Signal transduction in response to growth factor receptor activation in tumors is a complex process that involves downstream signaling through the RAS (reviewed in ) and PI3K (reviewed in ) signaling pathways. These pathways are among the best characterized in cancer biology, involve a network of protein and lipid kinases working in concert to regulate diverse biological outputs, and can be activated by multiple mechanisms including gene amplification and somatic mutation. Understanding the role of these pathways in cancer biology has been enabled through the characterization of alterations in component pathway nodes including amplification of receptor tyrosine kinases like Her and EGFR, and genetic changes in PTEN, PIK3CA, AKT, KRas and BRAF, all of which have been shown to contribute to the cancer phenotype. The RAS and PI3K pathways are thought to work in parallel and/or through cross-talk such that optimal therapeutic benefit can be achieved only through inhibition of both pathways. As AKT is a central node in the PI3K pathway and MEK is a central node in the RAS pathway, developing inhibitors of AKT and MEK is a strategy being pursued by the pharmaceutical industry .
Recent clinical data have emerged demonstrating that activating mutations in the KRAS gene predict resistance to treatment with inhibitors of the epidermal growth factor receptor (EGFR). For example, KRAS mutations are associated with decreased disease control rate, shorter progression-free survival and reduced overall survival in patients with advanced or metastatic colorectal cancer treated with the EGFR-targeting antibodies cetuximab or panitumumab [4–6]. In non-small cell lung cancer, the relationship between KRAS mutation and response to EGFR inhibitors is less clear. Response rates in patients that do not harbor an activating mutation in EGFR are low, and mutations in KRAS and EGFR rarely occur in the same tumor. As such, there has been no clear relationship between KRAS mutation status and clinical outcomes in patients treated with the EGFR tyrosine kinase inhibitors gefitinib or erlotinib . Therefore, while alterations in specific RAS pathway components have lead to an increased understanding of the molecular drivers of response to EGFR inhibition in colorectal cancer, the relationship between KRAS mutation, RAS pathway dependence, and drug response is less clear in NSCLC and other tumor types.
Given the importance of KRAS activation for the selection of targeted cancer therapies, it is crucial that optimal methods are developed to measure the activation state of RAS in tumors. Due to the numerous genetic changes in tumors and the complexity of mechanisms underlying RAS pathway activation, a more comprehensive means of assessing RAS pathway activation status would be preferable. One way to enable a more comprehensive readout of pathway activity is to identify gene expression profiles that are indicative of pathway activation status. A gene expression signature-based pathway readout may be more appropriate than relying on a single indicator of pathway activity, as alterations in multiple signaling components could lead to pathway activation and result in similar downstream effects (for example, mutations in B-raf also lead to pathway activation and may lead to resistance to therapies targeting EGFR in colorectal cancer [4, 5, 8]).
Recent gene expression profiling efforts have identified pathway signatures that can be applied broadly across different datasets to monitor pathway activity. Moreover, recent studies have shown that pathway signatures can predict drug response in vitro and stratify tumors according to predicted pathway status [9–13]. Gene expression signatures could have additional benefit as pathway biomarkers, as these signatures could be used for both pretreatment patient stratification (i.e. prospectively identifying patients harboring tumors that are dependent on RAS signaling) and pharmacodynamic evaluation (i.e. monitoring pathway inhibition post-treatment). However, comparing pathway signatures to one another and assessing their robustness in independent datasets can be hampered by the use of heterogeneous microarray profiling and analysis methods [14–16]. Given the clinical importance of understanding RAS pathway activation and its relationship to drug response, our main goal was to develop a gene expression signature indicative of RAS pathway activity in human tumors that is robust and translatable across multiple tumor types and datasets. With such a tool in hand, it would be possible to assign a RAS activation score to tumors for the purposes of drug response prediction and pharmacodynamic assessment.
Through an integrated analysis of literature data and internal datasets incorporating both cell line models and human tumors, we identified a RAS pathway signature consisting of 147 genes that is coherently expressed across multiple datasets. The RAS pathway signature has a high sensitivity for detecting KRAS mutant cell lines and human tumors, but also identifies samples that have apparent RAS pathway activation in the absence of a KRAS mutation. We show that baseline levels of the RAS pathway signature predict resistance to AKT inhibition and sensitivity to MEK inhibition in cell line panels independent of KRAS mutation status, that the signature is downregulated by MEK inhibition, and that the signature is a better predictor of RAS pathway dependence compared to KRAS mutation status in lung cancer cell lines. In human tumors, the RAS signature is coherent across multiple tumor types, is elevated in clinical subtypes not known to harbor KRAS mutations (i.e. ER negative breast cancer), and predicts resistance to cetuximab in metastatic colorectal cancer. These data demonstrate that the RAS signature significantly expands the population of human tumors exhibiting RAS pathway deregulation, and has potential clinical utility in identifying lung and breast tumors where RAS pathway dependence should be considered when choosing appropriate targeted therapies.