or signaling pathways with similar effect as TGF is an important next step in designing targeted therapy for BRCA WT ovarian cancer patients.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 06/25/2100

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