The Gene Cluster Instability Assay as ClinicalLung Cancer Biomarker

Our strategy for finding novel therapeutics for lung cancer started with the exploration of the expression of drug uptake genes in NSCLC tumors, reasoning that drug uptake proteins might be targeted in NSCLC with novel compounds that are both cytotoxic and specific substrates for those transporters. In published studies, we identified a cation transporter OA TP I B3 that showed increased expression in NSCLC lung tumors relative to adjacent non-malignant tissue. Microcystin LR is specific and toxic substrate ofOATPIB3 and the closely related (>80% homology) OA TP 1B 1 transporter. We expressed the cloned OA TPI B3 and OA TP 18 1 genes in Hela cells, demonstrated functional expression of this transporter, and found that the transfected cells were over 1,OOO-foidmore sensitive to microcystin LR than control cells. In addition, our data indicate that other microcystin analogs are extremely potent toxins in these cells, with IC50s less than 1 nM, that cytotoxicity is associated with inhibition of the target phosphatase PP2A, and that microcystin LR may circumvent apoptosis resistance by causing autophagic cell death. In preliminary studies, we also show that xenografts of cancer cells stably transfected with OATPIBI respond to microcystin LR therapy, thereby demonstrating the proof-of-principle that microcystins might be used to treat tumors over-expressing OATPIBI or OATPIB3. Therefore, we hypothesize that microcystin analogs can be developed as novel agents with a novel mechanism of action for lung cancer therapy. To explore this hypothesis, we propose three specific aims: 1) Identify a microcystin analog that is the most potent toxin in a tumor cell xenograft that expresses OA TP 1B3. 2) Test liver-protective strategies in xenograft models that will further enhance the therapeutic window of the microcystin analogs showing the most promise in Specific Aim 1. 3) Determine the mechanism of cell death in tumor cells exposed to microcystin analogs. The goal of these studies is to identifY a microcystin analog that may be used as a lead compound to submit to the NCI RAID program for preclinical development as a novel approach to lung cancer.