Date of Award

Fall 2018

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

David H. Townson

Second Advisor

Feixia Chu

Third Advisor

Vernon Reinhold

Abstract

Hyper-O-GlcNAcylation of proteins is a subsequent artifact of metabolic disorder and is indicative of many cancers, including cancers of the female reproductive tract. While the incidence of most cancer types has been declining in the U.S., endometrial and cervical cancer remain among the most common cancers diagnosed in women. Diabetic women have a 2-3 fold increased risk of developing endometrial cancer, and tend to have more aggressive cases of cervical cancer, however, the molecular aspects of these risks are not fully understood. This study investigated the alteration of cellular O-GlcNAcylation of proteins as the potential mechanistic connection between diabetes and tumorigenicity in cancers of the female reproductive tract. The cervical cancer cell line (SiHa) and the endometrial cancer cell line (Ishikawa) were utilized to study the effect of dysregulation of O-GlcNAcylation on the proliferation, migration, invasion, and related molecular mechanisms. In cervical cancer, O GlcNAcylation was found to be an important regulator of tumorigenicity. Overall, inhibition of O-GlcNAcylation (via the inhibitor, OSMI-1) in SiHa cells impaired cell proliferation (p<0.01) and invasion (p<0.01) yet did not affect cell cycle progression. These effects occurred concomitantly with an alteration of cellular morphology, principally the disruption/decline of K8/18 and β-actin filament expression. The results suggest O-GlcNAcylation regulates several aspects of tumorigenesis in cervical cancer cells, and cytoskeletal proteins are among the targets. Similarly, in endometrial cancer cells, hyper-O-GlcNAcylation (via 1μM Thiamet-G/ThmG or 25mM Glucose) enhanced the expression of EMT-associated genes (WNT5B and FOXC2), and the E-Cadherin suppressor, Snail. Reorganization of actin filaments into stress filaments, consistent with EMT, was also noted in ThmG-treated cells. Interestingly, Hypo-O GlcNAcylation (via 50 μM OSMI-1) also upregulated WNT5B, inferring that any disruption to O-GlcNAc cycling impacts EMT. However, Hypo-O-GlcNAcylation reduced cellular proliferation/migration and the expression of the pro-EMT genes (AHNAK, TGFB2, FGFBP1, CALD1, TFPI2). Finally, Ishikawa cells were used to investigate the effect hyper-O GlcNAcylation on the efficacy of progesterone (P4) in therapy for endometrial cancer proliferation and invasion. Ishikawa cells were exposed to ThmG, to induce hyper-O GlcNAcylation, and 100nM P4. P4 alone, significantly decreased cell proliferation, however, the addition of ThmG, and subsequent hyper-O-GlcNAcylation, negated this affect, returning the cells to control level proliferation (p<0.05). A similar pattern was noted in Matrigel invasion assays, where Hyper-O-GlcNAcylation augmented invasion compared to P4 treatment alone, both with and without progesterone treatment (p<0.05). Progesterone treatment has been shown to induce the expression of p21 and p27, reducing cell growth. In this study, P4 maintained p21 expression and increased p27 expression, however, ThmG decreased p27 expression and the expression of endogenous progesterone receptor B (PR B) despite P4 treatment. These results suggest that hyper-O-GlcNAcylation, common in obese and diabetic patients, may promote tumorigenicity in female cancers and could impair the efficacy of progesterone treatment. O GlcNAcylation has the potential to serve as a biomarker for early diagnosis and could predict treatment success. O-GlcNAc cycling enzyme inhibitors could prove to be useful tools for providers when treating cancer patients with metabolic disorders.

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