Cell adhesion molecules play a vital role in modulating both normal and tumor cell behavior. The focus of my research has been to investigate the contribution of Activated Leukocyte Cell Adhesion Molecule (ALCAM) to tumor progression. The functional contribution of ALCAM to metastasis was investigated using murine models of prostate cancer while a retrospective cohort of colorectal cancer patients was used to determine whether ALCAM has a clinical prognostic value in distinguishing aggressive cancer types. In addition to the function contribution and clinical relevance I searched for the modulatory cytokine responsible for promoting ALCAM ectodomain shedding via ADAM17. Our results, presented within this dissertation, explore the contribution of tumor-derived ALCAM in prostate tumorigenesis in vivo and the clinical application of intratumoral ALCAM shedding in colorectal cancer progression. We have now shown that the loss of tumor-specific ALCAM expression results in decreased metastasis to skeletal sites and reduced tumor cell survival in the bone microenvironment through enhanced apoptosis. In addition, we identified transforming growth factor beta-1 (TGFβ), a key tumor-associated cytokine in bone metastasis, as a major driver of ALCAM ectodomain shedding. In addition to enhanced ectodomain shedding and mRNA expression, ALCAM is required for the enhanced migratory behavior of tumor cells in response to TGFβ. Moreover, the development and application of a novel antibody specific to the intracellular domain of ALCAM has allowed for the investigation of intratumoral ALCAM shedding in retrospective clinical studies. Enhanced intratumoral ALCAM shedding in early colorectal disease, stage II specifically, is associated with decreased patient survival. In conclusion, we have been able to demonstrate that ALCAM shedding, driven by pro-tumorigenic cytokines, contributes to cancer metastasis and correlates clinically to poor patient outcome.

Inflammatory bowel disease (IBD), which affects 1 in 600 Americans, is characterized by severe and chronic inflammation, a known contributor to cancer. As such, the risk for cancer is increased in patients with IBD compared with the general population, and cancer is the most significant cause of mortality in IBD. This thesis will identify modifiers of colitis-associated cancer (CAC) utilizing a murine CAC model applied to models of dietary and genetic deficiencies. First, this thesis will focus on dietary selenium and two plasma selenoproteins as suppressors of tumorigenesis. Selenium deficiency results in increased inflammation in response to an inflammatory model utilizing dextran sodium sulfate (DSS) which, when coupled with the use of an initiator, azoxymethane (AOM/DSS inflammatory carcinogenesis model), leads to an increase in tumorigenesis concomitant with an increase in DNA damage. Knockout of the plasma selenoprotein glutathione peroxidase-3 (Gpx3) also results in increased tumor number as well as a higher degree of dysplasia. Finally, I demonstrate that selenoprotein P (Sepp1), which is considered to have both selenium transport and antioxidant properties, is a bimodal modifier of CAC. Complete loss of Sepp1 leads to decreased tumorigenesis as the result of clearance of initiated cells while haploinsufficiency leads to an increase in tumorigenesis which is mimicked by loss of either the putative selenium transport or antioxidant domains of Sepp1. A second set of CAC modifiers identified in this thesis are the myeloid translocation genes (MTGs). The MTGs are a family of transcriptional corepressors that were originally identified as targets of chromosomal translocations in acute myeloid leukemia. This thesis will first identify MTG, related-1 (MTGR1) as a modifier of inflammation which, upon its loss, leads to a decrease in inflammatory carcinogenesis. Moreover, I identify an interaction between MTG16 and the transcriptional repressor Kaiso. Knockout of MTG16 alone leads to increased tumorigenesis while knockout of Kaiso leads to unaltered tumorigenesis. Interestingly, absence of both proteins leads to rescue of the MTG16 phenotype, suggesting that Kaiso is epistatic to MTG16.

CANCER BIOLOGY BCL-2/BCL-XL REGULATES CELL CYCLE THROUGH A NOVEL MECHANISM IN ADDITION TO CELL SURVIVAL YELENA MELKUM JANUMYAN Dissertation under the direction of Professor Elizabeth Yang Bcl-2 and Bcl-xL are anti-apoptotic and inhibit proliferation. During cell cycle arrest, Bcl-2 or Bcl-xL expression causes elevation of the CDK inhibitor p27, decrease in cell size and RNA content. This enhanced state of G0 results in delay of G0-G1 transition and S phase entry upon cell cycle stimulation. We investigated the mechanism of the Bcl-2/Bcl-xL cell cycle function and the relationship between apoptosis regulation and cell cycle control. To assess the role of mitochondrial functions in Bcl-2/Bcl-xL-mediated cell cycle delay, we monitored ATP levels and found that cells expressing Bcl-2 or Bcl-xL exhibit a delay in peak ATP during cell cycle entry; however, exogenous elevation of ATP did not reverse the delay in cell cycle entry. In mtDNA-free 143B ?0 cells defective in electron transport, Bcl-xL is still able to delay cell cycle entry but not enhance G0 arrest. To determine whether enhanced G0 and subsequent delay in cell cycle are consequences of increased survival, we inhibited cell death by means other than Bcl-2/Bcl-xL expression. Increasing survival by caspase inhibition partially recapitulated the cell cycle arrest phenotype of Bcl-2/Bcl-xL, while cells expressing the survival kinase Akt exhibited none of the cell cycle phenotypes of Bcl-2/Bcl-xL. These data indicate that the cell cycle delay by Bcl-2/Bcl-xL is not mediated through the regulation of ATP/ADP exchange, and does not require normal mitochondrial membrane potential. Bcl-2/Bcl-xL may regulate quiescence partially through the inhibition of caspase-dependent apoptosis, but cell survival is not the sole mechanism of the cell cycle regulation by Bcl-2/Bcl-xL. In addition to inhibition of mitochondrial apoptosis, mechanisms requiring an intact electron transport chain are necessary for the full cell cycle function of Bcl-2/Bcl-xL.

Contribution of stromal TGF-b to prostate cancer progression Human prostate carcinoma-associated fibroblasts (CAF) induce malignant transformation in an initiated but non-malignant human prostatic epithelial cell line (BPH1). This study elucidates the interplay between TGF-b and CXCL12/SDF1 in tumorigenesis. Both TGFb and CXCL12 are elevated in CAF. The CXCL12 receptor, CXCR4, is not expressed in benign prostate tissue or in BPH1 cells in culture. In tissue recombinants with benign rat urogenital sinus mesenchyme (rUGM) epithelial CXCR4 titers are low, expression of the receptor is markedly upregulated by CAF mirroring findings in human prostate cancer progression. TGFb induces expression of CXCR4 in the epithelium. Loss of either TGFb signaling or CXCR4 expression abrogates the tumorigenic response to CAF. The present study also examines the effects of TGF-b on the non-tumorigenic human prostatic epithelial cell line BPH1 and on three derivatives tumorigenic sublines BPH1CAFTD-1, -3 and -5. The data demonstrate that TGF-b has different effects on the non-tumorigenic and tumorigenic cells. The non-tumorigenic cells are growth inhibited by TGF-b. In contrast the tumorigenic sub-lines are not growth inhibited but instead undergo an epithelial to mesenchymal transformation (EMT) in response to TGF-b. In vivo, tumorigenic cells with constitutively active TGF-b signaling show increased invasion with EMTs, which express vimentin, located specifically at the invasive front of the tumor. These data indicate that following malignant transformation TGF-b can play a direct role in promoting prostatic cancer and further that these responses are context specific in vivo.

Integration of receptor tyrosine kinase, integrin, and cadherin activities is crucial for normal cell growth, motility and adhesion. Here, we describe novel roles for p120 catenin (p120) and p190RhoGAP that coordinate crosstalk between these systems and regulate cadherin function. Surprisingly, PDGFR induced actin remodeling in NIH3T3 cells is blocked in the absence of p120, and the cells are partially transformed via constitutive activation of Rho. We have traced the mechanism to unexpected codependent roles for p120 and p190RhoGAP in regulating Rac-dependent antagonism of Rho. Receptor-induced Rac activity causes translocation of p190RhoGAP to adherens junctions (AJs), where it couples to the cadherin complex via interaction with p120. AJ formation is dependent on this p120-p190RhoGAP interaction and fails altogether if either of these proteins are compromised. We propose that Rac activation links diverse signaling systems to AJ assembly by controlling transient p190RhoGAP interactions with p120 and localized inhibition of Rho.

Although many studies have investigated the pro- and anti-apoptotic functions of the Bcl-2 family of proteins, it has been found that the anti-apoptotic Bcl-2 and Bcl-xL molecules also have a role in the cell cycle. It has been shown that cells that overexpress either Bcl-2 or Bcl-xL exhibit enhanced cell cycle arrest upon serum starvation or contact inhibition. The characteristic cell cycle arrest phenotype observed in Bcl-2 or Bcl-xL expressing cells resembles the autophagy-induced cell cycle arrest. In this study we investigated whether the enhanced arrest phenotype observed in Bcl-2 and Bcl-xL expressing cells is in part due to an enhanced autophagic response. During arrest conditions we treated cells with 3-methyladenine (3-MA), commonly used to inhibit autophagy, to determine whether Bcl-2 and Bcl-xL could still induce an enhanced arrest. We found that Bcl-xL expressing cells are not able to arrest effectively in G0 in the presence of 3-MA. This finding did not appear to be true for Bcl-2. These studies suggest that Bcl-xL mediates enhanced arrest in part through autophagy.

Although the signaling mechanisms of the apoptotic pathway have been extensively studied, there is still much left unknown. A key regulator protein, 14-3-3, is known to bind and protect BAD phosphorylation sites serine 112 and 136, sequestering BAD in the cytoplasm. Under conditions of cell death, 14-3-3 becomes loose and exposes the phosphorylated S112 site to phosphatases, promoting the sequential dephosphorylation of BAD at sites 112, 136, and 155, thereby inducing apoptosis. The examination of the BAD-14-3-3 complex under conditions of cell death is of acute interest to this thesis. Three possibilities were examined that may aid in the dissociation of 14-3-3 to BAD under conditions of cell death: 1) interaction of an unknown binding partner 2) novel BAD modification or phosphorylation 3) 14-3-3 modification or phosphorylation. Methods used in these investigations included: Mass Spectrometry, kinase assays, pulldown experiments, and western blots. Several novel binding partners to BAD were identified in either a condition of cell life or cell death, including the following: p38, CDC2, Beclin-1, BAX, cytochrome c1 oxidase, and voltage-dependent anion-selective channel proteins 1, 2, and 3. 32P kinase assays suggested that p38 phosphorylates a novel site on BAD, serine 6. The phosphorylation state of BAD S128 was observed 3-7 hours after cell death stimulation and was independent of p38 activation. Under cell life and death conditions, no new phosphorylation sites or monomeric changes on 14-3-3 were observed; p38 was concluded not to phosphorylate 14-3-3.

Transforming Growth Factor-β (TGF-β) is a cytokine involved in the regulation of multiple cellular responses, and it is accepted that the TGF-β signaling pathway is implicated in both tumor-suppressing and tumor-promoting processes. To assess the role of TGF-β signaling in intestinal cancer, we generated a mouse model that recapitulates two common genetic events observed in colon cancer. Hence, we mated Apc1638N/wt mice with mice that are null for Tgfbr2 in the intestinal epithelium, here called Tgfbr2IEKO. We observed a dramatic increase in the number of intestinal adenocarcinomas in the Apc1638N/wt; Tgfbr2IEKO mice compared to those mice with intact Tgfbr2. Additionally, analyses of tumor-derived epithelial cells and primary tumor tissues showed elevated amounts of TGF-β1 as well as higher MMP-2 activity in specimens from Apc1638N/wt; Tgfbr2IEKO mice in comparison to those that maintained Tgfbr2. These results indicate that inactivation of TGFBR2 in intestinal epithelial cells promotes the malignant transformation of neoplasms initiated by Apc mutation, thus providing in vivo evidence of cooperation between canonical Wnt signaling upregulation and TGF-β signaling inactivation to drive tumor progression. We also evaluated the effects of TGF-β signaling on advanced colorectal cancer, particularly on the acquisition of resistance to anoikis or cell death induced by inappropriate cell-cell or cell-extracellular matrix attachment. Thus, we assessed the effect of TGF-β1 on anoikis in established colorectal human cancer cell lines. We found that TGF-β1-treated HCT116+Chr3 and CBS showed a significant reduction of cell death upon loss of anchorage, and that suppression of autocrine TGF-β signaling in HCT116+Chr3 inhibited this effect. We found that TGF-β1 can promote the activation of the PIK3/AKT and MAPK/ERK pathways, and it modulates the expression of Bim and Bcl-2. Therefore, TGF-β paradoxically promotes the malignant behavior of a subset of TGF-β responsive colon cancer cell lines by blocking anoikis through cell-autonomous mechanisms. In aggregate, these studies show that TGF-β signaling plays a complex and context-dependent role in intestinal cancer development. TGF-β's cell-autonomous and non-autonomous functions are involved in intestinal tumor progression and should be thoroughly considered when planning therapeutic intervention through TGF-β signaling manipulation.

Kaposi's sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma (PEL) and multicentric Castlemen's disease (MCD). This ×-herpesvirus infects endothelial cells lining the vascular spaces of KS lesions, the so-called spindle cells and B cells in PEL. KSHV encodes proteins that mimic the actions of cellular proteins and override normal cell cycle checkpoints. Viral Bcl-2 and Flice-like inhibitory protein (v-FLIP) contribute to increased cell survival, while a virally-encoded G protein-coupled receptor and viral IL-6 regulate cell growth. K-cyclin, the cyclin D2 homolog, has non-cell cycle-related transcriptional regulatory properties. Data from our laboratory demonstrates that K-cyclin associates with the transcriptionally active S1 fraction of chromatin along with other proteins like p300, polymerase II and transcription factor IID (TFIID) known to be involved in transcription. K-cyclin/cdk6 complexes can bind and phosphorylate p300, and by doing so activate NF-ÛB dependent transcription. This is significant because our lab's work shows that K-cyclin's activity contributes to the constitutive activation of the NF-kappaB transcriptional pathway in HHV8-associated tumors. K-cyclin contains a protein docking site near the amino terminus, termed the MRAIL motif. This conserved site allows cellular cyclins to interact with proteins containing RXL motifs. We questioned whether the MRAIL motif was essential for the K-cyclin/p300 interaction and the subsequent activation of NF-kappaB-dependent transcription. To answer this question, using site-directed mutagenesis we generated alanine mutants of the amino acid residues comprising the MRAIL motif. We have found that the mutation of any single amino acid in the MRAIL motif is sufficient to disrupt the ability of K-cyclin to activate NF-kappaB-dependent transcription using an NF-kappaB luciferase reporter assay. The three most important latency genes, v-FLIP, K-cyclin and LANA are under the control of the same promoter, with both LANA and K-cyclin having emerging roles as transcriptional regulators. As K-cyclin and LANA are both transcribed from a single promoter, are expressed at the same time in the viral life cycle and are both found in the nucleus, we sought to determine whether they physically interact. We have identified a novel association between K-cyclin and LANA, a multifunctional viral protein involved in transcription by interacting directly with DNA and through protein-protein interactions with other transcription factors like AP-1, Sp1 and pRb. This interaction was detected at endogenous levels of both proteins in a PEL cell line by coimmunoprecipitation analysis in both directions. Finally, studies in transfected COS-7L cells suggest that K-cyclin-LANA interation is not dependent on any other viral proteins. These findings suggest that K-cyclin is a transcriptional regulator and that this association may play a role in the signature NF-kappaB activation found in KSHV-infected cells.

Cyclooxygenase-2 (COX-2) has long been known to be a facilitator of colorectal neoplasia, specifically in the development and progression of adenomatous polyps to colorectal carcinoma. The purpose of the studies conducted and reported hereafter in this thesis was to evaluate biologic changes in patients and their colorectal disease after pharmacologic inhibition of COX-2 with a selective inhibitor, celecoxib. In this thesis, I report our experience both with urine levels of the protstaglandin E2 metabolite (PGE-M) among patients with colorectal disease and changes in gene expression in rectal carcinomas after treatment with celecoxib. This research has led to the discovery of a potential biomarker of colorectal neoplasia and identified several specific genes and biologic pathways that are differentially expressed when COX-2 is pharmacologically inhibited.