Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Clinton Chichester

Abstract

Epithelial ovarian cancer (EOC) is the most common gynecologic malignancy worldwide. EOC has a notably poor prognosis, owing to the fact that patients are frequently diagnosed at a late stage after the disease has significantly progressed. While many patients typically respond well to frontline platinum-based chemotherapy, the tumor becomes chemoresistant when a recurrence follows within five years. Therefore, there is an urgent need for the discovery of non-invasive early detection biomarkers and novel targeted therapies.

Human Epididymis Protein 4 (HE4) is a secretory protein that is encoded by the gene whey acidic protein (WAP)-four disulfide core domain protein 2. The WAP domain family is a conserved motif that is inherit of many antiproteases. HE4 was initially found to be a component of the innate immune defenses of multiple epithelia and to function in epithelial host defense, through the promotion of mucosal surfaces first line of defense. HE4 is highly overexpressed in EOC and has been identified as a novel clinical biomarker. Clinical and translational studies have established HE4 as a contributor to tumorigenesis and chemoresistance in EOC. However, the exact processes in which HE4 promotes pathogenesis is unclear. The driving hypothesis of this thesis is that HE4 represents a novel targeted therapy due to its established role EOC tumorigenesis and suggested function in innate immunity. This evidence underlies the goals of this dissertation which are to elucidate the precise mechanisms of HE4’s contribution in EOC pathogenesis and establish HE4’s role in tumor immune invasion. It is hoped that results from this investigation will ultimately aide in the development of a novel targeted therapy against HE4 that can modulate tumor pathogenesis as well as the tumor immune response.

In manuscript I, subtractive hybridization revealed that HE4 significantly suppresses expression of osteopontin (OPN) in peripheral blood mononuclear cells (PBMCs) which ultimately compromised their cytotoxicity against ovarian cancer cells. Ovarian cancer cells exhibited enhanced proliferation in conditioned media from HE4-exposed PBMCs and this effect was attenuated by the addition of recombinant OPN and OPN -inducible cytokines (IL-12 and IFN-y). In addition, ovarian cancer cells and PBMCs with HE4 downregulation via short hairpin RNA (shRNA) were found to be increasingly more susceptible to cell death.

In manuscript II, subtractive hybridization identified dual specificity phosphatase 6 (DUSP6) as the most upregulated gene upon treatment with recombinant HE4 in PBMCs. Flow cytometry revealed that recombinant HE4 significantly upregulated DUSP6 levels specifically in CD8+ (cytotoxic T cell) and CD56+ (NK cell) populations. Exposure of these cells to HE4 led to an increase in ERK ½ phosphorylation, which was subsequently decreased upon DUSP6 inhibition. These results show that DUSP6 suppression of CD8+ and CD56+ lymphocyte toxicity is strongly enhanced by HE4. In co-culture of PBMCs and ovarian cancer cells, DUSP6 inhibition attenuated the enhanced proliferation noted upon stimulation with HE4. The effect of DUSP6 inhibition was obliterated in CD8+ and CD56+ devoid PBMCs.

In manuscript III, the role of DUSP6 and its relationship to HE4 in EOC was further elucidated. Increased DUSP6 levels were observed in ovarian cancer cells overexpressing HE4. siRNA-mediated downregulation of both HE4 and DUSP6 revealed a corresponding decrease of either factor. Treatment with an allosteric DUSP6 inhibitor in combination with chemotherapeutic agents produced synergistic effects on the reduction of cell viability. These effects correlated with alterations in expression of ERK pathway mediated genes. Finally, it was found that DUSP6 was significantly overexpressed in serous EOC patient tissue compared to normal adjacent tissue.

In manuscript IV it was determined from a small-scale proteomics study that 63 proteins were found to interact more strongly with HE4, in HE4 overexpressing clones compared to null vector control. The protein found to exhibit the highest interaction in the HE4 clones was Septin-2, a GTP binding protein. Immunohistochemical analysis of Septin-2 in EOC patient tissue revealed that levels were overexpressed in cancer compared to normal and benign controls. To identify Septin-2’s role in EOC, stable knockdown cell lines were constructed using the ovarian cancer cell line SKOV3. Septin-2 knockdown cells demonstrated a significantly lowered proliferation rate compared wild-type (WT) and Plasmid C control cells. To better define the role of Septin-2 in EOC, proteomics was employed. Pathway analysis showed an enrichment in autosphorylation, citric acid cycle, acetyl CoA/energy, and proteasomal/ubiquitin processes in Septin-2 knockout cells.

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