Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis.

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

Ras signaling in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is important in immunologic cytotoxicity, autoimmunity, sepsis, normal embryonic development, and wound healing. TNF exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. We found that enforced expression of an activated H-ras oncogene converted the non-tumorigenic TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells (10TEJ) that also became very sensitive to TNF-induced apoptosis. This finding suggested that the oncogenic form of H-Ras, in which the p21 is locked in the GTP-bound form, could play a role in TNF-induced apoptosis of these cells. To investigate whether Ras activation is an obligatory step in TNF-induced apoptosis, we introduced two different molecular antagonists of Ras, namely the Rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras transformed 10TEJ cells. Expression of either Rap1A or RasN17 in 10TEJ cells resulted in abrogation of TNF-induced apoptosis. Similar results were obtained by expression of either Ras antagonist in L929 cells, a fibroblast cell line that is sensitive to TNF-induced apoptosis but does not have a ras mutation. The effects of Rap-1A and RasN17 appear to be specific to TNF, since cytotoxicity induced by doxorubicin and thapsigargin are unaffected. Additionally, constitutive apoptosis sensitivity in isolated nuclei, as measured by activation of Ca$\sp{2+}$-dependent endogenous endonuclease, is not affected by Rap-1A or RasN17. Moreover, TNF treatment of L929 cells increased Ras-bound GTP, indicating that Ras activation is triggered by TNF. Thus, Ras activation is required for TNF-induced apoptosis in mouse cells. ^

K1 protein of human herpesvirus 8 suppresses lymphoma cell Fas-mediated apoptosis.

Expression of the K1 gene of human herpesvirus 8 activates nuclear factor-kappaB and induces lymph node hyperplasia and lymphomas in transgenic mice. To further delineate its role in cell survival, we determined whether K1 altered apoptosis of lymphoma cells. K1 protein is expressed in Kaposi sarcoma and primary effusion lymphoma. We retrovirally transfected BJAB lymphoma, THP-1, U937, and Kaposi sarcoma SLK cells to express K1 and a K1 mutant with the deleted immunoreceptor tyrosine-based activation motif (K1m). We challenged cells with an agonistic anti-Fas antibody, Fas ligand, irradiation, and tumor necrosis factor-related apoptosis-inducing ligand. K1 transfectants but not K1m transfectants exhibited reduced levels of apoptosis induced by the anti-Fas antibody but not apoptosis induced by the tumor necrosis factor-related apoptosis-inducing ligand or irradiation. K1 expression resulted in reduced apoptosis rates as shown in several assays. K1 induced a modest reduction in levels of Fas-associated death domain protein, and procaspase 8 recruited to the death-inducing signaling complex. Finally, K1 transfectants cleaved procaspase 8 at significantly lower rates than did K1m transfectants. K1-transfected mice, compared with vector-transfected mice, showed lower death rates after challenge with anti-Fas antibody. K1 may contribute to lymphoma development by stimulating cell survival by selectively blocking Fas-mediated apoptosis.

TYPE I INSULIN-LIKE GROWTH FACTOR RECEPTOR TYROSINE KINASE AS A MOLECULAR TARGET IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoid malignancy representing 5-10% of all non-Hodgkin’s lymphomas. It is distinguished by the t(11;14)(q13;q32) chromosomal translocation that juxtaposes the proto-oncogene CCND1, which encodes cyclin D1 at 11q13 to the IgH gene at 14q32. MCL patients represent about 6% of all new cases of Non-Hodgkin’s lymphomas per year or about 3,500 new cases per year. MCL occurs more frequently in older adults – the average age at diagnosis is the mid-60s with a male-to-female ratio of 2-3:1. It is typically characterized by the proliferation of neoplastic B-lymphocytes in the mantle zone of the lymph node follicle that have a prominent inclination to disseminate to other lymphoid tissues, bone marrow, peripheral blood and other organs. MCL patients have a poor prognosis because they develop resistance/relapse to current non-specific therapeutic regimens. It is of note that the exact molecular mechanisms underlying the pathogenesis of MCL are not completely known. It is reasonable to anticipate that better characterization of these mechanisms could lead to the development of specific and likely more effective therapeutics to treat this aggressive disease. The type I insulin-like growth factor receptor (IGF-IR) is thought to be a key player in several different solid malignancies such as those of the prostate, breast, lung, ovary, skin and soft tissue. In addition, recent studies in our lab showed evidence to support a pathogenic role of IGF-IR in some types of T-cell lymphomas and chronic myeloid leukemia. Constitutively active IGF-IR induces its oncogenic effects through the inhibition of apoptosis and induction of transformation, metastasis, and angiogenesis. Previous studies have shown that signaling through IGF-IR leads to the vi activation of multiple signaling transduction pathways mediated by the receptor-associated tyrosine kinase domain. These pathways include PI3K/Akt, MAP kinase, and Jak/Stat. In the present study, we tested the possible role of IGF-IR in MCL. Our results demonstrate that IGF-IR is over-expressed in mantle cell lymphoma cell lines compared with normal peripheral blood B- lymphocytes. Furthermore, inhibition of IGF-IR by the cyclolignan picropodophyllin (PPP) decreased cell viability and cell proliferation in addition to induction of apoptosis and G2/M cell cycle arrest. Screening of downstream oncogenes and apoptotic proteins that are involved in both IGF-IR and MCL signaling after treatment with PPP or IGF-IR siRNA showed significant alterations that are consistent with the cellular changes observed after PPP treatment. Therefore, our findings suggest that IGF-IR signaling contributes to the survival of MCL and thus may prove to be a legitimate therapeutic target in the future.

Stromal derived growth factor alpha (SDF-1α) induces the differentiation of bone marrow progenitor cells (BMCs) into pericytes by regulating platelet derived growth factor B (PDGF-B) transcription

We previously demonstrated that bone marrow cells (BMCs) migrate to TC71 and A4573 Ewing’s sarcoma tumors where they can differentiate into endothelial cells (ECs) and pericytes and, participate in the tumor vascular development. This process of neo-vascularization, known as vasculogenesis, is essential for Ewing’s sarcoma growth with the soluble vascular endothelial growth factor, VEGF165, being the chemotactic factor for BMC migration to the tumor site. Inhibiting VEGF165 in TC71 tumors (TC/siVEGF7-1) inhibited BMC infiltration to the tumor site and tumor growth. Introducing the stromal-derived growth factor (SDF-1α) into the TC/siVEGF7-1 tumors partially restored vasculogenesis with infiltration of BMCs to a perivascular area where they differentiated into pericytes and rescued tumor growth. RNA collected from the SDF-1α-treated TC/siVEGF7-1 tumors also revealed an increase in platelet-derived growth factor B (PDGF-B) mRNA levels. PDGF-B expression is elevated in several cancer types and the role of PDGF-B and its receptor, PDGFR-β, has been extensively described in the process of pericyte maturation. However, the mechanisms by which PDGF-B expression is up-regulated during vascular remodeling and the process by which BMCs differentiate into pericytes during tumor vasculogenesis remain areas of investigation. In this study, we are the first to demonstrate that SDF-1α regulates the expression of PDGF-B via a transcriptional mechanism which involves binding of the ELK-1 transcription factor to the pdgf-b promoter. We are also first to validate the critical role of the SDF-1α/PDGF-B pathway in the differentiation of BMCs into pericytes both in vitro and in vivo. SDF-1α up-regulated PDGF-B expression in both TC/siVEGF7-1 and HEK293 cells. In contrast, down-regulating SDF-1α, down-regulated PDGF-B. We cloned the 2 kb pdgf-b promoter fragment into the pGL3 reporter vector and showed that SDF-1α induced pdgf-b promoter activity. We used chromatin immunoprecipitation (ChIP) and demonstrated that the ELK-1 transcription factor bound to the pdgf-b promoter in response to SDF-1α stimulation in both TC/siVEGF7-1 and HEK293 cells. We collected BMCs from the hind femurs of mice and cultured the cells in medium containing SDF-1α and PDGF-B and found that PDGFR-β+ BMCs differentiated into NG2 and desmin positive pericytes in vitro. In contrast, inhibiting SDF-1α and PDGF-B abolished this differentiation process. In vivo, we injected TC71 or A4573 tumor-bearing mice with the SDF-1α antagonist, AMD3100 and found that inhibiting SDF-1α signaling in the tumor microenvironment decreased the tumor microvessel density, decreased the tumor blood vessel perfusion and, increased tumor cell apoptosis. We then analyzed the effect of AMD3100 on vasculogenesis of Ewing’s sarcoma and found that BMCs migrated to the tumor site where they differentiated into ECs but, they did not form thick perivascular layers of NG2 and desmin positive pericytes. Finally, we stained the AMD3100-treated tumors for PDGF-B and showed that inhibiting SDF-1α signaling also inhibited PDGF-B expression. All together, these findings demonstrated that the SDF-1α/PDGF-B pathway plays a critical role in the formation of BM-derived pericytes during vasculogenesis of Ewing’s sarcoma tumors.

Antibodies targeting hepatoma-derived growth factor as a novel strategy in treating lung cancer.

Hepatoma-derived growth factor (HDGF) is overexpressed in lung cancer and the overexpression correlates with aggressive biological behaviors and poor clinical outcomes. We developed anti-HDGF monoclonal antibodies and tested their antitumor activity in lung cancer xenograft models. We also determined biological effects in tumors treated with the antibody alone or in combination with bevacizumab/avastin (an anti-vascular endothelial growth factor antibody) and/or gemcitabine (a chemotherapeutic agent). We found the anti-HDGF was effective to inhibit tumor growth in non-small cell lung cancer xenograft models. In the A549 model, compared with control IgG, tumor growth was substantially inhibited in animals treated with anti-HDGF antibodies, particularly HDGF-C1 (P = 0.002) and HDGF-H3 (P = 0.005). When HDGF-H3 was combined with either bevacizumab or gemcitabine, we observed enhanced tumor growth inhibition, particularly when the three agents were used together. HDGF-H3-treated tumors exhibited significant reduction of microvessel density with a pattern distinctive from the microvessel reduction pattern observed in bevacizumab-treated tumors. HDGF-H3-treated but not bevacizumab-treated tumors also showed a significant increase of apoptosis. Interestingly, many of the apoptotic cells in HDGF-H3-treated tumors are stroma cells, suggesting that the mechanism of the antitumor activity is, at least in part, through disrupting formation of tumor-stroma structures. Our results show that HDGF is a novel therapeutic target for lung cancer and can be effectively targeted by an antibody-based approach.

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.

Mechanisms of apoptosis in chronic lymphocytic leukemia (CLL)

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by the accumulation of terminally differentiated, mature B cells that do not progress beyond the G1 stage of cell cycle, suggesting that these cells possess intrinsic defects in apoptosis. Treatment relies heavily on chemotherapy (primarily nucleoside analogs and glucocorticoids) that may initially be effective in patients, but ultimately give rise to refractory, untreatable disease. The purpose of this study was to determine whether key components of the apoptotic machinery were intact in CLL lymphocytes, especially in patients refractory to therapy. ^ Activation of proteases has been shown to be at the core of the apoptotic pathway and this work demonstrates that protease activation is required for glucocorticoid and nucleoside analog-induced apoptosis in CLL cells. Inhibitors of serine proteases as well as caspase inhibitors blocked induced DNA fragmentation, and a peptide inhibitor of the nuclear scaffold (NS) protease completely suppressed both induced and spontaneous apoptosis. However, the NS protease inhibitor actually promoted several pro-apoptotic events, such as caspase activation, exposure of surface phosphatidylserine, and loss of mitochondrial membrane potential. These results suggested that the NS protease may interact with the apoptotic program in CLL cells at two separate points. ^ In order to further investigate the role of the NS protease in CLL, patient isolates were treated with proteasome inhibitors because of previous results suggesting that the ISIS protease might be a β subunit of the proteasome. Proteasome inhibitors induced massive DNA fragmentation in every patient tested, even in those resistant to the effects of glucocorticoid and nucleoside analogs in vitro. Several other features of apoptosis were also promoted by the proteasome inhibitor, including mitochondrial alterations such as release of cytochrome c and drops in mitochondrial membrane potential. Proteasome inhibitor-induced apoptosis was associated with inhibition of NFκB, a proteasome-regulated transcription factor that has been implicated in the suppression of apoptosis in a number of systems. The NS protease inhibitor also caused a decrease in active NFκB, suggesting that the proapoptotic effects of this agent might be due to depletion of NFκB. ^ Given these findings, the role of NFκB, in conferring survival in CLL was investigated. Glucocorticoid hormone treatment was shown to cause decreases in the activity of the transcription factor, while phorbol dibutyrate, which blocks glucocorticoid-induced DNA fragmentation, was capable of upregulating NFκB. Compellingly, introduction of an undegradable form of the constitutive NFκB inhibitor, IκB, caused DNA fragmentation in several patient isolates, some of which were resistant to glucocorticoid in vitro. Transcription of anti-apoptotic proteins by NFκB was postulated to be responsible for its effects on survival, but Bcl-2 levels did not fluctuate with glucocorticoid or proteasome inhibitor treatment. ^ The in vitro values generated from these studies were organized into a database containing numbers for over 250 patients. Correlation of relevant clinical parameters revealed that levels of spontaneous apoptosis in vitro differ significantly between Rai stages. Importantly, in vitro resistance to nucleoside analogs or glucocorticoids predicted resistance to chemotherapy in vivo, and inability to achieve remission. ^

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. ^

POU domain transcription factor Brn3b is critical for the normal development and survival of retinal ganglion cells

The POU domain transcription factor Brn3b/POU4F2 plays a critical role regulating gene expression in mouse retinal ganglion cells (RGCs). Previous investigations have shown that Brn3b is not required for initial cell fate specification or migration; however, it is essential for normal RGC differentiation. In contrast to wild type axons, the mutant neurites were phenotypically different: shorter, rougher, disorganized, and poorly fasciculated. Wild type axons stained intensely with axon specific marker tau-1, while mutant projections were weakly stained and the mutant projections showed strong labeling with dendrite specific marker MAP2. Brn-3b mutant axonal projections contained more microtubules and fewer neurofilaments, a dendritic characteristic, than the wild type. The mutant neurites also exhibited significantly weaker staining of neurofilament low-molecular-weight (NF-L) in the axon when compared to the wild type, and NF-L accumulation in the neuron cell body. The absence of Brn-3b results in an inability to form normal axons and enhanced apoptosis in RGCs, suggesting that Brn-3b may control a set of genes involved in axon formation. ^ Brn3b contains several distinct sequence motifs: a glycine/serine rich region, two histidine rich regions, and a fifteen amino acid conserved sequence shared by all Brn3 family members in the N-terminus and a POU specific and POU homeodomain in the C-terminus. Brn3b activates a Luciferase reporter over 25 fold in cell culture when binding to native brn3 binding sites upstream of a minimal promoter. When fused to the Gal4 DNA Binding domain (DBD) and driven by either a strong (CMV) or weaker (pAHD) promoter, the N-terminal of Brn3b is capable of similar activation when binding to Gal4 UAS sites, indicating a presumptive activator of transcription. Both full length Brn3b or the C-terminus fused to the Gal4DBD and driven by pCMV repressed a Luciferase reporter downstream of UAS binding sites. Lower levels of expression of the fusion protein driven by pADH resulted in an alleviation of repression. This repression appears to be a limitation of this system of transcriptional analysis and a potential pitfall in conventional pCMV based transfection assays. ^

The transcription factor nuclear factor kappa B (NFkappaB) maintains TRAIL resistance in human pancreatic cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) is a member of the TNF family of cytokines that induces apoptosis in a variety of tumor cells while sparing normal cells. However, many human cancer cell lines display resistance to TRAIL-induced apoptosis and the mechanisms contributing to resistance remain controversial. Previous studies have demonstrated that the dimeric transcription factor Nuclear Factor kappa B (NFκB) is constitutively active in a majority of human pancreatic cancer cell lines and primary tumors, and although its role in tumor progression remains unclear it has been suggested that NFκB contributes to TRAIL resistance. Based on this, I examined the effects of NFκB inhibitors on TRAIL sensitivity in a panel of nine pancreatic cancer cell lines. I show here that inhibitors of NFκB, including two inhibitors of the proteasome (bortezomib (Velcade™, PS-341) and NPI-0052), a small molecule inhibitor of IKK (PS1145), and a novel synthetic diterpene NIK inhibitor (NPI-1342) reverse TRAIL resistance in pancreatic cancer cell lines. Further analysis revealed that the expression of the anti-apoptosic proteins BclXL and XIAP was significantly decreased following exposure to these inhibitors alone and in combination with TRAIL. Additionally, treatment with NPI0052 and TRAIL significantly reduced tumor burden relative to the control tumors in an L3.6pl orthotopic pancreatic xenograft model. This was associated with a significant decrease in proliferation and an increase in caspase 3 and 8 cleavage. Combination therapy employing PS1145 or NPI-1342 in combination with TRAIL also resulted in a significant reduction in tumor burden compared to either agent alone in a Panc1 orthotopic xenograft model. My studies show that combination therapy with inhibitors of NFκB alone and TRAIL is effective in pre-clinical models of pancreatic cancer and suggests that the approach should be evaluated in patients. ^

Mechanism of IFN-induced apoptosis and sensitization by the proteasome inhibitor bortezomib in bladder cancer cells

Bladder cancer is the fifth most common cancer with more than 50,000 cases diagnosed each year. Interferon-α (IFNα) is mostly used in combination with BCG for the treatment of transitional cell carcinoma (TCC). To examine the effects of IFNα on bladder cancer cells, I analyzed a panel of 20 bladder cancer cell lines in terms of their sensitivity to IFNα-induced apoptosis and the underlying mechanisms. I identified three categories: cells that die after 48hr, after 72h, and cells resistant even after 72hr of IFNα treatment. Examination of the IFN-signal transduction pathway revealed that the defect was not due to abrogation of IFN signaling. Further analysis demonstrated dependency of IFN-induced apoptosis on caspase-8, implicating the role of death receptors in IFN-induced cell death. Of the six most-IFN-sensitive cell lines, the majority upregulated Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) at the mRNA and protein level and IFN-induced cell death was mediated through TRAIL, while a minority of the most IFN-sensitive cells undergo apoptosis through a TNFα-dependent mechanism. IFNα resistance was due to either absence of TRAIL upregulation at the mRNA or protein level, resistance to exogenous rhTRAIL itself or lack of sensitization to IFN-induced cell death. Downregulation of XIAP, or XIAP inactivation through its regulator NFκB has been reported to sensitize tumor cells to death receptor-induced cell death. Baseline and IFN-inducible XIAP levels were examined in the most and least IFN-sensitive cells, knocking down XIAP and the p65 subunit of NFκB enhanced IFN-induced cell death, implicating XIAP downregulation as a mechanism through which bladder cancer cells are sensitized to IFN-induced apoptosis. To determine whether or not the proteasome inhibitor Bortezomib (BZ) sensitizes bladder cancer cells to IFN-induced cell death, the combined effects of IFN+BZ and the underlying molecular mechanisms were examined both in vitro and in vivo using two bladder xenograft models. In both models, tumor growth inhibition was the result of either increased cell death of tumor cells exerted by the two agents and/or inhibition of angiogenesis. In vitro, MAP downregulation in response to the combined treatment of IFN+BZ accounts for one of the mechanisms mediating IFN+BZ cell death in bladder cancer cells. ^

Reactive oxygen species-dependent mechanisms of N-(4-hydroxyphenyl)retinamide (4HPR)-induced apoptosis in human carcinoma cells

4HPR is a synthetic retinoid that has shown chemopreventive and therapeutic efficacy against premalignant and malignant lesions including oral leukoplakia, ovarian and breast cancer, and neuroblastoma. 4HPR induces apoptosis in various cancer cells and production of reactive oxygen species (ROS) has been suggested as a possible cause underlying these effects. However, the mechanisms governing these effects by 4HPR are not fully elucidated. In this study, we explored the mechanisms of 4HPR-induced ROS increase and apoptosis in human cancer cells. ^ First, we identified genes modulated by 4HPR using oligonucleotide gene expression arrays and found that they fall into specific functional canonical pathways and gene networks using Ingenuity Pathways Analysis®. Further analysis has shown that 4HPR induced up-regulation of Endoplasmic Reticulum (ER)-related genes such as Heat shock proteins 70 and 90 and the transcriptional factor, GADD153. These findings were validated using quantitative real-time PCR. ^ Second, we found that 4HPR induced extensive ER stress evidenced by dilation of the ER and endoribonuclease-mediated splicing and activation of the transcriptional factor, XBP-1. In addition, 4HPR induced the up-regulation of various ER stress-related genes and their protein products, as well as cleavage and activation of the ER specific Caspase-4. Concomitantly with XBP-1 splicing, all of these effects were dependent on ROS generation by 4HPR. Furthermore, chemical inhibition and RNA interference studies revealed a novel pro-apoptotic role for HSP70/A1A in 4HPR-mediated apoptosis. ^ Third, we observed rapid activation of the small GTPase Rac by 4HPR which was upstream of ROS generation. Inhibition of Rac activity or silencing of its expression by RNA interference inhibited ROS generation and apoptosis induction by 4HPR. siRNA targeting PAK1 and expression of a dominant negative Rac, decreased 4HPR-mediated ROS generation, while expression of a constitutive active Rac increased basal and 4HPR-induced ROS generation and PARP cleavage. Furthermore, metastatic cancer cells exhibited higher Rac activation, ROS generation, and cell growth inhibition due to 4HPR exposure compared to their primary cancer cell counterparts. ^ These findings provide novel insights into 4HPR-mediated ROS generation and apoptosis induction and support the use of ROS inducing agents such as 4HPR against metastatic cancer cells. ^