Growth factor-induced migration is a rate-limiting step in tumor invasiveness. The molecules that regulate this cellular behavior would represent novel targets for limiting tumor cell progression. Epidermal growth factor (EGF) receptor (EGFR)-mediated motility, present in both autocrine and paracrine modes in prostate carcinomas, requires de novo transcription to persist over times greater than a few hours. Therefore, we sought the specific signaling pathways that directly alter cellular transcription. We confirmed that STAT3 directly associates with, and is activated by EGFR in DU-145 and PC3 human prostate carcinoma cells in addition to the model NR6 fibroblast cell line. This correlated with electrophoretic motility shift of STAT3-selective oligonucleotides. Inhibition of STAT3 activity by antisense or siRNA down-regulation or expression of a dominant-negative construct limited cell motility as determined by an in vitro wound healing assay and invasiveness through a matrix barrier. The expression of constitutively activated STAT3 in the absence of EGF did not increase the migration. Together these data indicate that STAT3 is necessary but not sufficient for EGFR-mediated migration. An initial gene array detected a number of candidate operative molecules; the protein levels of both ENA/VASP, a repressor of cell motility, and caspase 3, a nexus of apoptotic signaling, were down regulated by EGF in a STAT3-dependent manner. Preliminary data show that EGF requires STAT3 functioning to inhibit the induction of apoptosis in the two human prostate cancer cell lines. This suggests that STAT3 signaling may be contributing to tumor progression in a second manner by rendering the cells resistant to death. Together, the sum of these findings suggest that STAT3 signaling may be a new target for both limiting prostate tumor cell invasion and enabling the tumor cells to be killed.

Melanoma, which accounts for only 4% of all skin cancers, but 75% of skin cancer-related deaths, continues to rise at an alarming rate worldwide. When a melanoma is detected and resected at an early stage, the cure rate for patients is favorable. However, the response rate of patients with metastatic melanoma to chemotherapy is less than 15%, and biological therapies have limited efficacy. Therefore, identification of genes that can serve as therapeutic targets for advanced-stage melanoma is crucial. The cell adhesion molecules N-cadherin, MCAM, and Beta3 integrin have been postulated to represent melanoma progression markers; yet, little is known regarding whether they may constitute valuable therapeutic targets for the disease. Furthermore, no studies conducted to date have examined the expression and function of these three molecules in concert in melanoma. The results of our whole-genome and tissue microarray profiling illustrate N-cadherin, MCAM, and Beta3 integrin expression in the distinct stages of melanoma progression. We demonstrate that N-cadherin and Beta3 integrin are melanoma progression markers, but MCAM is not. Furthermore, greater than 95% of metastatic melanomas analyzed in our study express at least one of the three adhesion molecules, and 50% express all three. Our next objective was to determine whether inhibition of N-cadherin, MCAM, or Beta3 integrin impairs melanoma cell proliferation, migration, and/or invasion. We hypothesized that due to redundancy in the functions of N-cadherin, MCAM, and Beta3 integrin, simultaneous inhibition of all three molecules may elicit the most effective therapeutic response. We demonstrate that inhibiting expression of N-cadherin, MCAM, or Beta3 integrin decreases melanoma cell proliferation. However, inhibiting their expression in parallel does not augment the anti-proliferative effect. In contrast, downregulation of N-cadherin, MCAM, and Beta3 integrin in parallel inhibits melanoma cell migration and invasion to a significantly greater extent than targeting each gene alone. Our results indicate that of the three adhesion molecules, MCAM and Beta3 integrin play the most pronounced role in migration and invasion, and therefore, in combination, may represent the most promising therapeutic targets. The data presented in this dissertation provide the foundation for future clinical studies that target adhesion molecules in advanced-stage melanoma patients.

Cell-matrix and cell-cell adhesion proteins are of great significance for many fundamental cellular processes such as survival, differentiation, spreading, adhesion, migration as well as oncogenic transformation. In the present dissertation study, the role of different integrin-proximal protein complexes was investigated in vitro in cancer cells and in primary rat hepatocytes and in vivo in whole animals. First it was shown that migfilin, a newly identified cell-matrix adhesion protein, is also an important component of cell-cell junctions critical for the organization and strengthening of the adherens junctions. Next, Ras-Suppressor-1 (RSU-1), which interacts with the focal adhesion protein PINCH, was shown to regulate cell spreading and adhesion, although the exact mechanism is yet unclear. Furthermore, the role of Integrin-Linked Kinase (ILK) was investigated in vitro in the model system of matrix-induced hepatocyte differentiation. It was shown that ILK along with its binding partners PINCH and Ñ-parvin are dramatically down-regulated during the matrix-induced re-differentiation of hepatocytes. Thus, ILK and its binding partners likely play an important role in matrix-induced-hepatocyte differentiation. Finally, the role of ILK was examined in vivo by removing the protein from the whole animal or specifically from the liver. First ILK was removed from ILK-floxed mice following Cre-recombinase-adenoviral injections giving rise to animals with fulminant hepatitis characterized by massive apoptosis, abnormal mitoses, fatty change and necrosis in the liver. Then, ILK-floxed animals were crossbred with alpha-fetoprotein(AFP)-albumin, albumin, or Foxa3-Cre transgenic mice and thus ILK was genetically removed specifically from the liver. In all cases, the livers of the animals had disorganized liver architecture, absence of hepatocyte plates, increased fibrosis, absence of microvilli in the canaliculi, different degrees of malformations in the biliary system, apoptosis and compensatory proliferation. The present findings therefore, clearly show that ILK is critical for hepatocyte differentiation and survival and more importantly, this holds true in vivo where ILK is crucial for the maintainance of normal liver architecture and function. Thus, the present dissertation work highlights the importance of cell-matrix adhesion proteins in vitro and in vivo and enhances the scientific knowledge in the field of molecular, cellular, hepatocyte and liver biology.

Caveolin-1 (cav-1) has been reported to regulate apoptosis, lipid metabolism and endocytosis. In the present study, we demonstrate that cav-1 can act as a potent immunomodulatory molecule in murine macrophages and play an important role in the development of fibrosis. In murine alveolar and peritoneal macrophages, loss of function experiments using siRNA showed that down-regulating cav-1 expression increased lipopolysaccharide (LPS)-induced proinflammatory cytokine tumor necrosis factor-alpha (TNF-á) and interleukin-6 (IL-6) production but decreased anti-inflammatory cytokine interleukin-10 (IL-10) production. Gain of function experiments demonstrated that overexpression of cav-1 in RAW264.7 decreased LPS-induced TNF-á and IL-6 production and augmented IL-10 production. Cav-1 interacted with TLR4 as revealed by co-immunoprecipitation in peritoneal macrophages. Overexpressing cav-1 in RAW264.7 disrupted Toll like receptor 4 (TLR4) MyD88 and TRIF complex formation; regulated mitogen-activated protein kinase (MAPK) phosphorylation; and inhibited NF-êB activation. Furthermore, the anti-inflammatory modulation by cav-1 involved p38, since the administration of SB203580 significantly abrogated the effects of cav-1, and peritoneal macrophages isolated from MKK3 null mice did not demonstrate any modulation of cav-1. Interestingly, HO-1 translocated into caveolae after LPS stimulation. Carbon monoxide (CO), the gaseous byproduct of HO activity responsible for the anti-inflammatory effects of HO-1, did not regulate cytokines production in cav-1 null macrophages. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients, compared to controls. Transforming growth factor-â1 (TGF-â1), the well-known pro-fibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. Cav-1 was able to suppress TGF-â1-induced extracellular matrix (ECM) production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF and bleomycin (BLM)-instilled lung tissue samples, which was dramatically suppressed by adenovirus cav-1 infection. Moreover, JNK1 null fibroblasts showed reduced Smads cascades signaling, mimicking the effects of cav-1. We also demonstrated that cav-1 markedly ameliorated BLM-induced pulmonary fibrosis as evidenced by histological analysis, hydroxyproline content and immunoblot analysis. In summary, our data suggest that cav-1 acts as a potent immunomodulatory and anti-fibrotic effector molecule. Cav-1 may mediate the anti-inflammatory effects of HO-1/CO in immune cells involving the MKK3/p38 MAPK pathway. Cav-1 also plays a pivotal role in ECM regulation and the development of fibrosis, possibly through the MAPK and Smads pathway. This study suggests cav-1 as a novel therapeutic target for patients with fibrosis and inflammation.

Disruption in important cell survival signaling pathways may represent a central mechanism in neurodegenerative disease processes. 6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin that is commonly used to injure catecholaminergic cells of the central and peripheral nervous systems, and has been used extensively to model Parkinsons Disease. Although it has been documented that 6-OHDA elicits phosphorylation of several kinases, downstream transcriptional effects that influence neuronal cell death are not well defined. The cAMP response element (CRE) is present in the promoter sequences of several important neuronal survival factors. Treatment of catecholaminergic neuronal cell lines (B65 and SH-SY5Y) with 6-OHDA resulted in repression of basal CRE transactivation, and message levels of CRE-mediated genes such as brain derived neurotrophic factor and the survival factor Bcl-2 were decreased in 6-OHDA-treated cells. Message levels of genes lacking CRE sequences were not affected. Interestingly, repression of CRE could be reversed by delayed treatment with cAMP several hours after initiation of 6-OHDA injury. Furthermore, this restoration of CRE-driven transcription, even up to 2 hours post 6-OHDA treatment, was associated with significant neuroprotection. In contrast to observations in other model systems, the mechanism of CRE repression did not involve decreased phosphorylation of its binding protein CREB. Instead, increased phospho-CREB was observed in 6-hydroxydopamine-treated cells, as both total CREB and phospho-CREB were markedly increased in the cytoplasm after treatment. Nuclear expression of p-CREB showed a different pattern, and was decreased in the nucleus of 6-hydroxydopamine-treated cells. 6-OHDA also decreased nuclear phospho-CREB in dopaminergic neurons of primary mouse midbrain cultures. Co-treatment with cAMP promoted/restored nuclear localization of phospho-CREB in both immortalized and primary culture systems, a trend that was associated with protection in both B65 and SY5Y cell lines. Additionally, when human Parkinsons/Lewy body brain tissue was examined, an intense clumped or granular distribution of cytoplasmic phospho-CREB was observed in degenerating substantia nigra neurons, with little to no cytoplasmic staining seen in age-matched controls. Overall, these studies suggest a common theme of impaired nuclear-cytoplasmic trafficking during oxidative neuronal injury processes, with disruption in CREB sub-cellular localization being a recurring trend. It is interesting to note that cytoplasmic accumulation of upstream CREB kinases has previously been observed in both 6-hydroxydopamine-treated cells and in degenerating Parkinson's disease neurons, further supporting a potential role for impaired nuclear import of phosphorylated signaling proteins in neuronal injury processes. These studies present important insight into oxidant-mediated modulation of survival signaling pathways in neuronal cells, may offer potential relevance to the pathogenic mechanisms underlying the progression of neurodegenerative disease.

Squamous cell carcinoma of the head and neck (SCCHN) is the 5th most common cancer worldwide. Signal transducer and activator of transcription 3 (STAT3) is overexpressed in SCCHN and associated with decreased survival. A transcription factor decoy was designed to bind to the DNA binding domain of STAT3, abrogating expression of downstream target genes. The antitumor mechanisms of transcription factor decoys, including the STAT3 decoy, are incompletely understood. STAT3 forms heterodimers with STAT1 suggesting that the STAT3 decoy may interact with STAT1. We determined that the STAT1 pathway was functional in SCCHN cell lines. The STAT3 decoy inhibited STAT1-mediated expression of the target gene, IRF-1. Stimulation of the STAT1 pathway with IFN-× did not mitigate STAT3 decoy-mediated growth inhibition. STAT3 decoy-mediated inhibition of STAT1 signaling did not abrogate its antitumor effects in vitro. Studies using STAT3 knockout cells indicated that STAT3 is necessary for decoy-mediated growth inhibition. The STAT3 decoy was then studied in combination with an EGFR inhibitor and/or a Bcl-XL inhibitor as a therapeutic strategy for SCCHN. Targeting this pathway at several levels¡Xthe upstream receptor (EGFR), the intracellular transcription factor (STAT3), and the downstream target gene (Bcl-XL)¡Xhas not been previously investigated. Combined targeting of EGFR and STAT3 using erlotinib and the STAT3 decoy enhanced growth inhibition of SCCHN cells in vitro. The STAT3 decoy in combination with gossypol, a Bcl-XL inhibitor, resulted in enhanced growth inhibition. The triple combination of all 3 agents enhanced growth inhibition in vitro. These results indicate that targeting the EGFR-STAT3-Bcl-XL pathway at three distinct levels may be a promising treatment strategy for SCCHN.

Select epidermal growth factor (EGF)-like (EGFL) repeats of human tenascin cytotactin can stimulate EGF receptor (EGFR) signaling, but activation requires micromolar concentrations of soluble EGFL repeats in contrast to subnanomolar concentrations of EGF. Using in silico homology modeling techniques, we generated a structure for one such repeat, the 14th EGFL repeat (Ten14). Ten14 assumes a tight EGF-like fold with truncated loops, consistent with circular dichroism studies. We generated bound structures for Ten14 with EGFR using two different approaches, resulting in two distinctly different conformations. Normal mode analysis of both structures indicated that the binding pocket of EGFR exhibits significantly higher mobility in Ten14-EGFR complex compared to the EGF-EGFR complex; we attributed this to loss of key high-affinity interactions within the Ten14-EGFR complex. We proved the efficacy of our in silico models by in vitro experiments. Surface plasmon resonance measurements yielded equilibrium constant KD of 74µM for Ten14, approximately three orders of magnitude weaker than that of EGF. In accordance with our predicted bound models, Ten14 in monomeric form does not bind EGFR with sufficient stability to induce degradation of receptor, or undergo EGFR-mediated internalization. This transient interaction of Ten14 with the receptor on the cell surface is in marked contrast to other EGFR ligands which cause EGFR transit through, and signaling from intracellular locales in addition to cell surface signaling. We investigated whether Ten14-mediated surface restriction of EGFR resulted in altered cellular responses compared to EGF. Activation of PLCã and m-calpain, molecules associated with migration, were noted even at sub-saturating doses of Ten14. However, activation of ERK/MAPK, p90RSK and Elk1, factors affecting proliferation, remained low even at high Ten14 concentrations. Similar activation profiles were observed for EGF-treated cells at 4°C, a maneuver that limits receptor internalization. We demonstrated a direct concurrent effect of such altered signaling on overall biophysical responses - sustained migration was observed at lower levels of Ten14 that activated PLCã, but proliferation remained basal. We present a novel class of EGFR ligands that can potentially signal as a part of the matrix, triggering select signaling cascades leading to a directed cellular response from an otherwise pleiotropic receptor.

Background. Androgen deprivation has been in use for the treatment of advanced prostate cancer since 1941; however, most patients develop resistance to treatment leading to incurable, androgen-independent disease. Previous reports have correlated endothelin A receptor (ETA) expression with increasing prostate cancer grade and stage, and have shown that endothelin-1 (ET-1) treatment of ETA-expressing prostate cancer cells inhibits apoptosis. ETA blockade has emerged as a potential strategy in the treatment of advanced prostate cancer. Here, the potential role of endothelin signaling in promoting prostate cancer cell survival during androgen ablation therapy is evaluated in efforts to establish the potential value of ETA blockade in improving hormone therapy. Methodology and Principle Findings. Androgen-dependent human prostate cancer cells were androgen deprived and evaluated for expression changes in ET-1, ETA, ETB, and AR. Ligand binding, real time quantitative PCR, and immunohistochemical studies show that androgen deprivation increased ET-1, ETA, ETB, and AR expression in prostate cancer cell lines, and ETA expression in human prostate tissue. Using the specific AR inhibitor bicalutamide, acute androgen receptor blockade increased prostate cancer cell ET-1 secretion. Following androgen deprivation, LNCaP cells acquired androgen independence (LNCaP-AI), but retained sensitivity to androgens. ET-1 treatment of ETA over-expressing prostate cancer cells induced a more rapid and sustained activation of Akt, and ETA blockade significantly reduced Akt activation. In vivo ETA blockade, in combination with castration, significantly reduced LNCaP xenograft cell growth, compared to either treatment alone. Affymetrix GeneChip HG-U133 Plus 2 expression array analysis of androgen deprived prostate cancer cells discovered dramatic changes in gene expression patterns throughout the transition to androgen independence. Lastly, the role of ETB signaling in prostate cancer cell apoptosis was examined but remains to be further elucidated. Conclusions and Significance. During androgen deprivation, prostate cancer cells up-regulate ET-1 and ETA expression. Upon engagement of ET-1, ETA invokes activation of the survival factor Akt. In vivo, ETA blockade plus castration inhibits prostate cancer growth. Collectively, these results implicate endothelin survival signaling in promoting progression to androgen-independent disease, and lend support to the targeted disruption of endothelin survival signaling in treating advanced, metastatic prostate cancer.

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) have a critical role in tumor vasculogenesis, mobilizing to tumors and supporting de novo formation of blood vessels essential for tumor growth and metastasis. Vascular endothelial growth inhibitor (VEGI; TL1A) is a member of the tumor necrosis superfamily (TNFSF15) and is produced predominantly by endothelial cells (ECs). VEGI has been shown to act in an autocrine manner by specifically targeting ECs to inhibit their proliferation and induce apoptosis, resulting in elimination of ECs in established tumor vasculature and inhibition of angiogenesis. However, it remains unclear whether VEGI exerts its function solely on fully differentiated ECs or if it is able to modulate BM-derived EPCs as well. Here, the effect of recombinant VEGI on BM-derived EPC function is evaluated in an effort to establish the potential therapeutic value of VEGI. We found that VEGI inhibits the differentiation of EPCs from murine BM under EC stimulating culture conditions. Consistently, VEGI treatment decreases the capability of the cells to adhere, migrate and form capillary-like structures necessary for vascular formation. Additionally, differentiated BM-derived EPCs in cultures underwent apoptosis in response to VEGI treatment. To investigate the impact of VEGI on BM-derived EPC-supported tumor vasculogenesis, mice bearing Lewis lung carcinoma (LLC) tumors were treated with intraperitoneal injection of recombinant VEGI. VEGI treatment significantly decreased the population of BM-derived EPCs found in the tumors while increasing their population in the bone marrow. Furthermore, an overall increase in apoptosis of BM-derived cells at the tumor site was observed after VEGI treatment. Our results indicate VEGI prevents incorporation of BM-derived EPCs into LLC tumors, resulting in the inhibition of EPC-supported tumor vasculogenesis and tumor growth. Together, these findings suggest that VEGI takes part in the modulation of tumor vasculogenesis by inhibiting BM-derived EPC differentiation and mobilization as well as inducing apoptosis. These studies yield important insights into the function of VEGI in postnatal vasculogenesis, helping to facilitate the development of therapeutic uses of VEGI in cancer.

Dendritic cells(DC) are professional antigen presenting cells bridging the innate and adaptive immune systems by detecting pathogen- and- damage associated molecular pattern(PAMP, DAMP) molecules. This triggers maturation and migration to regional lymph nodes where they stimulate T lymphocytes. In tissues normally exposed to relatively high level of PAMP molecules, such as the liver, DC have a higher threshold to stimulation and therefore maintain an immature phenotype under conditions that would stimulate DC at other sites. In these studies we tested the hypothesis that interleukin-6(IL-6)/Signal Transducer and Activation of Transcription-3(STAT3) activity increases the activation/maturation threshold of hepatic and bone marrow(BM) DC towards innate immune signals. Results show that liver nuclear STAT3 activity is significantly higher than other organs and is IL-6-dependent. Hepatic DC in normal wild-type(IL-6+/+) mice are phenotypically and functionally less mature than DC from IL-6-deficient(IL-6-/-) or STAT3 inhibited IL-6+/+ mice, as determined by surface marker expression, pro-inflammatory cytokine secretion, and allogenic T-cell stimulation. IL-6+/+ liver DC produce IL-6 in response to exposure to PAMPs, but resist maturation compared to IL-6-/- liver DC. Conversely, exogenous IL-6 inhibits LPS-induced IL-6-/- liver DC maturation. Oral antibiotic depletion of commensal gut bacteria in IL-6+/+ mice decreased portal blood endotoxin levels, lowered IL-6/STAT3 activity and significantly increased liver DC maturation. BM derived IL-6+/+DC with elevated STAT3 activity are also significantly less mature than IL-6-/- BMDC. The reduced maturation was especially pronounced when IL-6+/+ BMDC when cultured in elevated IL-6 conditions. IL-6 neutralization increased BMDC maturation. Blocking STAT3 activity increases maturation in IL-6+/+ BMDC but not in IL-6-/- BMDC, which have low basal STAT3 activity. Compared to IL-6-/- BMDC, IL-6+/+ BMDC significantly resisted maturation in response to low concentrations of the PAMP molecules. At higher concentrations of these same ligands stimulation of both IL-6+/+ and IL-6-/- BMDC induced maturation. In Conclusion, gut-derived bacterial products, by stimulating hepatic IL-6/STAT3 signaling, inhibit hepatic DC activation/maturation. Elevated IL-6/STAT3 activity raises the threshold needed for DC to translate triggers of innate immunity into adaptive immune responses. Manipulating gut bacteria or IL-6/STAT3 activity may therefore be an effective strategy to alter intra-hepatic immune responses.