The objective of our laboratory is to understand the molecular determinants of cellular survival that allow tumor cells to escape programmed cell death (apoptosis) when they are exposed to chemotherapy or irradiation. Identifying specific molecules that promote survival will provide new, attractive targets for the development of compounds that abrogate survival signals and enhance therapeutic effectiveness. In addition, we will extend in vitro studies by evaluating survival pathways in tumor specimens and designing clinical trials that rationally incorporate inhibitors of specific survival pathways with traditional forms of cancer therapy. Cellular survival is determined by factors both within the cell and outside the cell, including the contribution of extracellular influences such as soluble growth factors and extracellular matrix molecules. Both growth factors and extracellular matrix molecules stimulate survival through activation of enzymatic pathways within the cell that involve proteins that either add phosphate to downstream substrates (kinase) or remove phosphate (phosphatases). The best described survival pathways depend on activity from kinases such as P13K, Akt, PKC, p38, and MAPK, that become activated when they themselves are phosphorylated. Activation can occur after binding of extracellular growth factors to their cognate receptors, or in the case of some tumor cells, activation is independent of extracellular growth factors and is constitutive. Recently, studies from other laboratories have begun to identify downstream substrates of these kinases as well as different means of regulating these kinases' activity. In our laboratory, previous studies in a neuroblastoma cell system have demonstrated that, although multiple growth factors promote survival through different pathways, points of convergence exist in these pathways such that, when specific kinases are inhibited pharmacologically or genetically, all growth factor protection is abrogated, and irradiation and chemotherapy become much more effective. Specifically, inhibition of the P13K/Akt pathway and p38 resulted in complete abrogation of growth factor protection in the SH-SY5Y cell line. Based on these results, we have now chosen to evaluate survival signals in one of the most resistant tumor types, non-small cell lung cancer (NSCLC). Our approach has been to evaluate whether growth factors can be identified that protect NSCLC cells from apoptosis, to identify survival pathways utilized by theses cells, to correlate therapeutic resistance with activation of specific pathways, and to attempt to abrogate signals from these pathways and increase apoptosis caused by chemotherapy and irradiation. Preliminary data using some of the over 200 available NSCLC cell lines has revealed, not surprisingly, that all cell lines tested are quite resistant to chemotherapy and irradiation as measured by traditional apoptotic assays. Because all growth factors tested offered no additional protection, even under severe conditions, this suggested that NSCLC cells may constitutively activate survival pathways. Immunoblotting with antibodies directed against phosphorylated forms of kinases previously shown to be important in survival, we found that 10 of 10 NSCLC lines examined constitutively phosphorylate at least one well described kinase in survival pathways. Furthermore, using pharmacologic inhibitors directed against specific kinases, we have shown that these inhibitors inhibit their substrates' phosphorylation and are more toxic to the cells than either traditional chemotherapy or irradiation alone and also add significantly to the apoptosis observed with chemotherapy or irradiation. We are now using genetic approaches to confirm these data and abrogate specific kinase activity in order to measure apoptosis with and without traditional cancer therapy. Although preliminary, these studies are providing exciting insights into the means by which NSCLC lines survive DNA damage caused by chemotherapy and irradiation. Moreover, they suggest that approaches that target survival pathways might increase the effectiveness of chemotherapy and irradiation in patients with NSCLC.
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