Upregulation of defensins in burn sheep small intestine.

OBJECTIVE: The aim of this study was to visualize and localize the sheep antimicrobials, beta-defensins 1, 2, and 3, (SBD-1, SBD-2, SBD-3), sheep neutrophil defensin alpha (SNP-1), and the cathelicidin LL-37 in sheep small intestine after burn injury, our hypothesis being that these compounds would be upregulated in an effort to overcome a compromised endothelial lining. Response to burn injury includes the release of proinflammatory cytokines and systemic immune suppression that, if untreated, can progress to multiple organ failure and death, so protective mechanisms have to be initiated and implemented. METHODS: Tissue sections were probed with antibodies to the antimicrobials and then visualized with fluorescently labeled secondary antibodies and subjected to fluorescence deconvolution microscopy and image reconstruction. RESULTS: In both the sham and burn samples, all the aforementioned antimicrobials were seen in each of the layers of small intestine, the highest concentration being localized to the epithelium. SBD-2, SBD-3, and SNP-1 were upregulated in both enterocytes and Paneth cells, while SNP-1 and LL-37 showed increases in both the inner circular and outer longitudinal muscle layers of the muscularis externa following burn injury. Each of the defensins, except SBD-1, was also seen in between the muscle layers of the externa and while burn caused slight increases of SBD-2, SBD-3, and SNP-1 in this location, LL-37 content was significantly decreased. CONCLUSION: That while each of these human antimicrobials is present in multiple layers of sheep small intestine, SBD-2, SBD-3, SNP-1, and LL-37 are upregulated in the specific layers of the small intestine.

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction.

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

Upregulation of Reactive Oxygen Species During the Retrovirus Life Cycle and Their Roles in a Mutant of Moloney Murine Leukemia Virus, ts1-Mediated Neurodegeneration

Viral invasion of the central nervous system (CNS) and development of neurological symptoms is a characteristic of many retroviruses. The mechanism by which retrovirus infection causes neurological dysfunction has yet to be fully elucidated. Given the complexity of the retrovirus-mediated neuropathogenesis, studies using small animal models are extremely valuable. Our laboratory has used a mutant moloney murine leukemia retrovirus, ts1-mediated neurodegneration. We hypothesize that astrocytes play an important role in ts1-induced neurodegeneration since they are retroviral reservoirs and supporting cells for neurons. It has been shown that ts1 is able to infect astrocytes in vivo and in vitro. Astrocytes, the dominant cell population in the CNS, extend their end feet to endothelial cells and neuronal synapse to provide neuronal support. Signs of oxidative stress in the ts1-infected CNS have been well-documented from previous studies. After viral infection, retroviral DNA is generated from its RNA genome and integrated into the host genome. In this study, we identified the life cycle of ts1 in the infected astrocytes. During the infection, we observed reactive oxygen species (ROS) upregulations: one at low levels during the early infection phase and another at high levels during the late infection phase. Initially we hypothesized that p53 might play an important role in ts1-mediated astrocytic cell death. Subsequently, we found that p53 is unlikely to be involved in the ts1-mediated astrocytic cell death. Instead, p53 phosphorylation was increased by the early ROS upregulation via ATM, the protein encoded by the ataxia-telangiectasia (A-T) mutated gene. The early upregulation of p53 delayed viral gene expression by suppressing expression of the catalytic subunit of NADPH oxidase (NOX). We further demonstrated that the ROS upregulation induced by NOX activation plays an important role in establishing retroviral genome into the host. Inhibition of NOX decreased viral replication and delayed the onset of pathological symptoms in ts1-infected mice. These observations lead us to conclude that suppression of NOX not only prevents the establishment of the retrovirus but also decreases oxidative stress in the CNS. This study provides us with new perspectives on the retrovirus-host cell interaction and sheds light on retrovirus-induced neurodegeneration as a result of the astrocyte-neuron interaction.

Effects of butyrate on the colon cancer cell phenotype-chemosensitization and upregulation of proteins implicated in cancer progression

Colon cancer is the second leading cause of cancer mortality in the U.S. Surgery is the only truly effective human colon cancer (HCC) therapy due to marked intrinsic drug resistance. The inefficacy of therapies developed for metastatic HCC suggests that advances in colon cancer chemoprevention and chemotherapy will be needed to reduce HCC mortality. The dietary fiber metabolite butyrate (NaB) is a candidate cancer chemopreventive agent that inhibits growth, promotes differentiation and stimulates apoptosis of HCC cells. Epidemiological and experimental studies suggest that dietary fiber protects against the development of HCC, however, recent large prospective trials have not found significant protection. ^ The first central hypothesis of this dissertation project is that the diversity of phenotypic changes induced by NaB in HCC cells includes molecular alterations that oppose its chemopreventive action and thereby limit its efficacy. We investigated the effect of NaB on the expression/activity of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) in HCC HT29 cells. NaB treatment induced a 13-fold increase in EGFR expression in concert with its chemopreventive action in vitro, i.e., induction of growth suppression and G1 arrest, apoptosis and a differentiated phenotype. NaB-induced EGFR was active based on multiple lines of evidence. The EGFR was: (1) heavily phosphorylated at Tyrosine (P-Tyr); (2) associated with the cytoskeleton; (3) localized at the cell surface, and activated in response to EGF; and (4) NaB treatment of the cells induced activation of the EGFR effector Erk1/2. NaB treatment also induced a 7-fold increase in COX-2 expression. The NaB-induced COX-2 was active based on significantly increased PGE2 production. ^ The second central hypothesis is that NaB treatment would render HCC cells more chemosensitive to chemotherapy agents based on the increased apoptotic index induced by NaB. NaB treatment chemosensitized HT29 cells to 5-FU and doxorubicin, despite increases in the expression of P-glycoprotein and a related drug resistance protein (MRP). ^ These results raise the intriguing possibility that the chemopreventive effects of fiber may require concomitant treatment with EGFR and/or COX-2 inhibitors. Similarly, NaB may be a rational drug to combine with existing chemotherapeutic agents for the management of advanced HCC patients. ^

Transcriptional upregulation of the p21Cip1 promoter by STAT3 and nuclear ErbB2

Over-expression of the receptor tyrosine kinase ErbB2 is prevalent in approximately 30% of human breast carcinomas and confers Taxol resistance. In breast cancer cells, Taxol induces tubulin polymerization and hyperstable microtubule formation. This in turn prematurely activates Cdc2 kinase allowing early entry into the G2/M phase of the cell cycle resultant in mitotic catastrophe followed by apoptosis. Over-expression of ErbB2 upregulates p21Cip1, which inhibits Cdc2 activation, and leads to Taxol resistance in patients. However, the mechanism of ErbB2-mediated p21 Cip1 upregulation is unclear. Here in this study, we investigated the mechanism of ErbB2 downstream signaling events leading to upregulation. The CDKN1A (p21Cip1) gene promoter contains numerous cis-elements including a Signal transducer and activator of transcription (STAT) Inducable Element (SIE) located at -679 kb. Our studies showed ErbB2 overexpressing cells had increased activated levels of STAT3, and therefore we hypothesized that STAT3 is responsible for the upregulation of the p21Cip1 promoter by ErbB2. EMSA and ChIP assays confirmed the binding of STAT3 to the p21Cip1 promoter and luciferase assays showed higher p21 Cip1 promoter activity in ErbB2 over-expressing transfectants when compared to parental cells, in a STAT3 binding site dependant manner. Additionally, reduced level of STAT3 led to reduced p21Cip1 protein expression and promoter activity indicating that both the STAT3 binding site and STAT3 protein are required for ErbB2-mediated p21Cip1 upregulation. Further investigation of ErbB2 downstream signaling showed increased Src kinase activity in ErbB2 over-expressing cells which was required for ErbB2-mediated STAT3 activation and p21Cip1 increase. Treatment of ErbB2 over-expressing resistant cells with STAT3 inhibitor peptides sensitized the cells to Taxol. In addition to classical signal transduction pathways, I identified a novel ErbB2 mediated regulatory mechanism of p21Cip1. I found that a nuclear ErbB2 and STAT3 complex binds directly to the p21Cip1 promoter offering a non-classical mechanism of p21Cip1 promoter regulation. These data suggest that ErbB2 over-expression can confer Taxol resistance of breast cancer cells by transcriptional upregulation of p21 Cip1 via activation of STAT3 by Src kinase and also by cooperation with nuclear ErbB2. The data suggest a potential clinical mechanism for STAT3 inhibitors in sensitizing ErbB2 over-expressing breast cancers to Taxol. ^

Upregulation of VEGF through p70S6K in ErbB2 -mediated angiogenesis and inhibition by combined herceptin plus taxol therapy

ErbB2 overexpression in breast tumors increases metastasis, angiogenesis, and reduces survival. To study ErbB2 signaling mechanisms in metastasis and angiogenesis, a spontaneous metastasis assay was performed using human breast cancer cells transfected with constitutively active ErbB2 kinase (V659E), an ErbB2 kinase-dead mutant (K753M), or vector control. Mice injected with V659E had increased metastasis and tumor microvessel density; and the increased angiogenesis in vivo from the V659E transfectants paralleled increased angiogenic potential in vitro, which resulted from increased VEGF by increased protein synthesis. This appeared to be mediated through a PI3K, Akt, mTOR, p70S6K-signaling pathway. Furthermore, V659E xenografts had significantly increased phosphorylated Akt, phosphorylated p70S6K, and VEGF compared with control. To validate the clinical relevance of these findings, human breast tumor samples were examined. Tumors overexpressing ErbB2 correlated with p70S6K phosphorylation and VEGF expression, which significantly correlated with higher levels of Akt and mTOR phosphorylation. Additionally, patients with tumors having increased p70S6K phosphorylation showed a trend for worse disease-free survival and increased metastasis. Together, ErbB2 increases VEGF expression by activating the p70S6K signaling pathway, which may serve as targets for antiangiogenic and antimetastatic therapies. ^ Herceptin is an anti-ErbB2 antibody that demonstrated anti-tumor function, especially in combination with other chemotherapies such as Taxol, in patients with ErbB2-overexpressing tumors. Since the repeated administration of low-dose chemotherapy endorsed an antiangiogenic effect in vitro, and Herceptin was shown to inhibit angiogenesis in tumor xenografts, I investigated whether combined Taxol plus Herceptin treatment inhibits ErbB2-mediated angiogenic responses more effectively. Mice with ErbB2-overexpressing xenografts were treated with control, Herceptin, Taxol, or combination Herceptin plus Taxol. Mice treated with the combination exhibited reduced tumor volumes, tumor microvessel densities, and lung metastasis; and ErbB2-overexpressing cells treated with the combination secreted less VEGF, and stimulated less endothelial cell migration. Furthermore, Akt phosphorylation contributed to VEGF upregulation and was most effectively reduced by combination treatment. ^ In summary, ErbB2 activates signaling to Akt and p70S6K leading to increased VEGF and angiogenesis. Combination Herceptin plus Taxol treatment most effectively inhibited ErbB2-mediated angiogenesis, resulting in pronounced tumoricidal effects, and may be mediated through reduction of phosphorylated Akt, a positive regulator in the p70S6K pathway. ^

IL-2 REGULATION OF IL-24 EXPRESSION LEADS TO GROWTH SUPPRESSION OF MELANOMA CELLS

Melanoma is known to be highly resistant to chemotherapy. Treatment with high dose IL-2 has shown significant clinical benefit in a minority of metastatic melanoma patients and has lead to long term survival in a few cases. However, this treatment is associated with excessive multiorgan toxicities, which severely limits its use. We hypothesize that one mechanism of effective IL-2 therapy is through the direct upregulation of IL-24 production in melanoma tumors and subsequent IL-24 mediated tumor growth suppression. Five melanoma cell lines were treated with high dose recombinant hIL-2 at 1000U/ml. Three of the cell lines (A375, WM1341, WM793) showed statistically significant increases in their levels of IL-24 protein when measured by Western blotting, while the remaining two lines (WM35, MeWo) remained negative for IL-24 message and protein. This increase in IL-24 was abolished by either preincubating with an anti-IL-2 antibody or by blocking the IL-2 receptor directly with antibodies against the receptor chains. We also demonstrated by ELISA that these three cell lines secrete IL-24 protein in higher amounts when stimulated with IL-2 than do untreated cells. These cells were found to contain IL-2R beta and gamma message by RT-PCR and also expressed higher levels of IL-24 when treated with IL-15, which shares the IL-2R beta chain. Thus we propose that IL-2 is signaling through IL-2R beta on some melanoma cells to upregulate IL-24 protein expression. To address the biological function of IL-2 in melanoma cells, five cell lines were treated with IL-2 and cell viability determined. Cell growth was found to be significantly decreased by day 4 in the IL-24 positive cell lines while no effect on growth was seen in WM35 or MeWo. Incubating the cells with anti-IL-24 antibody or transfecting with IL-24 siRNA effectively negated the growth suppression seen with IL-2. These data support our hypothesis that in addition to its immunotherapeutic effects, IL-2 also acts directly on some melanoma tumors and that the IL-24 and IL-2R beta status of a tumor may be useful in predicting patient response to high dose IL-2.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

An Innovative Phase I Trial Design Allowing for the Identification of Multiple Potential Maximum Tolerated Doses with Combination Therapy of Targeted Agents

Treatment for cancer often involves combination therapies used both in medical practice and clinical trials. Korn and Simon listed three reasons for the utility of combinations: 1) biochemical synergism, 2) differential susceptibility of tumor cells to different agents, and 3) higher achievable dose intensity by exploiting non-overlapping toxicities to the host. Even if the toxicity profile of each agent of a given combination is known, the toxicity profile of the agents used in combination must be established. Thus, caution is required when designing and evaluating trials with combination therapies. Traditional clinical design is based on the consideration of a single drug. However, a trial of drugs in combination requires a dose-selection procedure that is vastly different than that needed for a single-drug trial. When two drugs are combined in a phase I trial, an important trial objective is to determine the maximum tolerated dose (MTD). The MTD is defined as the dose level below the dose at which two of six patients experience drug-related dose-limiting toxicity (DLT). In phase I trials that combine two agents, more than one MTD generally exists, although all are rarely determined. For example, there may be an MTD that includes high doses of drug A with lower doses of drug B, another one for high doses of drug B with lower doses of drug A, and yet another for intermediate doses of both drugs administered together. With classic phase I trial designs, only one MTD is identified. Our new trial design allows identification of more than one MTD efficiently, within the context of a single protocol. The two drugs combined in our phase I trial are temsirolimus and bevacizumab. Bevacizumab is a monoclonal antibody targeting the vascular endothelial growth factor (VEGF) pathway which is fundamental for tumor growth and metastasis. One mechanism of tumor resistance to antiangiogenic therapy is upregulation of hypoxia inducible factor 1α (HIF-1α) which mediates responses to hypoxic conditions. Temsirolimus has resulted in reduced levels of HIF-1α making this an ideal combination therapy. Dr. Donald Berry developed a trial design schema for evaluating low, intermediate and high dose levels of two drugs given in combination as illustrated in a recently published paper in Biometrics entitled “A Parallel Phase I/II Clinical Trial Design for Combination Therapies.” His trial design utilized cytotoxic chemotherapy. We adapted this design schema by incorporating greater numbers of dose levels for each drug. Additional dose levels are being examined because it has been the experience of phase I trials that targeted agents, when given in combination, are often effective at dosing levels lower than the FDA-approved dose of said drugs. A total of thirteen dose levels including representative high, intermediate and low dose levels of temsirolimus with representative high, intermediate, and low dose levels of bevacizumab will be evaluated. We hypothesize that our new trial design will facilitate identification of more than one MTD, if they exist, efficiently and within the context of a single protocol. Doses gleaned from this approach could potentially allow for a more personalized approach in dose selection from among the MTDs obtained that can be based upon a patient’s specific co-morbid conditions or anticipated toxicities.

Killing of human melanoma cells induced by activation of class I interferon-regulated signaling pathways via MDA-7/IL-24.

Restoration of the tumor-suppression function by gene transfer of the melanoma differentiation-associated gene 7 (MDA7)/interleukin 24 (IL-24) successfully induces apoptosis in melanoma tumors in vivo. To address the molecular mechanisms involved, we previously revealed that MDA7/IL-24 treatment of melanoma cells down-regulates interferon regulatory factor (IRF)-1 expression and concomitantly up-regulates IRF-2 expression, which competes with the activity of IRF-1 and reverses the induction of IRF-1-regulated inducible nitric oxide synthase (iNOS). Interferons (IFNs) influence melanoma cell survival by modulating apoptosis. A class I IFN (IFN-alpha) has been approved for the treatment of advanced melanoma with some limited success. A class II IFN (IFN-gamma), on the other hand, supports melanoma cell survival, possibly through constitutive activation of iNOS expression. We therefore conducted this study to explore the molecular pathways of MDA7/IL-24 regulation of apoptosis via the intracellular induction of IFNs in melanoma. We hypothesized that the restoration of the MDA7/IL-24 axis leads to upregulation of class I IFNs and induction of the apoptotic cascade. We found that MDA7/IL-24 induces the secretion of endogenous IFN-beta, another class I IFN, leading to the arrest of melanoma cell growth and apoptosis. We also identified a series of apoptotic markers that play a role in this pathway, including the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas-FasL. In summary, we described a novel pathway of MDA7/IL-24 regulation of apoptosis in melanoma tumors via endogenous IFN-beta induction followed by IRF regulation and TRAIL/FasL system activation.

KEAP1 E3 ligase-mediated downregulation of NF-kappaB signaling by targeting IKKbeta.

IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.

Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Ketamine-induced hepatoprotection: the role of heme oxygenase-1.

Lipopolysaccharide (LPS) causes hepatic injury that is mediated, in part, by upregulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Ketamine has been shown to prevent these effects. Because upregulation of heme oxygenase-1 (HO-1) has hepatoprotective effects, as does carbon monoxide (CO), an end product of the HO-1 catalytic reaction, we examined the effects of HO-1 inhibition on ketamine-induced hepatoprotection and assessed whether CO could attenuate LPS-induced hepatic injury. One group of rats received ketamine (70 mg/kg ip) or saline concurrently with either the HO-1 inhibitor tin protoporphyrin IX (50 micromol/kg ip) or saline. Another group of rats received inhalational CO (250 ppm over 1 h) or room air. All rats were given LPS (20 mg/kg ip) or saline 1 h later and euthanized 5 h after LPS or saline. Liver was collected for iNOS, COX-2, and HO-1 (Western blot), NF-kappaB and PPAR-gamma analysis (EMSA), and iNOS and COX-2 mRNA analysis (RT-PCR). Serum was collected to measure alanine aminotransferase as an index of hepatocellular injury. HO-1 inhibition attenuated ketamine-induced hepatoprotection and downregulation of iNOS and COX-2 protein. CO prevented LPS-induced hepatic injury and upregulation of iNOS and COX-2 proteins. Although CO abolished the ability of LPS to diminish PPAR-gamma activity, it enhanced NF-kappaB activity. These data suggest that the hepatoprotective effects of ketamine are mediated primarily by HO-1 and its end product CO.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

DOWNREGULATION OF PAX2 SUPPRESSES OVARIAN CANCER CELL GROWTH

PAX2 is one of nine PAX genes regulating tissue development and cellular differentiation in embryos. PAX2 promotes cell proliferation, oncogenic transformation, cell-lineage specification, migration, and survival. Unattenuated PAX2 has been found in several cancer types. We therefore sought to elucidate the role of PAX2 in ovarian carcinomas. We found that PAX2 was expressed in low-grade serous, clear cell, endometrioid and mucinous cell ovarian carcinomas, which are relatively chemoresistant compared to high grade serous ovarian carcinomas. Four ovarian cancer cell lines, RMUGL (mucinous), TOV21G (clear cell), MDAH-2774 (endometrioid) and IGROV1 (endometrioid), which express high-levels of PAX2, were used to study the function of PAX2. Lentiviral shRNAs targeting PAX2 were used to knock down PAX2 expression in these cell lines. Cellular proliferation and motility assays subsequently showed that PAX2 stable knockdown had slower growth and migration rates. Microarray gene expression profile analysis further identified genes that were affected by PAX2 including the tumor suppressor gene G0S2. Reverse phase protein array (RPPA) data showed that PAX2 knockdown affected several genes that are involved in apoptosis, which supports the fact that downregulation of PAX2 in PAX2-expressing ovarian cancer cells inhibits cell growth. We hypothesize that this growth inhibition is due to upregulation of the tumor suppressor gene G0S2 via induction of apoptosis. PAX2 represents a potential therapeutic target for chemoresistant PAX2-expressing ovarian carcinomas.