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.

Epithelial-cadherin downregulation enables cancer cells to escape from the primary mass; however, E-cadherin has been found to be expressed on metastatic foci, bringing into question the role of this molecule in tumor progression. We define a novel role for the cellular adhesion molecule E-cadherin, in which the proteins re-emergence promotes carcinoma-parenchymal interactions in ectopic sites. Non-metastatic E-cadherin positive MCF7 breast cancer cells form heterotypic cohesions mediated by E-cadherin, and in invasive and metastatic MDA-MB-231 cells, the E-cadherin promoter hypermethylation that prevents endogenous E-cadherin expression is reversed when these cells are cultured with hepatocytes. The function of this re-expression is suggested by the E-cadherin-dependent sustained activation of Erk-MAP kinase and Akt in these breast carcinoma cells. Thus, we propose that E-cadherin expression and subsequent heterocellular interactions direct cell fate decisions that may ultimately enable colonization of a secondary site by an invasive cancer cell.

Environmental triggers, such as viral infection and environmental toxins, have been proposed to initiate the autoimmune disease of Type 1 Diabetes (T1D), however, the mechanism is unknown. The identification of novel autoantigens may provide insight to the mechanism of environmental triggers and pathogenesis of T1D. I identified the antigen recognized by the diabetogenic BDC- 2.5 T cell clone using a novel in vivo reconstitution system, Restricted Immune System via Adoptive Transfer (RISAT). In RISAT, immunodeficient mice are adoptive transferred with a single T cell clone and an open repertoire of B cells. Reconstituted mice are immunized with an antigenic protein preparation. This system will drive an antibody response to the cognate antigen for the T and B cell through the co-stimulatory pathways involved in linked recognition. For the BDC-2.5 RISAT, non-obese diabetic (NOD).Rag-/- mice were adoptive transferred with the diabetogenic BDC-2.5 T cells and NOD B cells and then immunized with an antigenic beta cell membrane preparation (βmem) to drive an antibody response. The resulting antibodies recognized the endoplasmic reticulum (ER) stress associated protein glucose regulated protein 78 (GRP78) from βmem. To determine if ER stress plays a role in the antigenic response of the BDC-2.5 T cell clone, the non-antigenic NOD insulinoma cell line, NIT-1, were treated with thapsigargin, which induces ER stress. The treatment of NIT-1 with thapsigargin led to increased GRP78 synthesis, correlating with antigenic recognition by the BDC-2.5 T cell clone. The antibodies from the BDC-2.5 TCR-Tg recognizes a subset of GRP78 which is modified with phosphoserine. The data presented in this thesis demonstrates a mechanistic link between ER stress and environmental triggers leading to the initiation of TID through the novel autoantigen, GRP78. Also the technique, RISAT, can be used to identify additional potential autoantigens of isolated T cell clones in both T1D and other autoimmune diseases.

Cardiac allograft arteriosclerosis (CAA) is the leading cause of death in cardiac transplantation recipients. Nitric oxide (NO) produced by NO donors or nitric oxide synthase (NOS) inhibits smooth muscle cell proliferation and migration, promotes endothelial cell proliferation, blocks endothelial cell apoptosis, prevents platelet and leukocyte adhesion and platelet aggregation. NO has been shown to inhibit intimal hyperplasia and suppress the development of cardiac allograft arteriosclerosis. Conversely, induction of NO in cardiomyocytes leads to myocardial apoptosis and suppresses cardiac function, mainly represented by decreased cardiac contractility. Based on these studies, we hypothesize that cell type-specific overexpression of eNOS or iNOS, in smooth muscle or endothelial cells, will inhibit the development of cardiac allograft arteriosclerosis without suppressing cardiac function. To test this hypothesis, we developed two gene delivery systems that specifically express NO in smooth muscle or endothelial cells. One system we constructed is adenoviral vectors that employed the fms-like tyrosine kinase-1 (Flt-1, -748 ~ +284 bp), or the intercellular molecule 2 (ICAM2, -292 ~ +44 bp) promoters to drive the human iNOS specific expression in endothelial cells. Another system we developed is vascular-specific iNOS inducible transgenic mice that employed the GeneSwitch system (Invitrogen) and vascular specific promoter. We obtained two iNOS transgenic lines, in which iNOS can be induced by the ligand mifepristone with the supply of the Switch protein. To determine the efficacy of eNOS endothelial-specific expression on suppression of cardiac allograft arteriosclerosis, we employed eNOS transgenic mice in which the human eNOS promoter was driving the human eNOS expression. In a mouse heterotopic transplantation model, the male transgenic and female wild-type counterparts were used as donor and recipient, respectively. The donor grafts were harvested 60 days after transplantation and the percentage of vessel occlusion was determined. The vessel occlusion developed in the transegenic donor did not show significant difference compared to the wild-type control. As an alternative approach, the eNOS/GTPCH double transgenic mice will be used as donor and its efficacy will be determined. We hope that these proof-of -principle studies will serve as the basis for the development of an effective treatment strategy to prevent cardiac allograft arteriosclerosis.

Phosphatidylinositol-3-kinase (PI3K) is a well-known regulator of cell division, motility, metabolism and survival in most cell types. Proper liver function and development highly depend on intact PI3K signal transduction. Aberrant PI3K pathway signaling in the liver is associated with hepatocellular carcinoma (HCC). In addition, PI3K signaling is involved in the homeostasis of lipid and glucose metabolism. Activation of the PI3K pathway induces lipogenesis and glycogenesis in the liver, since both Akt overexpressing transgenic mice and PTEN knockout mice develop fatty liver and hypoglycemia. Our laboratory characterized a novel protein that we call PI3K Interacting Protein 1 (PIK3IP1) which binds to the p110 catalytic subunit of PI3K and reduces its activity in vitro. Little is known about PIK3IP1s role in tumorigenesis and metabolism in vivo. Therefore we constructed PIK3IP1 transgenic mice (TG) which overexpress PIK3IP1 in hepatocytes under an albumin promoter in the C3H mouse strain to investigate the effect of PIK3IP1 on hepatocyte growth and metabolism, as well as HCC tumorigenesis. We detected a high expression level of PIK3IP1 in the livers from TG animals. The PI3K pathway was successfully suppressed both in liver tissues and isolated hepatocytes, which was confirmed by Western blots and phospho-protein array studies. Given the fact that PI3K signaling is associated with liver tumorigenesis, our next objective was to determine whether PIK3IP1 inhibits HCC development through PIK3IP1- mediated downregulation of the PI3K pathway. In vivo, spontaneous liver tumorigenesis was significantly dampened in the transgenic animals. This was accompanied by decreased hepatic PI3K activity and reduced hepatocyte proliferation in the transgenics as compared to controls. Isolated PIK3IP1 transgenic mouse hepatocytes showed blunted PI3K signaling, DNA synthetic activity, motility and survival as compared to controls. We then investigated the effect of PIK3IP1 on the maintenance of whole-body glucose and fat homeostasis. We observed that mice overexpressing PIK3IP1 have increased body weight, hyperglycemia, as well as increased visceral fat deposition. This suggests PIK3IP1 is an important regulator of metabolism. In conclusion, we successfully generated a transgenic mouse model with PIK3IP1 overexpression in hepatocytes to assess the biological functions of PIK3IP1, an important negative regulator of PI3K, in liver tumorigenesis and insulin signaling. A high level expression of PIK3IP1 suppressed PI3K signaling pathway in vivo and in vitro, which curbs hepatic tumorigenesis. Furthermore, we show that PIK3IP1 overexpression can contribute to glucose homeostasis and fatty deposition.

Vascular Endothelial Growth Inhibitor (VEGI) is an endothelial cell autocrine factor and a member of the tumor necrosis family of ligands. VEGI is able to specifically inhibit endothelial cell growth and is an efficient inhibitor of angiogenesis. The molecular mechanisms of VEGI activity on endothelial cells remain undefined. Here we focused on two important steps in the signal transduction of VEGI. We first determined a role of NF-κB in VEGI-induced apoptosis. We found that inhibition of the NF-κB pathway resulted in an increased apoptotic potential of VEGI. We conclude that the NF-κB pathway plays a role in suppressing the apoptotic potential of VEGI. We next investigated the receptor responsible for VEGI-induced endothelial cell apoptosis. DR3 is a receptor for VEGI and thus we first focused on confirming if DR3 is the receptor responsible for VEGI-mediated endothelial cell death. We determined VEGI had diminished apoptotic activity in endothelial cells that are depleted of DR3 by siRNA. However, it was determined that the apoptotic stimuli, LPS and TNFα, were also unable to mediate cell death in DR3-depleted endothelial cells. We conclude that DR3 is mediating an intracellular event that is involved in controlling the apoptotic pathway. This is a novel role of DR3 that is yet to be described. However, this role of DR3 interferes with our analysis of the ligand/receptor relationship and therefore we were unable to confirm that DR3 is the receptor responsible for VEGI-induced apoptosis. We also provide preliminary evidence that VEGI is utilizing an unknown receptor to mediate NF-κB activation. We therefore provide several mechanisms to control VEGI-mediated endothelial cell death, one being the activation of NF-κB to suppress the apoptotic potential of VEGI and the needed presence of DR3 for VEGI to initiate apoptosis, a role that is possibly independent of ligand binding.

The class of proteins known as immunophilins, that are cis-trans prolyl isomerases perform diverse chaperone roles. The immunophilins FKBP52 and FKBP51 (FK506 Binding Proteins) are adapter proteins involved in the trafficking of the glucocorticoid receptor (GR), in which FKBP52 facilitates binding of retrograde molecular motor protein dynein and the GR, while FKBP51 binds only the GR. This body of work presents: 1. An analysis of the FKBP family of proteins and their potential for involvement of neuropathogenesis and identifies FKBP52 and FKBP51 as an evolutionarily divergent duo in mammals, 2. The changes in gene and protein levels of these immunophilins in the frontal cortex of patients with HIV and Major Depressive Disorder (MDD), and 3. A role for FKBP52 in the ligand-activated redistribution of GR in neurons. Using primary human mixed neuron-glia cultures, we tested the hypothesis that immunophilin ligands, like FK506, may alter the kinetics of FKBP52 or FKBP51-mediated trafficking of the GR in neurons. We treated the neuron-glia cultures with cortisol with or without FK506 pretreatment, and found that FK506 altered the distribution of GR. By knocking down expression of FKBP52 using siRNA in a differentiated neuroblastoma cell line, hydrocortisone-mediated nuclear translocation of GR was slowed. Treatment of neuroblastoma cells media supplemented with 10% conditioned media of HIV-infected microglia lead to increased expression of both immunophilins. In a parallel study, we assessed the transcriptional and postranscriptional levels of the GR adapter proteins FKBP52 and FKBP51 in autopsy tissues from the frontal cortex of patients with MDD with and without HIV. We found increased expression of both proteins in HIV infected patients. FKBP51 was increased in MDD while expression of FKBP52 was the highest in the HIV population with MDD. These data support the hypothesis that the immunophilins described here modulate the cellular function of the GR in the brain and expression levels may be related to mood disorders. In general, viral infection and inflammation increase expression, of both immunophilins, which may alter the cortisol-induced trafficking of GR.

Head and neck squamous cell carcinoma (HNSCC) is a commonly occurring malignancy associated with severe morbidity, persistently high mortality rates, frequent recurrence, and the appearance of second primary tumors (SPTs). A great need exists, therefore, for new therapies, including complementary and preventive approaches to treating HNSCC. Signal transducer and activator of transcription (STAT)-3, an oncogenic transcription factor, shows promise as an important therapeutic target in the treatment of HNSCC. The current study focuses on the STAT3-targeting activities of two natural compounds, guggulsterone and honokiol, and investigation of their antitumor activity in HNSCC. Guggulsterone, a compound contained in the resin of the Commiphora mukul plant, used in Indian Ayurvedic medicine, is widely available as a dietary supplement and associated with few side effects. Honokiol is a naturally-occurring compound that has been used in traditional Chinese medicine and is derived from the plant, Magnolia officinalis. Both compounds have been shown to have anticancer activity in various models and to inhibit nuclear factor kappa B (NF kappa B), an oncogenic transcription factor. NF kappa B and STAT3 interact with one another in various ways. Therefore, we hypothesized that guggulsterone and/or honokiol might be useful in targeting STAT3. Both compounds inhibited growth and invasiveness and induced apoptosis in HNSCC cell lines, in addition to decreasing levels of phosphotyrosine STAT3, and, for guggulsterone, total STAT3. Guggulsterone was also found to cause cell cycle arrest and to target hypoxia-inducible factor (HIF)-1 alpha, a potential therapeutic target whose expression is correlated with poor clinical outcome in HNSCC. Guggulsterone-induced growth inhibition relied partly on its ability to inhibit STAT3. Both compounds enhanced the activities of current HNSCC therapies and modestly inhibited tumor growth in the xenograft model of HNSCC. To test the chemopreventive potential of STAT3 and epidermal growth factor receptor (EGFR) inhibition, a study administering Guggulipid, a guggulsterone-containing nutraceutical, or erlotinib, an EGFR-targeting tyrosine kinase inhibitor (TKI) to mice treated orally with a carcinogen is currently underway. Our results so far suggest that guggulsterone and honokiol-mediated inhibition of STAT3 and guggulsterone-mediated inhibition of HIF-1 alpha provide a biologic rationale for further clinical investigation of these compounds as complementary and preventive treatments for HNSCC.