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| Research |
Dao M. Nguyen, M.D.
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Surgery Branch | |||||||||||||||||
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| Dr. Nguyen is a graduate of the Faculty of Medicine, McGill University (Montreal, Quebec, Canada). After completing residencies in general surgery (1986-1992)and cardiothoracic surgery (1992-1994)at McGill University, he did a 2-year research/clinical fellowship in thoracic oncology at MD Anderson Cancer Center, Houston, Tx(1994-1996). Prior to joining the Surgery Branch of NCI, he was appointed as an assistant professor of surgery at McGill University (1996-1998). He is currently a tenure-track principal investigator in the Section of Thoracic Oncolgy of the Surgery Branch at NCI. | ||||||||||||||||||||
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Research
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| The focus of my laboratory research efforts is the development of molecularly targeted therapeutics to treat cancers that arise from the chest namely primary cancer of the lung, of the esophagus and of the pleura (malignant pleural mesothelioma). To achieve this objective, we develop clinically applicable strategies to induce apoptosis of malignant cells while minimally impacting normal tissues by targeting either the receptor-mediated apoptosis-inducing signal transduction pathways (Fas/FasL, DR4/DR5/TRAIL) or the oncogene/protooncogene growth factor receptor-mediated signal transduction pathways (EGFR, erbB2 and others).
RESEARCH RELATED TO GROWTH-FACTOR RECEPTOR SIGNAL TRANSDUCTION PATHWAYS. Inhibition of the EGFR-mediated signalings by the selective EGFR–tyrosine kinase inhibitor PD153035 (PD) in esophageal cancer cells that express high levels of EGFR (>5-fold of EGFR levels of normal cells) resulted in significant downregulation of the pro-metastasis phenotypes of cancer cells namely cell motility, cell invasion through the Matrigel extracellular matrix membrane, cell proliferation and clonogenic property, production of pro-angiogenic cytokines VEGF, MMP-9. Moreover, PD-treated cells were sensitized to the cytotoxic effects of DNA-damaging agents (Gemcitabine, Cisplatin, radiation). The chemo- and radio-sensitization effect of PD-mediated EGFR blockade was associated with the significant reduction of the DNA repair capacity in these cells. Ongoing experiments are being performed to further study the effect of EGFR blockade on the expression of similar pro-metastasis phenotypes in cultured cancer cells that express normal levels of EGFR. We also evaluated the cytotoxic effect of combining the heat shock protein (HSP) active compound 17AA Geldanamycin (17AAG) with the tubular active agent Taxol (Paclitaxel) in cultured lung and esophageal cancer cells in vitro and in vivo animal model. 17AAG, via its ability to interact with the chaperone protein HSP90, induces rapid depletion of oncoproteins EGFR, erbB2, Raf-1, mutated p53 as well as others such as AKT, and possibly p21. Taxol and 17AAG combinations synergistically induced profound cytotoxicity and apoptosis of cultured cancer cells in a sequence-dependent manner. Moreover, 17AAG treatments diminished the expression of prometastasis phenotypes of cultured lung cancer cells expressing high levels of erbB2. The combinations of Taxol and 17AAG induced synergistic antitumor effects in an animal model of nude mice bearing H358 NSCLC human xenografts. Immunohistochemical analysis of tumors harvested from mice treated with the drug combinations indicated significant reduction of erbB2 expression, VEGF expression and tumor capillary densities (inhibition of angiogenesis) and increased apoptosis. Ongoing experiments are being performed to further elucidate the molecular mechanisms responsible for the synergistic antitumor effect of this drug combination. 17AAG in combination with Taxol is currently evaluated in a phase I clinical trial. We further evaluated the effect of blocking the PI3K-mediated signaling on tumor cell response to Taxol. PI3K plays an essential role of intracellular propagation the mitogenic/survival signals originated from receptors at the cell surface to multiple parallel downstream signaling cascades. Inhibition of PI3K activity by LY294002 (LY) resulted in diminished phosphorylated AKT, inhibition of NF-kB transcriptional activity and significant reduction of expression of NF-kB-dependent proteins (BclXL, cIAP-1,cIAP-2). These were associated with enhancement of the intrinsic sensitivity of cancer cells to the cytotoxic effect of Taxol. Direct inhibition of NF-kB using BAY 11-0782 also resulted in profound induction of apoptosis of cancer cells treated with Taxol. These findings suggest PI3K as well as NF-kB as targets for further development of strategies to enhance the cytotoxic effect of conventional chemotherapeutics. Works are in progress to further elucidate the exact pathway downstream of PI3K (AKT vs. others) as the one responsible for the enhanced cytotoxicity described above. More recently, our laboratory has focused on the development of rational strategies to potentiate the ability of Histone Deacetylase Inhibitors (HDACi) to induce apoptosis in cancer cells. It has been reported that activation of NF-kB and upregulation of p21 following HDACi exposure may contribute to the resistance of cancer cells to the apoptosis-inducing effect of HDACi. Protein kinase C (PKC) has been shown to suppress downregulate NF-kB activity as well as HDCi-mediated upregulation of p21. We have recently demonstrated that PKC inhibitor Calphostin C (CC) significantly suppresses HDACi Trichostatin A (TSA)-induced NF-kB activation and p21 upregulation (both at transcriptional and translational levels). This was associated with a profound synergistic induction of apoptosis of cells treated with TSA and CC combinations but not in cells treated with either drug alone. Ongoing works are being performed to further characterize clinically relevant drug combinations and conditions for potential development of a clinical trial to evaluate the antitumor efficacy of HDAi and PKC inhibitor combinations. RESEARCH RELATED TO RECEPTOR-MEDIATED APOPTOSIS-INDUCING SIGNAL TRANSDUCTION PATHWAYS. We have observed that the majority of cultured cancer cells (derived from lung, esophagus, colon, mesothelioma) express death receptors (Fas or DR4/DR5) yet only a small fraction of them are intrinsically sensitive to apoptosis-inducing ligands FasL or TRAIL. Cotreatment of Fas-positive cells with cycloheximide significantly sensitizes these cells to recombinant soluble FasL (sFasL), indicative of the presence of functional intrinsic death-signaling cascades. Exposure of FasL-refractory cells to sublethal concentrations of cisplatin prior to sFasL treatment results in a profoundly synergistic induction of cytotoxicity and apoptosis. The apoptosis induced by this combination (CDDP + sFasL) is caspase dependent. More importantly, selective caspase 9 inhibitor significantly suppressed cytotoxicity and apoptosis induced by this combination, suggestive of the essential role of the mitochondria-mediated death-signaling cascade (type II pathway). Reactive oxygen radicals (ROS) also appeared to play a crucial role in the process as the ROS scavenger N-acetyl cystein (N-Ac) completely abrogates the cytotoxicity and apoptosis induced by CDDP+sFasL combinations. To extend this study further, we evaluated the combination of CDDP with Apo2L/TRAIL on esophageal cancer cells in vitro. We observed that in association with its ability to mediate synergistic induction of cytotoxicity and apoptosis, CDDP+TRAIL combination rapidly and synergistically activated caspase 8, 9 and 3 proteolytic activity. More importantly, the caspase 8 activation by this combination was completely abrogated by the selective caspase 9 inhibitor as well as by overexpression of Bcl2. These findings confirm the crucial role and the important involvement of the mitochondria-mediated type II pathway in generating a positive feedback loop to promote and to amplify caspase 8 activation leading to further downstream caspase activation (eg. Caspase 3) and induction of apoptosis. We also employed the tumor-selective adenoviral vector to transduce membrane-bound TRAIL to cancer cells but not normal cells to induce apoptosis of cancer cells but not normal cells by TRAIL expression. We further enhanced the efficacy of adenovirus TRAIL in mediating cytotoxicity and apoptosis by pretreating cancer cells with cisplatin. CDDP has been shown to increase the expression of adenovirally mediated gene transfer and, as described above, CDDP also sensitized cancer cells to TRAIL-mediated signaling and induction of apoptosis. More recently, we have evaluated the ability of HDACi TSA on sensitizing cancer cells to death-inducing proteins such as FasL or Apo2L/TRAIL . HDACi has been shown to affect the expression of mitochondria-associated pro- and antiapoptotic protein leading to an increase of the Bax/Bak to Bcl2/BclXL ratios. Such an effect may prove to be beneficial in enhancing Apo2L/TRAIL-mediated apoptosis. Treating cancer cells (malignant pleural mesothelioma) but not normal cells with either concurrent (TSA+Apo2L/TRAIL) or sequential (TSA+Apo2L/TRAIL) results in profound cytotoxicity and induction of apoptosis. Ongoing experiments are underway to further elucidate the molecular mechanisms responsible for such a profound and synergistic drug interactions leading to complete induction of apoptosis of treated cells. Finally, in vivo animal experiments are in progress to validate the synergistic tumoricidal effect of chemotherpy+Apo2/TRAIL or MegaFasL combinations. Reverse phase protein microarray will be utilized to study apoptosis signal transduction in treated cells with the vision of using this technology to track molecular response in microscopic tumor samples obtained by minimally invasive biopsies in preclinical animal models as well as in patients undergoing clinical trials with Apo2L/TRAIL-based biologic therapy. |
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| This page was last updated on 4/15/2004. | ||||||||||||||||||||
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