I have been focused on development of a translational research program of targeted molecular therapy for thoracic cancers. The main approach of my research efforts is to design clinically applicable strategies to induce and/or to enhance the induction of apoptosis of cancer cells by targeting either the receptor-mediated growth/survival signaling pathways or the receptor-mediated apoptosis-inducing pathways. This report summarizes research achievement in the last 12 months of the 2004 fiscal year.1. Targeting receptor-mediated mitogenic/survival signaling pathways - Suppression of the prometastasis phenotype expression in malignant pleural mesothelioma (MPM) by the selective EGFR tyrosine kinase inhibitor PD153035 (PD).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. We have previously demonstrated that treating cultured esophageal cancer cells TE2, TE3 and TE12 that express high levels of EGFR with PD resulted in profound inhibition of cell proliferation, cell motility and invasion through the extracellular matrix Matrigel, production of angiogenic cytokines such as VEGF and MMP-9 in conjunction with mild to moderate induction of apoptosis of certain cultured cell lines. We have extended that observation to other cultured thoracic cancer cell lines, namely MPM cells, expressing either normal levels or moderately elevated levels of EGFR. PD-mediated inhibition of cell proliferation and production of VEGF at baseline or under hypoxic condition (at drug concentrations that are clinically achievable) was only observed in cell lines expressing elevated (2- to 4-fold) levels of EGFR but not in normal EGFR-expressing cell lines. On the other hand, the motogenic activity (cell motility and Matrigel invasion) of all MPM cells, regardless of their intrinsic EGFR levels was profoundly suppressed by the EGFR inhibitor. Further works are in progress to elucidate which downstream EGFR-dependent signaling pathways (Raf-MEK-ERK1/2 or PI3K-AKT or PLC-gamma) regulate the expression of prometastasis phenotypes. The preliminary data indicated that selective targeting PI3K by using LY294002 or MEK using UO-126 in MPM cells results in profound cytotoxicity that is mediated by both cell cycle arrest at the G1/S checkpoint and by pronounced dose-dependent induction of apoptosis (this is in contrast to the effect of EGFR inhibitor PD153035 that does not induce apoptosis of MPM cells). Either LY294002 or UO126 also suppresses the motogenic activity as well as production of the angiogenic cytokine VEGF in cultured MPM cells. This kind of experimental approach enables us to evaluate not only the growth inhibitory effect of selective pathway kinase inhibitors but also their ability to modulate the cellular phenotypes that play crucial roles in the process of carcinogenesis and metastasis formation. The translational values of our findings underscore the fact that selective pathway kinase inhibitors may be of greater values as chemopreventive agents or when utilized in the postoperative adjuvant settings to target microscopic disease when they are least susceptible to conventional cytotoxic chemotherapeutic drugs.2. Targeting the receptor-mediated apoptosis-inducing signaling pathway - Development of strategies to enhance the cytotoxic potency of TRAIL and FasL.We have previously demonstrated that cisplatin (CDDP), when administered at sublethal concentrations prior to TRAIL or FasL exposure profoundly enhances the susceptibility of cancer cells to the cytotoxic effect of these death-inducing ligands. We have further demonstrated that recruitment of the type II mitochondria-dependent death signaling cascade and amplification of caspase 8 activation by the feedback loop mediates potentiation of cytotoxicity and apoptosis of the CDDP/FasL or CDDP/TRAIL combination. Based on that observation, we hypothesized that any therapeutic maneuver that activates the mitochondria would enhance the potency of death-inducing ligands. Histone deacetylase inhibitors (HDACIs) are novel anticancer agents that are currently being evaluated in early phase clinical trials. HDACIs induce elevation of the ratios of Bax, Bak vs. Bcl2, BclXL thus priming the mitochondria to a second apoptosis signal. Concurrent exposure of cultured thoracic cancer cells to the HDACi Trichostatin A (TSA 16 nM to 1000nM) and Apo2L/TRAIL (Zn++-containing trimer of recombinant extracellular domain of TRAIL, Genentech, Inc.) for 12 hours results in 4- to >50 fold reduction of TRAIL IC50 values, indicative of profound enhancement of cancer cell susceptibility to TRAIL. In a cell line-dependent manner, concurrent TSA+TRAIL or sequential TSA/TRAL combination are equally effective in inducing synergistic cytotoxicity. While TSA alone or TRAIL alone induces minimal (<20%) cell death, >80% of cancer cells treated with the TSA+TRAIL combination undergo apoptosis. Cytotoxicity and apoptosis induced by this combination is sensitive to either the selective caspase 9 inhibitor or overexpression of Bcl2, indicating that the type II pathway is responsible for caspase activation and induction of apoptosis. Substantial caspase 8 activation in cells treated with TSA/TRAIL is blocked by Bcl2 overexpression, pointing to the amplification feedback loop mediating retrograde activation of caspase 8 by the type II pathway that is in operational in cells treated by this combination. Animal experiments designed to evaluate the in vivo antitumor effect of the TSA+TRAIL combinations are in progress. In addition to evaluating the synergistic induction of apoptosis by TSA and TRAIL, similar studies have been concurrently performed using other chemotherapeutic agent such as Paclitaxel (Taxol - a commonly used first-line chemotherapeutic agent for thoracic malignancies and known to induce cytotoxicity via activation of the mitochondria) or Valproic acid (a well characterized antiepileptic agent with HDACI activity). Future plans include evaluation of novel technology (tissue protein arrays, tissue and serum mass spectrometry) to detect and to track apoptosis in tumors treated with combinations of TRAIL and chemotherapeutic agent sensitizers. To better assist screening for TRAIL sensitizer and selection of the most optimal treatment sequence for the death ligand and sensitizer combinations, gene microarray will be utilized to provide a global assessment of alteration of genes that may be involved in receptor-mediated death signaling. In addition to evaluating Apo2L/TRAIL as a recombinant apoptosis-inducing ligand, we also direct our laboratory efforts to characterizing the therapeutic value of a novel form of soluble FasL also known as MegaFasL. This is a humanized covalently bonded hexamer of the extracellular domain of FasL. Similar experiments have been carried out to evaluate its intrinsic cytotoxic effect as well as the ability of chemotherapeutic agents to sensitize cultured thoracic cancer cells to this form of recombinant FasL.Another approach our laboratory has been taken is to promote TRAIL expression in cancer cells using recombinant tumor-selective adenovirus expression membrane-bound TRAIL (AdVgTRAIL). We evaluated the cytotoxic effect of combining CDDP with AdVgTRAIL as CDDP is known to enhance adenoviral transgene expression and also shown to sensitize cancer cells to TRAIL. We have demonstrated that CDDP increases AdVgTRAIL-mediated TRAIL expression by 2- to 10-fold when compared to that of cells infected with AdVgTRAIL alone. Profound synergistic cytotoxicity was observed in cells treated with CDDP/AdVgTRAIL combination. This combination is not toxic to normal cells. By normaliztion for the level of TRAIL
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