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Publication Date

2013-08-29

Availability

UM campus only

Embargo Period

2013-08-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Cancer Biology (Medicine)

Date of Defense

2013-08-26

First Committee Member

Vinata B. Lokeshwar

Second Committee Member

Priyamvada Rai

Third Committee Member

Murugesan Manoharan

Fourth Committee Member

Enrique A. Mesri

Fifth Committee Member

Xiangxi Xu

Abstract

Molecular determinants of genitourinary metastasis can potentially serve as accurate diagnostic and prognostic markers, and can also be targeted for therapy. Prostate cancer (PCa) is one of the leading cancers in men in the United States. Approximately 30% of the men with PCa eventually experience hormone refractory disease, which is the main cause of morbidity and mortality associated with PCa. Bladder cancer (BCa) is a common cancer of the urinary tract. Due to frequent recurrence, multi-focality and heterogeneity in tumor progression the clinical management of bladder cancer is the costliest among all cancer patients. When renal cell carcinoma (RCC) is initially diagnosed, ~25% of patients will present with metastatic disease, further, nearly a third of those treated with nephrectomy will eventually experience recurrence. Metastatic-RCC is insidious, as despite several FDA-approved treatments, the five-year survival of patients is less than 10%. Hyaluronic acid (HA) family members are associated with bladder, prostate and kidney cancer progression. HA, a non-sulfated glycosaminoglycan that is synthesized by HA-synthases: HAS1, HAS2 and HAS3 (Hyaluronidase (HAase) degrades HA into small angiogenic fragments. HYAL-1 is the major tumor-derived HAase. Both HA and angiogenic HA fragments induce intracellular signaling by binding to HA receptors, CD44 and RHAMM. Using genitourinary cancer cells as model systems our laboratory has established that the tumor-associated HA-HYAL-1 system promotes tumor growth, angiogenesis, and progression. Prior work from our laboratory has demonstrated that when HA or angiogenic HA fragments generated by degradation of tumor-associated HA by HYAL-1, bind to HA receptors CD44 or RHAMM, it induces HA signaling that ultimately stimulates tumor cell growth, invasion, motility and angiogenesis. Therefore, we hypothesized that targeting the HA-HAase system with HA & HAase inhibitors would inhibit tumor formation, growth and metastasis. In our laboratory we have established sulfated hyaluronic acid (sHA) as a specific inhibitor of HYAL-1 and 4-methylumbelliferone is known to be an inhibitor of HA synthesis. In my first project, I demonstrated that sHA inhibited proliferation, motility, and invasion of PCa cells that expressed HYAL-1 Furthermore, sHA inhibited Akt signaling and showed potent antitumor activity in xenografts, without significant toxicity. Addition of angiogenic HA fragments, as well as, overexpression of a constitutively active form of Akt (myr-Akt) inhibited sHA effects. Contrarily, downregulation of HA receptors, mimicked the effects of sHA. Therefore, the mechanism of the potent antitumor activity of sHA involves the inhibition of the generation of angiogenic HA fragments and subsequently inhibition of HA signaling. My second project was built on prior published results from the laboratory, which showed that 4-MU has potent anti-tumor activity in PCa models. To determine whether 4-MU has chemo-preventive, anti-metastatic properties, and the mechanism of such activities, I utilized three different pre-clinical models of PCa, and in vitro culture systems. In the first model, I utilized the TRAMP (Transgenic Adenocarcinoma of the Mouse Prostate) model to examine the chemo-preventive, anti-tumor and anti-metastatic activities of 4-MU in a Stage-Specific manner. The stage specific design showed that regardless of whether 4-MU treatment was started as late as 22-weeks, when invasive carcinoma is present in the prostate, 4-MU was able to block, and reverse tumor progression and completely inhibited metastasis to soft tissues, as it is observed in this model. I established an Intracardiac bone metastasis model using PC3 –ML cells, 4-MU treatment completely prevented and inhibited metastasis formation in bony tissues (tibia, spinal cord, mandible, etc). It is noteworthy that PC3-ML cells are Pten null and Pten is deleted at a frequency of 70% in PCa. In the subcutaneous xenograft model using DU145 cells, which are androgen receptor negative and Pten positive, 4-MU inhibited tumor growth. Further, although 4-MU demonstrated potent anti-tumor and anti-metastatic activities, there was no weight loss and no serum/organ toxicities observed. In mechanistic studies, I was able to link the potent anti-tumor and anti-metastatic activities of 4-MU to downregulation of EMT determinants β-Catenin, Zeb2, and E-Cadherin by inhibiting HA-HA receptor mediated activation of Akt signaling. In both the TRAMP model and PCa cell lines (PC3-ML and DU145), 4-MU downregulated β-Catenin, Zeb2, and Akt signaling, while upregulating E-Cadherin expression. Further, downregulation of HA-receptors (CD44 and RHAMM) mimicked the effects of 4-MU on Akt signaling and EMT determinants, whereas, addition of HA or overexpression of myr-Akt, attenuated effects of 4-MU. Taking the targeting of the HA-HAase system with 4-MU a step further, I investigated the synergistic effects of 4-MU and Sorafenib (SF) combination for controlling RCC growth. In cell culture and xenograft studies, I found that at 10-fold lower concentration, than when used as single agents, 4-MU+SF inhibited RCC cell growth, invasion, motility and endothelial functions. In fact, the 4-MU+SF blocked all three angiogenic signaling pathways. The combination was effective at concentrations at which 4-MU and SF were individually ineffective. Furthermore, the combination completely inhibited tumor growth in a SF-resistant xenograft without any toxicity. I have also developed a spontaneously metastatic RCC xenograft model and the research will continue to determine the efficacy of the combination to prevent/inhibit metastasis. Finally, I examined the utilization of molecular determinants of cancer progression as diagnostic and prognostic predictors. The prognostic benefit of C-X-C chemokine receptors were explored in the context of the genitourinary malignancies, bladder cancer. Via coimmunoprecipitation, it was shown that CXCR7 co-localized and formed a functional complex with epidermal growth factor receptor (EGFR). This was further confirmed by the establishment of In-Cell Coimmunoprecipitation technique that allowed for the visualization of the co-localization in BCa cells. CXCR7 promotes BCa cell proliferation and motility through the EGFR and Akt signaling. This was mechanistically proven, by knockdown and overexpression of CXCR7. CXCR7 expression was elevated in BCa tissues and exfoliated cells and is associated with high-grade. In a multivariate analysis, CXCR7 transcript levels and protein levels independently associated with metastasis and disease specific mortality. This research shows potentially insight into the tumor-associated HA–HAase system and a preclinical proof-of-concept of the safety and efficacy of targeting the system with an HAase inhibitor (sHA) to control PCa growth and progression. The research plausibly also offers mechanistic insights regarding the potent anti-tumor and anti-metastatic activities of both 4-MU and sHA. The research efforts on biomarkers show that CXCR7 is potentially an accurate biomarker for detecting BCa and for predicting its recurrence and metastasis. Furthermore, CXCR7 has biomarker potential because it promotes BCa growth, invasion and motility by activating EGFR-signaling. It is my hope that the findings of the research can aid in bringing sHA, 4-MU or other agents that target the tumor-associated HA-HAase system, to clinical trials and warrant multi-center studies on testing the biomarker potential of CXCR7.

Keywords

Prostate Cancer; Bladder Cancer; Kidney Cancer; TRAMP; Hyaluronic Acid; 4-MU

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