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Stromal-epithelial interactions are important in both prostate development and cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. Several molecules found to be aberrantly expressed in cancer associated fibroblasts (CAFs) (including cyclin D1 [CD1], stromal derived factor 1 [SDF-1]) contribute to tumorigenesis and malignant transformation in xenograft experiments. These molecules can be regulated by a number of different factors, but are both putative estrogen regulated genes. In this study, we show that dysregulation of ERα expression in cancer associated stroma results in the differential regulation of estrogen responsive genes that are key factors in enhancing the invasive potential of the epithelial tumor. The cell cycle regulator CD1 and the estrogen receptor are known to interact and can induce estrogenic gene transcription. Cathepsin D (CathD) is an estrogen regulated aspartic endopeptidase, known to be involved in a number of physiological processes as well as in the regulation of apoptosis. In this study, we highlight CathD as a mediator of cancer associated stromal promotion of prostate tumorigenesis. An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue in comparison to benign prostate tissue. Stromal specific overexpression of CathD in benign prostate stromal cells induced malignancy in adjacent epithelium through increased TGFβ signaling and responsive gene expression. The proteolytic function of stromally-derived CathD is dependent on the activity of hydrogen-proton pump activity on the surface of prostate epithelial cell lines. The study presented here indicates that CathD is not only an important mediator of stroma-epithelial cross talk, but also an essential component in promotion of tumorigenesis in vivo, and Inhibition of ER signaling in the cancer associated stroma inhibits malignant transformation in the adjacent epithelium.

Major Histocompatibility Complex (MHC) Class I molecules present peptides to CD8⁺ T cells and are essential for most adaptive immune responses. The first described-spliced tapasin (“isoform 1”) plays a critical role in MHC-I antigen presentation by facilitating peptide loading onto MHC-I molecules in the endoplasmic reticulum (ER). This thesis examines the expression, localization and function of two novel, alternatively-spliced isoforms of human tapasin that lack exon 7 (“isoform 2”) or both exons 6 and 7 (“isoform 3”). Isoform 1 contains a di-lysine ER-retention motif; the two novel isoforms encode different carboxy (C) termini that lack this motif. It was hypothesized that isoforms 2 and 3 would function in MHC-I cross-presentation of exogenous antigens in non-ER compartments. Isoform 2, like isoform 1, was found to be mainly ER-localized; however, both these isoforms were also found to co-localize in smaller amounts with the trans Golgi network and endo/lysosomes by confocal microscopy. Isoform 3 lacks a transmembrane domain and was found to be secreted from cells as well as being found within the ER. All isoforms were widely expressed at the RNA level in many tissues and cell types; however, mature dendritic cells (DCs) expressed the highest levels of all three isoforms, consistent with the high cross-presenting abilities of DCs. Both isoform 1 and 2 stabilized the transporters associated with antigen processing (TAP) in murine tapasin-/- cells, but isoform 3 did not due to its missing transmembrane domain. Isoform 1 and 2 mediated very similar effects on endogenous MHC-I presentation of self and viral peptides, on surface MHC-I thermostability, and on MHC-I maturation rates. Isoform 3 was found to decrease loading of exogenous peptides onto MHC-I. None of the isoforms influenced cross-presentation of the soluble antigen ovalbumin in a mouse dendritic cell line. This thesis also examines the effect of antigen presentation machinery (APM) re-expression in MHC-I-deficient tumor cell lines, B16F10 and CMT.64, which are deficient in TAP and tapasin. Virally-driven TAP1 and Tapasin expression increased MHC-I expression in the tumor cell lines, augmented tumor cell immunogenicity, and decreased tumor growth in vivo due to increased tumor cell elimination by the immune system.

Cutaneous malignant melanoma is an aggressive form of skin cancer, characterized by strong chemoresistance and poor patient prognosis. The molecular mechanisms underlying its resistance to chemotherapy remain unclear but are speculated to involve dysregulation of apoptosis and reinforcement of survival signaling. In this work, we show that aberrant expression of two key proteins, PUMA and p-Akt, is associated with melanoma tumor progression and poor patient survival. We report that PUMA expression is reduced in melanoma tumors compared to dysplastic nevi, while p-Akt expression is elevated in melanoma tissue compared to dysplastic nevi. We propose a two-pronged therapeutic strategy of (1) boosting PUMA expression and (2) inhibiting Akt phosphorylation. We demonstrate that exogenous overexpression of PUMA, via adenoviral-mediated gene expression (ad-PUMA), forces melanoma cells to undergo rapid mitochondrial-mediated apoptosis in vitro. We also report that a small molecule Akt inhibitor, API-2, greatly inhibits melanoma cell growth in vitro. Using a SCID mouse melanoma xenograft model, we show that combination treatment of ad-PUMA and API-2 dramatically suppresses tumor growth in an additive manner, leading to over 80% growth inhibition compared to controls. We also investigate the role of NF-κB overexpression in melanoma. Our lab previously reported that expression of the p50 subunit of NF-κB, in particular, correlates with melanoma progression and poor patient survival. Here, we use cDNA microarray analysis to show that p50 overexpression upregulates IL-6 in melanoma cells. We further demonstrate that p50-mediated IL-6 expression stimulates the growth of endothelial cells in vitro and promotes angiogenesis in vivo. This work supports the hypothesis that melanoma cells exploit multiple mechanisms to sustain a survival advantage, including: 1) suppression of apoptosis (via PUMA down-regulation), 2) increased activation of survival pathways (via increased p-Akt), and 3) upregulation of pro-angiogenic factors (via p50-mediated IL-6 induction). This work suggests that the specific targeting of one or more key mediators of these processes may be an effective therapeutic strategy for treating malignant melanoma.

Introduction: Radiotherapy utilization rates for cancer vary widely internationally. It has previously been suggested that approximately 50% of all cancer patients should receive radiation. However, this estimate was not evidence-based. The aim of this study was to estimate the ideal proportion of new cases of cancer that should receive radiotherapy at least once during the course of their illness based on the best available evidence. Materials and Methods: An optimal radiotherapy utilization tree was constructed for each cancer based upon indications for radiotherapy taken from evidence-based treatment guidelines. The proportion of patients with clinical attributes that indicated a possible benefit from radiotherapy was obtained by adding epidemiological data to the radiotherapy utilization tree. The optimal proportion of patients with cancer that should receive radiotherapy was then calculated using TreeAge software. Sensitivity analyses using univariate analysis and Monte Carlo simulations were performed. Results: The proportion of patients with cancer in whom external beam radiotherapy is indicated according to the best available evidence was calculated to be 52%. Monte Carlo analysis indicated that the 95% confidence limits were from 51.7% to 53.1%. The tightness of the confidence interval suggests that the overall estimate is robust. Comparison with actual radiotherapy utilization data suggests a shortfall in actual radiotherapy delivery. Conclusion: This methodology allows comparison of optimal rates with actual rates to identify areas where improvements in the evidence-based use of radiotherapy can be made. It provides valuable data for radiotherapy service planning. Actual rates need to be addressed to ensure better radiotherapy utilization.

Introduction: Radiotherapy utilization rates for cancer vary widely internationally. It has previously been suggested that approximately 50% of all cancer patients should receive radiation. However, this estimate was not evidence-based. The aim of this study was to estimate the ideal proportion of new cases of cancer that should receive radiotherapy at least once during the course of their illness based on the best available evidence. Materials and Methods: An optimal radiotherapy utilization tree was constructed for each cancer based upon indications for radiotherapy taken from evidence-based treatment guidelines. The proportion of patients with clinical attributes that indicated a possible benefit from radiotherapy was obtained by adding epidemiological data to the radiotherapy utilization tree. The optimal proportion of patients with cancer that should receive radiotherapy was then calculated using TreeAge software. Sensitivity analyses using univariate analysis and Monte Carlo simulations were performed. Results: The proportion of patients with cancer in whom external beam radiotherapy is indicated according to the best available evidence was calculated to be 52%. Monte Carlo analysis indicated that the 95% confidence limits were from 51.7% to 53.1%. The tightness of the confidence interval suggests that the overall estimate is robust. Comparison with actual radiotherapy utilization data suggests a shortfall in actual radiotherapy delivery. Conclusion: This methodology allows comparison of optimal rates with actual rates to identify areas where improvements in the evidence-based use of radiotherapy can be made. It provides valuable data for radiotherapy service planning. Actual rates need to be addressed to ensure better radiotherapy utilization.