The main focus of my research efforts is to develop novel anticancer therapeutic strategies for thoracic malignancies (lung, esophageal cancers and malignant pleural mesothelioma) by targeting either the growth-promoting pathways or the apoptosis-inducing pathways. Growth factor pathways: EGFR-dependent signal transduction pathways: EGFR is ubiquitously expressed in epithelium-derived cancer cells. Depending on tumor histology, 30% to 90% of these tumors overexpress EGFR. Selective targeting of EGFR as cancer therapeutics by using monoclonal antibodies or small molecule tyrosine kinase inhibitor (TKI) to inhibit its signal transduction activity has drawn considerable interest. Our laboratory has observed that inhibition of EGFR signaling of cultured esophageal cancer cells overexpressing EGFR (> 5 times the level of normal cells) by the selective TKI PD153035 results in significant suppression of DNA repair capacity that appears to be mediated by the PI3K/AKT and not the MAPK ERK1/2 pathway. This correlates with profound enhancement of apoptosis in cells treated with DNA-damaging agents in combination with this TKI. Moreover, PD153035 treatment of these EGFR-overexpressing cells significantly downregulates the expression of various cellular phenotypes that are essential to the process of angiogenesis/metastasis (cell proliferation, production of VEGF, MMP-9 proteolytic activity, cell motility and cellular invasion through the ECM Matrigel). There exists, however, little experimental data regarding the effect of selective EGFR TKI in cultured cancer cells expressing normal level of EGFR (similar to EGFR levels of cultured primary human skin keratinocytes or bronchial epithelial cells). Moreover, there are conflicting experimental data regarding the correlation between the growth inhibitory effect of EGFR-TKI and the levels of EGFR expression on target cells. Recent data from our laboratory have indicated that PD153035 appears to be more effective at downregulating """"""""pro-metastasis"""""""" phenotypes (cell motility, cell invasion through ECM Matrigel) but not at mediating cell growth arrest in vitro in cancer cells with normal EGFR expression. These findings suggest another mechanism (other than direct growth inhibitory property) by which EGFR TKI's exert their antitumor effect in vivo. Correlative experiments using animal model will be performed to properly evaluate the antimetastasis / antiangiogenic property of this EGFR-TKI. Further molecular and biochemical analysis are being performed to elucidate the effect of PD153035 on EGFR-dependent intracellular signaling pathways (PI3K/AKT and MAPK ERK1/2) and correlating these pathways with cellular expression of pro-metastasis phenotypes in cultured cancer cell lines expressing different levels of EGFR. Such study would allow identifying pathways that regulate the expression of these malignant phenotypes and provide us with new strategies for antimetastasis therapy. PI3K/AKT/NF-kB signal transduction pathways: The PI3K/AKT-mediated signaling pathway plays an important role in cell survival, protecting cells from stress-induced apoptosis. We demonstrated that specific inhibition of PI3K by LY294002 in lung or esophageal cancer cells results in dose-dependent suppression of AKT activation, NF-kB transcription activity and reduced expression of NF-kB-dependent antiapoptotic proteins (cIAP1/cIAP2, BclXL). These are correlated with enhancement of intrinsic sensitivity of LY294002-treated cancer cells to Taxol. Direct inhibition of NF-kB by the IKK inhibitor BAY 11-0782 also resulted in enhancement of Taxol-mediated induction of apoptosis in these cells, suggesting that NF-kB may be the downstream mediator of the PI3K/AKT-dependent regulation of Taxol sensitivity in cancer cells. Further works are being performed employing strategy of incremental and selective inhibition of AKT (adenovirus-mediated expression of dominant-negative AKT) or NF-kB (adenoviral vectors expressing super repressor IkB or the dominant negative IKK2) without altering constitutive PI3K kinase activity to definitely elucidate if AKT and NF-kB are the downstream mediators of PI3K-regulated intrinsic chemo-resistance in cancer cells. This work will allow further identification of intracellular targets for development of more effective and cancer cell-selective chemosensitization strategies. Apoptosis-inducing pathways. Strategies to enhance histone deacetylase (HDAC) Inhibitor-mediated apoptosis. HDAC inhibitors are potent anticancer agents that are currently in clinical trials. HDAC inhibitror-mediated upregulation of NF-kB and p21/WAF-1 has been shown to impede the ability of these agents to efficiently induce apoptosis. Our laboratory has recently focused on development of clinically relevant strategies to enhance the efficacy of HDAC inhibitor Depsipeptide (DP) or Trichostatin (TSA), the former is in phase II clinical trial for hematologic as well as solid cancers, in mediating apoptosis of thoracic malignancies. Combining DP with the cdk inhibitor Flavopiridol (FLA), also in early phase clinical trials, results in profound abrogation of DP-mediated upregulation of p21/WAF-1 which is associated with robust induction of apoptosis in cultured cancer cell lines derived from the lung, esophagus or the pleura. We further demonstrate that DP+FLA-induced apoptosis is mitochondria-dependent as it is completely inhibited by selective caspase 9 inhibitor. Moreover, overexpression of p21/WAF-1 using adenoviral vector also inhibits apoptosis induced by this drug combination affirming the regulatory role of this protein in this process. We further investigate the role of protein kinase C (PKC) as upstream regulator of NF-kB and p21/WAF-1 activation in TSA-treated lung or esophageal cancer cells. Concurrent treatment of these cultured cancer cells with Calphostin C (general PKC inhibitor) and TSA results in profound suppression of NF-kB activation and transcriptional upregulation of p21/WAF-1 gene that is paralleled with profound induction of apoptosis in these cells. More importantly, specific inhibition of NF-kB (by overexpression of the super repressor IkB using adenoviral vector) in TSA-treated cells not only prevents NF-kB transcriptional activation but also inhibits p21/WAF-1 gene transcription and protein expression. This indicates NF-kB as the upstream regulator of p21/WAF-1 activation in TSA-treated cells. To make the combination more clinically relevant, we are in the process of evaluating the cytotoxic effect of TSA or DP with staurosporine and its derivative UCN-01 (known cdk/PKC inhibitors with the latter already in early phase clinical trials for solid tumors) in cancer cells of diverse histology. FasL- or TRAIL-mediated apoptosis. The majority of malignant cell express functional death receptors Fas and/or DR4/DR5 yet they are frequently refractory to FasL and/or TRAIL. The intracellular death-signaling pathway appears to be phenotypically intact as cycloheximide sensitizes refractory cells to soluble multimerized sFasL or soluble trimerized sTRAIL (Genentech, CA). We are interested in developing clinically relevant strategy to exploit the apoptosis-inducing pathway as a novel anticancer therapeutic. Pretreating FasL- or TRAIL-refractory cells with Cisplatin (CDDP) profoundly sensitizes them to the cytotoxic effect of these ligands. More importantly, these combinations, particularly that of CDDP+sTRAIL, are not toxic to normal cells. Animal model of abdominal carcinomatosis demonstrates the efficacy of Cisplatin+sFasL combinations in eradicating tumor formation
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