Reduced vascular endothelial growth factor expression in contusive spinal cord injury.

Vascular endothelial growth factor (VEGF) is being investigated as a potential interventional therapy for spinal cord injury (SCI). In the current study, we examined SCI-induced changes in VEGF protein levels using Western blot analysis around the epicenter of injury. Our results indicate a significant decrease in the levels of VEGF(165) and other VEGF isoforms at the lesion epicenter 1 day after injury, which was maintained up to 1 month after injury. We also examined if robust VEGF(165) decrease in injured spinal cords affects neuronal survival, given that a number of reported studies show neuroprotective effect of this VEGF isoform. However, exogenously administered VEGF(165) at the time of injury did not affect the number of sparred neurons. In contrast, exogenous administration of VEGF antibody that inhibits actions of not only VEGF(165) but also of several other VEGF isoforms, significantly decreased number of sparred neurons after SCI. Together these results indicate a general reduction of VEGF isoforms following SCI and that isoforms other than VEGF(165) (e.g., VEGF(121) and/or VEGF(189)) provide neuroprotection, suggesting that VEGF(165) isoform is likely involved in other pathophysiological process after SCI.

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

rIFN-gamma-mediated growth suppression of platinum-sensitive and -resistant ovarian tumor cell lines not dependent upon arginase inhibition.

BACKGROUND: Arginine metabolism in tumor cell lines can be influenced by various cytokines, including recombinant human interferon-gamma (rIFN-gamma), a cytokine that shows promising clinical activity in epithelial ovarian cancer (EOC). METHODS: We examined EOC cell lines for the expression of arginase in an enzymatic assay and for transcripts of arginase I and II, inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO) by reverse transcription-polymerase chain reaction. The effects of rIFN-gamma on arginase activity and on tumor cell growth inhibition were determined by measuring [3H]thymidine uptake. RESULTS: Elevated arginase activity was detected in 5 of 8 tumor cell lines, and analysis at the transcriptional level showed that arginase II was involved but arginase I was not. rIFN-gamma reduced arginase activity in 3 EOC cell lines but increased activity in the 2008 cell line and its platinum-resistant subline, 2008.C13. iNOS transcripts were not detected in rIFN-gamma-treated or untreated cell lines. In contrast, IDO activity was induced or increased by rIFN-gamma. Suppression of arginase activity by rIFN-gamma in certain cell lines suggested that such inhibition might contribute to its antiproliferative effects. However, supplementation of the medium with polyamine pathway products did not interfere with the growth-inhibitory effects of rIFN-gamma EOC cells. CONCLUSIONS: Increased arginase activity, specifically identified with arginase II, is present in most of the tested EOC cell lines. rIFN-gamma inhibits or stimulates arginase activity in certain EOC cell lines, though the decrease in arginase activity does not appear to be associated with the in vitro antiproliferative activity of rIFN-gamma. Since cells within the stroma of EOC tissues could also contribute to arginine metabolism following treatment with rIFN-gamma or rIFN-gamma-inducers, it would be helpful to examine these effects in vivo.

LY2109761, a novel transforming growth factor beta receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis.

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.

The Role of Acidic Fibroblast Growth Factor and Fibroblast Growth Factor Receptor 4 in High Grade Serous Carcinoma

Background: High grade serous carcinoma whether ovarian, tubal or primary peritoneal, continues to be the most lethal gynecologic malignancy in the USA. Although combination chemotherapy and aggressive surgical resection has improved survival in the past decade the majority of patients still succumb to chemo-resistant disease recurrence. It has recently been reported that amplification of 5q31-5q35.3 is associated with poor prognosis in patients with high grade serous ovarian carcinoma. Although the amplicon contains over 50 genes, it is notable for the presence of several members of the fibroblast growth factor signaling axis. In particular acidic fibroblast growth factor (FGF1) has been demonstrated to be one of the driving genes in mediating the observed prognostic effect of the amplicon in ovarian cancer patients. This study seeks to further validate the prognostic value of fibroblast growth receptor 4 (FGFR4), another candidate gene of the FGF/FGFR axis located in the same amplicon. The emphasis will be delineating the role the FGF1/FGFR4 signaling axis plays in high grade serous ovarian carcinoma; and test the feasibility of targeting the FGF1/FGFR4 axis therapeutically. Materials and Methods: Spearman and Pearson correlation studies on data generated from array CGH and transcriptome profiling analyses on 51 microdissected tumor samples were used to identify genes located on chromosome 5q31-35.3 that showed significant correlation between DNA and mRNA copy numbers. Significant correlation between FGF1 and FGFR4 DNA copy numbers was further validated by qPCR analysis on DNA isolated from 51 microdissected tumor samples. Immunolocalization and quantification of FGFR4 expression were performed on paraffin embedded tissue samples from 183 cases of high-grade serous ovarian carcinoma. The expression was then correlated with clinical data to assess impact on survival. The expression of FGF1 and FGFR4 in vitro was quantified by real-time PCR and western blotting in six high-grade serous ovarian carcinoma cell lines and compared to those in human ovarian surface epithelial cells to identify overexpression. The effect of FGF1 on these cell lines after serum starvation was quantified for in vitro cellular proliferation, migration/invasion, chemoresistance and survival utilizing a combination of commercially available colorimetric, fluorometric and electrical impedance assays. FGFR4 expression was then transiently silenced via siRNA transfection and the effects on response to FGF1, cellular proliferation, and migration were quantified. To identify relevant cellular pathways involved, responsive cell lines were transduced with different transcription response elements using the Cignal-Lenti reporter system and treated with FGF1 with and without transient FGFR4 knock down. This was followed by western blot confirmation for the relevant phosphoproteins. Anti-FGF1 antibodies and FGFR trap proteins were used to attempt inhibition of FGF mediated phenotypic changes and relevant signaling in vitro. Orthotopic intraperitoneal tumors were established in nude mice using serous cell lines that have been previously transfected with luciferase expressing constructs. The mice were then treated with FGFR trap protein. Tumor progression was then followed via bioluminescent imaging. The FGFR4 gene from 52 clinical samples was sequenced to screen for mutations. Results: FGFR4 DNA and mRNA copy numbers were significantly correlated and FGFR4 DNA copy number was significantly correlated with that of FGF1. Survival of patients with high FGFR4 expressing tumors was significantly shorter that those with low expression(median survival 28 vs 55 month p< 0.001) In a multivariate cox regression model FGFR expression significantly increased risk of death (HR 2.1, p<0.001). FGFR4 expression was significantly higher in all cell lines tested compared to HOSE, OVCA432 cell line in particular had very high expression suggesting amplification. FGF1 was also particularly overexpressed in OVCA432. FGF1 significantly increased cell survival after serum deprivation in all cell lines. Transient knock down of FGFR4 caused significant reduction in cell migration and proliferation in vitro and significantly decreased the proliferative effects of FGF1 in vitro. FGFR1, FGFR4 traps and anti-FGF1 antibodies did not show activity in vitro. OVCA432 transfected with the cignal lenti reporter system revealed significant activation of MAPK, NFkB and WNT pathways, western blotting confirmed the results. Reverse phase protein array (RPPA) analysis also showed activation of MAPK, AKT, WNT pathways and down regulation of E Cadherin. FGFR trap protein significantly reduced tumor growth in vivo in an orthotopic mouse model. Conclusions: Overexpression and amplification of several members of the FGF signaling axis present on the amplicon 5q31-35.3 is a negative prognostic indicator in high grade serous ovarian carcinoma and may drive poor survival associated with that amplicon. Activation of The FGF signaling pathway leads to downstream activation of MAPK, AKT, WNT and NFkB pathways leading to a more aggressive cancer phenotype with increased tumor growth, evasion of apoptosis and increased migration and invasion. Inhibition of FGF pathway in vivo via FGFR trap protein leads to significantly decreased tumor growth in an orthotopic mouse model.

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

Integrin function in T cell activation recapitulated in tumor growth

An in vitro model using highly purified freshly isolated T cells demonstrated that immobilized ligands for the integrin $\alpha4\beta1$ could cooperate to enhance mitogen signals delivered by coimmobilized anti-CD3 specfic monoclonal antibody OKT3. Costimulation through $\alpha4\beta1$ integrin lead to enhanced proliferation which depended on expression of both IL-2 as well as IL-2 receptor. The transcription factors NF-AT, AP-1, and NF-$\kappa$B, which are involved in the regulation of IL-2 as well as other cytokine genes, were weakly induced by anti-CD3 stimulation alone in electromobility shift assays, but were augmented significantly with $\alpha4\beta1$ costimulation. These results suggested that $\alpha4\beta1$ ligands delivered a growth promoting signal which could synergize with signals induced by engagement of the TCR/CD3 complex, and also suggested a dual function for integrins in both localization and subsequent delivery of a growth promoting signal for T lymphocytes. Integrin involvement in lymphocyte trafficking has been employed as a model for understanding tumor cell metastasis. Therefore we have extended the duality of integrin function in both homing and subsequent delivery of a growth promoting signal to include a role for integrins in providing growth stimulation for tumor cells. Using a gastric derived tumor line, inhibition of adhesion to substrate leads to G0/G1 cell cycle arrest, reduced cyclin A expression, and reduced phospholipid synthesis. This effect could be reversed upon $\alpha2\beta1$ integrin mediated reattachment to collagen. These observations demonstrated a role for an integrin in the growth regulation of a tumor line. The small GTP-binding protein Rho, implicated in phospholipid synthesis, can be inactivated by the ADP-ribosylation exoenzyme C3 from C. botulinum. Addition of C3 to cell cultures inhibited the growth promoting effect due to integrin mediated adhesion. Taken together, these results are consistent with a model for cooperative interaction between integrins and Rho leading to enhanced phospholipid synthesis and mitogen signaling. This model may provide a basis for understanding the phenomena of integrin costimulation in T cell activation. ^

Integrin mediated growth and apoptosis in human gastric adenocarcinoma

Integrins are important as the primary cell adhesion molecule providing information about the extracellular microenvironment to the interior of the cell to influence cellular behavior such as differentiation, proliferation and apoptosis. Apoptotic death due to loss of adhesion is termed anoikis. In this study we have obtained a parental human gastric adenocarcinoma cell line that yielded two variant lines that had differing responses to lack of adhesion. The STAD.APO cell line undergoes apoptosis when denied adherence and the STAD.ARR cell line enters into cell cycle arrest under the identical suspended conditions. We have shown that cyclin A and cyclin D mRNA and protein are down regulated when cells are denied adherence for 24 hours in tissue culture wells previously coated with poly-HEMA. To test whether cyclin A was able to rescue cells from cell cycle arrest and/or anoikis by overriding the cell cycle machinery we transfected the full length cDNA in to each cell type. Surprisingly we found that anoikis and cell cycle arrest due to suspended conditions was not affected by overexpression of cyclin A protein, but that growth under adhered conditions was reduced compared to vector alone control transfectants. Further, we transfected other cell lines; ST7, gastric cancer, MDA-MB-4.35, breast cancer, and HPB T-cell leukemic and in no case were suspended culturing conditions overcome by cyclin A. This result indicates an additional level of regulation for the cell cycle machinery. Additionally, soluble collagen was shown to be able to save from anoikis and also from cell cycle arrest while the β1 specific mAb 33B6 was only able to save from anoikis. Immunofluorescent studies show that soluble collagen creates clusters of β1 with FAK and also β1 with actin in the STAD.ARR cells but does not in the STAD.APO cells. This result indicates that the phenotypes under suspended conditions between these cell lines may diverge at their requirements for integrin ligation. Additionally we characterized the nature of anoikis by showing cytochrome c release, caspase 3, p21 and p53 activation in STAD.APO cells. Thus, our results have implications in the understanding of integrin biology and neoplastic progression. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

Bone marrow-derived stem/progenitor cells play an important role in the growth of Ewing's sarcoma tumors

Both angiogenesis and vasculogenesis contribute to the formation and expansion of tumor neovasculature. We demonstrated that bone marrow (BM)-derived cells migrated to TC71 Ewing's tumors and differentiated into endothelial cells lining perfused, functional tumor neovessels. In addition, a substantial fraction of recruited, BM-derived cells resided in the vessel vicinity but did not demonstrate endothelial differentiation. Rather, these perivascular cells expressed desmin and PDGFR-β, implying pericyte-like/vascular smooth muscle cell differentiation. No defined, consensus set of markers exists for endothelial progenitor cells (EPCs) and the specific subsets of BM cells that participate in vessel formation are poorly understood. We used a functional in vivo assay to investigate the roles performed by specific human- and murine-derived stem/progenitor subpopulations within Ewing's sarcoma tumors. CD34 +45+, CD34+38-, VEGFR2 + and Sca1+Gr1+ cells were demonstrated to establish residence within the expanding tumor vascular network and differentiate into endothelial cells and pericytes. By constrast, CD34-45 + and Sca1-Gr1+ cells predominantly localized to sites outside the Ewing's tumor vasculature, and differentiated into macrophages. Cytokines, such as VEGF, influence the recruitment of BM cells and their incorporation into the tumor vasculature. VEGF165-inhibited TC/siVEGF7-1 Ewing's tumors showed delayed in vivo tumor growth, decreased vessel density, and reduced infiltration of BM progenitor cells. We tested whether another chemoattractant, Stromal Cell-Derived Factor-1 (SDF-1), could augment the growth of these VEGF165-inhibited TC/siVEGF 7-1 tumors by enhancing the recruitment of BM cells and stimulating neovasculature expansion. SDF-1 promoted progenitor cell chemotaxis and retainment of BM-derived pericyte precursors in close association with functional, perfused tumor blood vessels. Treatment of TC/siVEGF7-1 tumors with adenovirus-SDF-1α resulted in augmented tumor size, enhanced pericyte coverage of tumor neovessels, remodeling of vascular endothelium into larger, functional structures, and upregulation of PDGF-BB, with no effect on VEGF165. Taken together, these findings suggest that the recruitment of BM stem/progenitor cells plays an important role in the growth of Ewing's tumors. ^

The role of SRC family kinase activation in prostate cancer growth and lymph node metastasis

Aberrant expression and/or activation of Src Family of non-receptor protein tyrosine kinases (SFKs) occur frequently during progressive stages of multiple types of human malignancies, including prostate cancer. Two SFKs, Src and Lyn, are expressed and implicated in prostate cancer progression. Work in this dissertation investigated the specific roles of Src and Lyn in the prostate tumor progression, and the effects of SFK inhibition on prostate tumor growth and lymph node metastasis in pre-clinical mouse models. ^ Firstly, using a pharmacological inhibitor of SFKs in clinical trials, dasatinib, I demonstrated that SFK inhibition affects both cellular migration and proliferation in vitro. Systemic administration of dasatinib reduced primary tumor growth, as well as development of lymph node metastases, in both androgen-sensitive and -resistant orthotopic prostate cancer mouse models. Immunohistochemical analysis of the primary tumors revealed that dasatinib treatment decreased SFK phosphorylation but not expression, resulting in decreased cellular proliferation and increased apoptosis. For this analysis of immunohistochemical stained tissues, I developed a novel method of quantifying immunohistochemical stain intensity that greatly reduced the inherent bias in analyzing staining intensity. ^ To determine if Src and Lyn played overlapping or distinct roles in prostate cancer tumor growth and progression, Src expression alone was inhibited by small-interfering RNA. The resulting stable cell lines were decreased in migration, but not substantially affected in proliferation rates. In contrast, an analogous strategy targeting Lyn led to stable cell lines in which proliferation rates were significantly reduced. ^ Lastly, I tested the efficacy of a novel SFK inhibitor (KX2-391) targeting peptide substrate-binding domain, on prostate cancer growth and lymph node metastasis in vivo. I demonstrated that KX2-391 has similar effects as dasatinib, an ATP-competitive small molecular inhibitor, on both the primary tumor growth and development of lymph node metastasis in vivo, work that contributed to the first-in-man Phase I clinical trial of KX2-391. ^ In summary, studies in this dissertation provide the first demonstration that Src and Lyn activities affect different cellular functions required for prostate tumor growth and metastasis, and SFK inhibitors effectively reduce primary tumor growth and lymph node metastasis. Therefore, I conclude that SFKs are promising therapeutic targets for treatment of human prostate cancer. ^

Roles of insulin-like growth factor binding protein-3 in gastrointestinal stromal tumors

Imatinib mesylate, a selective inhibitor of KIT, PDGFR, and Abl kinases, has shown significant success as a therapy for patients with advanced gastrointestinal stromal tumors (GISTs). However, the underlying mechanisms of imatinib-induced cytotoxicity are not well understood. Using gene expression profiling and real-time PCR for target validation, we identified insulin-like growth factor binding protein-3 (IGFBP3) to be to be up-regulated after imatinib treatment in imatinib-sensitive GISTs. IGFBP3 is a multifunctional protein that regulates cell proliferation and survival and mediates the effects of a variety of anti-cancer agents through IGF-dependent and IGF-independent mechanisms. Therefore, we hypothesized that IGFBP3 mediates GIST cell response to imatinib. To test this hypothesis, we manipulated IGFBP3 protein levels in two KIT mutant, imatinib-sensitive GIST cell lines and assessed the resultant changes in cell viability, survival, and imatinib sensitivity. In GIST882 cells, endogenous IGFBP3 was required for cell viability. However, inhibiting imatinib-induced IGFBP3 up-regulation by RNA interference or neutralization resulted in reduced drug sensitivity, suggesting that IGFBP3 sensitizes GIST882 cells to imatinib. GIST-T1 cells, on the other hand, had no detectable levels of endogenous IGFBP3, nor did imatinib induce IGFBP3 up-regulation, in contrast to our previous findings. IGFBP3 overexpression in GIST-T1 cells reduced viability but did not induce cell death; rather, the cells became polyploid through a mechanism that may involve attenuated Cdc20 expression and securin degradation. Moreover, IGFBP3 overexpression resulted in a loss of KIT activation and decreased levels of mature KIT. Consistent with this, GIST-T1 cells overexpressing IGFBP3 were less sensitive to imatinib. Furthermore, as neither GIST882 cells nor GIST-T1 cells expressed detectable levels of IGF-1R, IGFBP3 is likely not exerting its effects by modulating IGF signaling through IGF-1R or IR/IGF-1R hybrid receptors in these cell lines. Collectively, these findings demonstrate that IGFBP3 has cell-dependent effects and would, therefore, not be an ideal marker for identifying imatinib response in GISTs. Nevertheless, our results provide preliminary evidence that IGFBP3 may have some therapeutic benefits in GISTs. ^

Activation of c -Met contributes to growth and metastasis of human colorectal carcinoma

c-Met is the protein tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) and mediates several normal cellular functions including proliferation, survival, and migration. Overexpression of c-Met correlates with progression and metastasis of human colorectal carcinoma (CRC). The goals of this study were to determine if overexpression of c-Met directly contributes to tumorigenicity and liver metastatic potential of colon cancer, and what are the critical pathways regulated by c-Met in this process. The studies used two colon tumor cell lines, KM12SM and KM20, which express high levels of constitutively active c-Met and are highly metastatic in nude mice. To examine the effects of c-Met overexpression, subclones of theses lines with reduced c-Met expression were obtained following transfection with a c-Met specific targeting ribozyme. Reduction of c-Met in KM12SM cells abolished liver metastases when cells were injected intrasplenically in an experimental metastasis assay. However, c-Met downregulation in theses clones was unstable. Three stable KM20 clones with a 25–35% reduction in c-Met protein levels but 60–90% reduction in basal c-Met autophosphorylation and kinase activity were obtained. While HGF increased c-Met kinase activity in the clones with reduced c-Met, the activity was less than that observed in parental or control transfected cells. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates, soft-agar colony forming abilities, and increased apoptosis. HGF/SF treatment did not affect anchorage-dependent growth or soft-agar colony forming abilities. Further, c-Met downregulation significantly impaired the ability of HGF/SF to induce migration. To examine the effects of reduced c-Met on tumor formation, parental and c-Met reduced KM20 cells were grown subcutaneously and intrahepatically in nude mice. c-Met downregulation delayed, but did not abolish growth at the subcutaneous site. When these cells were injected intrahepatically, both tumor incidences and size were significantly reduced. To further understand the molecular basis of c-Met in promoting tumor growth, the activation of several signaling intermediates that have been implicated in c-Met mediated growth, survival and migration were compared between KM20 parental cells and subclones with reduced c-Met expression levels. The expression and activity (as determined by phosphorylation) of AKT and Erk1/2 were unaltered. In contrast, Src kinase activity, as measured by immune complex kinase assay, was reduced 2–5 fold following c-Met downregulation. As Src has been implicated in growth, survival and migration, Src activation in c-Met overexpressing lines is likely contributing to the tumorigenic and metastatic capabilities of colon tumor cell lines that overexpress c-Met. Collectively, these results suggest that c-Met overexpression plays a causal role in the development of CRC liver metastases, and that c-Src and c-Met inhibitors may be of potential therapeutic benefit for late-stage colon cancer. ^