Reverse-phase protein array (RPPA) profiling of non-small cell lung cancer lines identifies tumor signatures for sensitivity and resistance to chemotherapy and targeted agents (2007)

B178 Introduction: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease, with a variety of cell signaling pathways driving progression and therapeutic resistance. Although agents are available that target many of these critical pathways, there is currently no biologically-based method for selecting the best drug for a patient. Using reverse phase protein array (RPPA), a high-throughput technology that systematically measures key signaling proteins, we profiled forty-four NSCLC cell lines for markers of sensitivity and resistance to fifteen cytotoxic and targeted agents. RPPA is a quantitative, antibody-based assay that allows broad and simultaneous profiling of numerous therapeutically relevant targets from small amounts of protein, as is obtained from routine biopsy. Unlike gene expression arrays, RPPAs directly measure protein levels and can discern specific post-translational modifications such as phosphorylation or cleavage. The ability to compare levels of phosphorylated versus non-phosphorylated proteins is particularly critical to determining the activity of signaling proteins such as tyrosine kinases. Methods: The drug concentration required for 50% growth inhibition (IC50) was determined for 44 cell lines using MTS assays. For the RPPA, serial dilutions of pre-treatment cell lysates were printed on nitrocellulose-coated glass slides, and each slide incubated with one of 59 validated, monospecific antibodies. A single, representative logarithmic value for the signal intensity-serial dilution curve was then determined for each sample, representing the quantitative measure of a specific protein. Results: Subsets of sensitive and resistant cell lines were identified for each drug, despite most cell lines having never been exposed to any treatment either in the patient from which they were derived or in vitro. When subjected to unsupervised clustering, mechanistically-related drugs grouped together (such as taxanes, platinum agents, and epidermal growth factor receptor inhibitors), with similar patterns of protein expression characterizing sensitivity or resistance. For example, resistance to cisplatin and carboplatin was associated with increased expression of cell adhesion molecules beta-catenin and E-cadherin and with Rab25, a GTPase previously correlated with worse outcome in breast and ovarian cancer. Cell lines treated with pemetrexed showed the greatest variation in IC50’s, with a 1000-fold difference between sensitivity and resistance in 42 of the cell lines. Pemetrexed resistance was strongly associated with increased phospho-Rb (pRb) relative to Rb expression and with increased PKCalpha. Elevated pRb and Rb levels were also strongly associated with resistance to etoposide, as was high levels of phospho-LKB1. Conclusions: RPPA proteomic profiling identified intracellular signaling pathways and proteins associated with sensitivity and resistance to chemotherapies and targeted agents in NSCLC cell lines. These results suggest biologic mechanisms of therapeutic resistance. Our findings will be further investigated by correlating RPPA of tumor samples with clinical outcomes, with the goal of developing predictive markers that can guide treatment selection and identify new targets in NSCLC.