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NUCLEAR RECEPTORS, ROS & CANCER BIOLOGY
RESEARCH FOCUS Diabetes and cancer are common diseases among adults worldwide. New research shows that both hyperinsulinaemia and hyperglycemia, characteristics of type 2 diabetes, are major risk factors for a number of cancer – promotes tumor development. My area of research focuses on exploring the role of nuclear receptors, such as PPARs (peroxisome proliferator-activated receptors), ERalpha, HNF4alpha, and DEAD-box protein DP103 (a HDAC- and SUMO-dependent transcriptional repressor) in the prevention and treatment of cancer and diabetes. Among the three PPAR isoforms, PPARgamma activation appears to play important role in diverse physiological and pathophysiological events including stimulation of adipocyte differentiation, stimulation of insulin action, and inhibition of tumor cell proliferation. My current research interest includes identification of novel PPARgamma target genes and to elucidate signalling pathways that targets PPARgamma activity in cancer and diabetes. We are currently exploring the mechanism(s) involved in the regulation of a few novel PPARgamma targets, a mechanism involving interplay between ERalpha, PPARgamma, and SUMO pathway. Understanding this mechanism could shed new light for clinical intervention. In addition to PPARgamma, we also discovered a novel role for DP103 in cancer cells involving the molecular aspects of tumor cell invasion and metastasis - a project with significant basic and translational potential. Since HNF4alpha has been reported to prevent cancer cell transmigration, I’m currently exploring the roles of HNF4alpha and DP103 in PPARgamma- and statin- induced decrease in tumor cells’ invasive property in cellular and later animal models. Loss-of-function mutations of HNF4alpha or its response element in target promoters result in Maturity-Onset Diabetes of the Young Type I (MODY I) in humans. While the role of HNF4alpha in regulating glucose metabolism in the liver and pancreas is already known, we are exploring how renal HNF4alpha contributes to glucose homeostasis, lipid mobilization, and mitochondrial beta-oxidation in cellular, animal models, and in various types of nephropathy. Incidentally, down-regulation of HNF4alpha is also reported to contribute to the progression of liver and kidney cancer. Because both PPARgamma and HNF4alpha recognize the same DNA-binding consensus sequence, we are currently exploring the mechanism(s) involved in the regulation of a few novel HNF4alpha targets and ways to re-induce its tumor suppressor activity.
DRUG DISCOVERY Another area of interest is to have a greater understanding of these nuclear receptors – aimed at developing newer selective PPARgamma modulators (SPPARgMs), drugs with more potent activity and less toxicity. Towards this end, I have (in USA-NUS collaboration) developed 16 novel and potent PPARgamma ligands, identified through in vitro screening. My goal is to use these new PPARgamma ligands to demonstrate its effectiveness in a variety of cancer cell lines, mouse xenograft, and diabetic models with intent to a pilot clinical trial here in Singapore. It is my hope that one of these new ligands could represent the next generation insulin sensitizer and anti-neoplastic agent. In addition, I’m also setting up research collaboration between NUS and my collaborator in the USA to identify novel synthetic ligands for HNF4alpha (an orphan receptor) to re-induce its suppressor activity in the clinic.
ON-GOING RESEARCH AREAS 1. In collaboration with Assoc. Prof. Marie V. Clement, Biochemistry, we recently identified regulation of Na+/H+ exchanger, NHE1 gene expression as a possible mechanism involved in reactive oxygen species (ROS)-mediated regulation of tumor cells’ response to apoptosis. The first area of research is to decipher the mechanism involved in hydrogen peroxide (H2O2) mediated inhibition of NHE1 gene expression and the relevance of this inhibition to cells’ response to apoptosis. In the present research we focus on understanding the mechanism involved in H2O2-mediated inhibition of NHE1 and the relevance of this regulation to cells’ sensitivity to apoptotic triggers. The study will first be done in cells without a tumorigenic background. Findings from this study will later be extended to tumor cells. 2. In collaboration with Assoc. Prof. Marie V. Clement, Biochemistry and Prof. Shazib Pervaiz, Physiology, given that expression of peroxisome proliferator-activated receptor gamma (PPARgamma) is increased in breast cancer cell lines, that PPARgamma activation generated intracellular reactive oxygen species (ROS) and induced cellular acidosis, together with our previous findings that ROS mediates expression of NHE1 gene, we plan to assess if the human NHE1 gene is a transcriptional target of PPARgamma and if ROS production can play a role in PPARgamma–mediated activation of NHE1 gene expression. This study will be done in breast carcinoma-derived cell lines, and results compared to normal breast epithelial cell lines. We anticipate that, this study will provide insight into the regulation of the human NHE1 gene transcription and address the role of PPARgamma as transcription factor in human NHE1 gene expression. Another area of research is to uncover the mechanism by which estrogen receptor alpha (ERalpha) binding plays a role in mediating PPARgamma responsiveness in ERalpha-positive breast cancer cells. These results may provide insight into development of new intervention strategies for the treatment of breast cancer at the level of ERalpha DNA binding, by designing transcription factor decoys rather than classical antagonism of estrogen binding. 3. In collaboration with Assoc. Prof. Marie V. Clement, Biochemistry and Prof. Shazib Pervaiz, Physiology,Tumor necrosis factor alpha (TNFalpha) induced signal transduction pathway involves the intracellular production of ROS. However, there is conflicting evidence regarding the source(s) and roles of these ROS in modulating TNF-induced apoptosis. Recently it has been shown that in MCF-7 cells, TNFalpha activates caspase 6 and preferentially cleaved a transcription factor, AP-2. An on-going project in our lab has suggested that H2O2 mediated repression of the NHE1 gene expression and this may be the function of caspase 6 activation, cleaving transcription factor AP-2. Incidentally, AP-2 transcription factor is required not only to maintain basal NHE1 expression but also to activate NHE1 expression. 4. In collaboration with Prof. Shazib Pervaiz, Physiology, statins by inhibiting 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase are thought to reduce the risk of cancer. The molecular mechanisms underlying antitumor activity of statins have not been fully elucidated but interference with the function of Ras and Rho family GTPases seem to participate in this activity. In this study, we will (a) investigate if statin-induced apoptosis in colorectal cancer cell line, HCT116 is dependent on its p53 status or (b) Ras status comparing HCT116 (expressing oncogenic Ras to HT29 (expressing normal Ras). (c) If statin-induced cell death is a function of lowering intracellular superoxide production or (d) is it a function of lowered NHE1 gene expression which then induces intracellular acidosis. If so, then (e) decipher the signal pathways involved in statin-induced downregulation of NHE1 gene expression. To do research for a reason, I have planned to extend findings from this work to human colorectal tissues. Clinical aspect of the project will be in collaboration with A/Prof. Robert Hewitt, NUH-Tissue Repository. 6. In collaboration with A/Prof. Martin Lee Beng Huat, Physiology, we seek to identify a role of DP103 in proliferation and apoptosis in cancer and developing kidney, using a human malignant melanoma cell line and a human embryonic kidney cell line as models through loss-of-function and gain-of-function experiments. We show that specific knockdown of DP103 in the malignant melanoma cell line increased cell viability and proliferation. Conversely, over-expression of DP103 decreased cell viability and proliferation and increased the percentage of cells entering apoptosis. Together, our results showed that DP103 has anti-proliferative and pro-apoptotic activity in cancer cells as well as in the developing kidneys. In our present study, we therefore ask if the cells’ fate to absence of over-expression of DP103 is a function of a ratio in intracellular O2.- to H2O2. 7. Hepatocyte Nuclear Factor 4 alpha (HNF4A, NR2A1) is an orphan nuclear receptor critical for development and function of the liver, pancreas and intestine. Pancreatic HNF4A helps regulate glucose metabolism by modulating insulin secretion, while liver HNF4A regulates glucose production by controlling expression of the key gluconeogenic enzymes PEPCK and glucose-6-phosphatase. Loss-of-function mutations of HNF4A or its response element in target promoters result in Maturity-Onset Diabetes of the Young Type I (MODY I) in humans. In the kidney, HNF4A is expressed only in the proximal tubule. This strikingly restricted expression strongly suggests that HNF4A regulates proximal tubule-specific functions such as reabsorption of filtered glucose, PEPCK-mediated gluconeogenesis and insulin degradation. Indeed, the proximal tubule produces up to 20% of circulating glucose in humans, and accounts for up to two-thirds of renal insulin clearance. Therefore, we will ask if renal HNF4A contributes to glucose homeostasis in cellular and animal models of the proximal tubule. This work will be done in collaboration with Dr Frances Sladek at the University of California Riverside, USA, A/Prof. Martin Lee Beng Huat, Physiology, NUS, and A/Prof. Thomas P. Thamboo, Pathology, NUS. 8. In collaboration with A/Prof. Lina Lim Hsiu Kim, Physiology, we hypothesize that Annexin1 (ANXA1) regulates COX2 expression through mechanisms which we intend to study in this proposal, leading to increased tumor growth when ANXA1 is lost. Using MCF7 human breast cancer cells, and primary breast cancer cells isolated from annexin-1 wild-type and deficient animals, we will study the mechanisms involved in annexin-1 mediated tumor cell proliferation and the regulation of COX-2 and PGE2. We also intend to study the mechanisms of ANXA1 regulation, as our preliminary data demonstrates that ANXA1 can be regulated by p53, estrogen and PPARgamma in breast cancer cells. The clinical significance of these experiments are clear, that inhibiting the loss of ANXA1 during cancer development using PPARgamma antagonists can inhibit cell growth and proliferation. These studies will lead us to a deeper understanding of tumor cell function and growth, and will be important in the development of new tools for the treatment or prevention of cancer. 9. In collaboration with Assoc/Prof Manuel Salto-Telleza, Pathology and A/Prof. Robert Hewitt, NUH-Tissue Repository, Assoc. Prof. Marie V. Clement, Biochemistry and Prof. Shazib Pervaiz, Physiology, in an effort to translate bench to bedside, we will assess if the levels of PPARgamma and NHE1 expression in different tissues and cell types are likely to have a significant influence on the in vivo response to treatment with PPARgamma agonists. For this reason, we aim to determine the histological distribution of PPARgamma and NHE1 expression in normal breast tissue and infiltrating ductal carcinoma using both immunohistochemistry (IHC) and in situ hybridization. IHCs on tumor breast tissues from an initial pilot study was very promising to provide evidence that cancer patients on glitazones should response better to chemotherapy. From our preliminary study, we show NHE1 gene expression as a novel gene signature predictive of responsiveness to chemotherapy and that expression of NHE1 levels could provide useful prognostic information for cancer patients. We are currently extending this study to more than 100 patients and in different cancer types using tissue microarrays (TMAs) in collaboration with Assoc/Prof Manuel Salto-Telleza, Pathology. 10. Studies by others have provided evidence that siRNA-suppression of mitochondrial manganese-dependent superoxide dismutase (MnSOD) expression leads to decreased breast cancer cells’ invasive property and sensitization to anti-cancer drugs. Clinically, it has been observed that a polymorphism in the MnSOD promoter resulting in decreased MnSOD expression correlates strongly to recurrence-free breast cancer survival. Having identified putative PPARgamma binding sites on the human MnSOD promoter, our preliminary results show that indeed activation of PPARgamma decreases MnSOD gene expression in a PPARgamma-dependent manner. Therefore, down-regulation of MnSOD expression by PPARgamma ligands could be a promising avenue as a new strategy to induce tumor cells-specific growth arrest and increase sensitivity to anticancer treatment. This project is in collaboration with Assoc/Prof Manuel Salto-Telleza, Pathology and A/Prof. Robert Hewitt, NUH-Tissue Repository, Assoc. Prof. Marie V. Clement, Biochemistry and Prof. Shazib Pervaiz, Physiology. 11. In collaboration with Professor Patrick Casey, Senior Vice-Dean for Research, Duke-NUS and Prof. Shazib Pervaiz, Physiology, we aim to (1) confirm the involvement of mevalonate, geranylgeranylation, or farnesylation pathways in statin-induced apoptosis of breast cancer cells, (2) to explore if statin treatment increases GTP loading and accumulation of cytosolic Rho GTPases (Rho, Rac and cdc42), despite the lack of prenylation, (3) to investigate the kinetics and intracellular source of ROS, the ROS species involved, and the cross talk between GTP loaded Rho GTPases and ROS in statininduced apoptosis, (4) to elucidate if the growth inhibitory effect is mediated via Rho GTPases-dependent inhibition of Akt phosphorylation and to identify the Rho family protein involved using pharmacological inhibitors as well as siRNA knockdown strategy, (5) to investigate physical interaction between GTP-loaded Rho GTPases and Akt or the Akt activating kinase PDK1 as a mechanism of inhibition of Akt phosphorylation, and (6) to decipher if inhibition of Akt activation results in Foxo-3a activation and induction of the pro-apoptotic protein Bim. This study will not only enhance understanding of statin-induced cell death, but also uncover mechanisms by which unprenylated GTPases mediate statin sensitivity with the potential for developing novel strategies for effective therapeutic management of breast cancer. 12. In collaboration with A/Prof. Javi Piedrafita, San Diego, USA and Prof. Angel R. de Lera, Departamento de Química Física e Química Orgánica, Universidade de Vigo, Spain, we are currently in midst of generating new derivative ligands of PPARgamma. In March 2000, PPARgamma ligand, troglitazone was withdrawn from the market following FDA warnings over rare but life-threatening risk of hepatotoxicity. Recently, in May 2007, another PPARgamma ligand, rosiglitazone was reported to be associated with significant increase in the risk of myocardial infarction and with an increase in the risk of death from cardiovascular causes that had borderline significance. Since then, the use of TZD class of drugs has triggered much controversy due to its possible side effects. However, in the light of the tremendous clinical potential of PPARgamma activation (alone and in combination therapy), it is highly desirable to discover novel and safer molecules with strong PPARgamma agonist activity. Therefore, selective PPARgamma modulators (SPPARgMs or partial agonists) as opposed to full agonists may be desired PPARgamma ligands for the long-term management of cancer. Towards this end, a series of structurally novel and potent PPARgamma ligands were identified through in vitro screening. Our goal is to use these new PPARgamma ligands to demonstrate its effectiveness using various cancer cell lines and mouse xenograft and mouse diabetic models with intent to a pilot clinical trial here in Singapore as a Spain-USA-Singapore collaboration (Singapore collaborators include Assoc. Prof. Marie V. Clement, Biochemistry and Prof. Shazib Pervaiz, Physiology).
TEAM MEMBERS Dr. Alan Prem Kumar, NUMI, Principal Investigator Prof. Shazib Pervaiz, Physiology, Collaborator Assoc. Prof. Marie Veronique Clement, Biochemistry, Collaborator Professor Patrick Casey, Senior Vice-Dean for Research, Duke-NUS, Collaborator Professor Edison Tak-Bun Liu, Executive Director, Genome Institute of Singapore, Collaborator A/P Javier Piedrafita, Sidney Kimmel Cancer Center, San Diego, California, USA, Collaborator Professor Angel R. de Lera, Departamento de Química Física e Química Orgánica, Universidade de Vigo, Spain, Collaborator A/P Alan Lee Yiu Wah, Physiology, Collaborator Assoc. Prof Manuel Salto-Telleza, Pathology, Collaborator Assoc. Prof. Lim Chwee Teck, Deputy Director, NUS Life Sciences Institute, Faculty of Engineering, Collaborator A/P Robert Hewitt, NUH Tissue Repository, Collaborator A/P Lina Lim Hsiu Kim, Physiology, Collaborator A/P Martin Lee Beng Huat, Physiology, Collaborator Dr. Balakrishnan Ramanathan, Biochemistry, Collaborator Dr. Sotirios Paul Georgantopoulos, Biochemistry, Collaborator Ms. Michelle Chang Ker Xing, Postgraduate student (Supervisor: Assoc Prof Marie V. Clement) Ms. Charis Chua Sui Huay, NGS Postgraduate student (Supervisor: Assoc Prof Marie V. Clement) Ms. Wong Chew Hooi, NGS Postgraduate Student (Supervisor: Prof Shazib Pervaiz) Ms. Zhou Ting, NGS Postgraduate student (Supervisor: Prof Shazib Pervaiz) Ms. Angeline Zhu Yan, NGS Postgraduate student (Supervisor: Prof Shazib Pervaiz) Ms. Carolyn Ng Chun Chi, NGS Postgraduate student (Supervisor: Prof Shazib Pervaiz) Ms. Diana Hay Hui Sin, Postgraduate student (Supervisor: Dr. Alan P. Kumar) Ms. Chen Luxi, Postgraduate student (Supervisor: Dr. Alan P. Kumar) Ms. Loo Ser Yue, Honor’s student (Supervisor: Prof Shazib Pervaiz) Ms. Kirthan Shenoy, Postgraduate Student (Supervisor: Prof Shazib Pervaiz)
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