Functional signaling pathway analysis of lung adenocarcinomas identifies novel therapeutic targets for KRAS mutant tumors

Abstract

Little is known about the complex signaling architecture of KRAS and the interconnected RAS-driven protein-protein interactions, especially as it occurs in human clinical specimens. This study explored the activated and interconnected signaling network of KRAS mutant lung adenocarcinomas (AD) to identify novel therapeutic targets.Thirty-four KRAS mutant (MT) and twenty-four KRAS wild-type (WT) frozen biospecimens were obtained from surgically treated lung ADs. Samples were subjected to Laser Capture Microdissection and Reverse Phase Protein Microarray analysis to explore the expression/activation levels of 150 signaling proteins along with co-activation concordance mapping. An independent set of 90 non-small cell lung cancers (NSCLC) was used to validate selected findings by immunohistochemistry (IHC).Compared to KRAS WT tumors, the signaling architecture of KRAS MT ADs revealed significant interactions between KRAS downstream substrates, the AKT/mTOR pathway, and a number of Receptor Tyrosine Kinases (RTK). Approximately one-third of the KRAS MT tumors had ERK activation greater than the WT counterpart (p<0.01). Notably 18% of the KRAS MT tumors had elevated activation of the Estrogen Receptor alpha (ER-α) (p=0.02).This finding was verified in an independent population by IHC (p=0.03).KRAS MT lung ADs appear to have a more intricate RAS linked signaling network than WT tumors with linkage to many RTKs and to the AKT-mTOR pathway. Combination therapy targeting different nodes of this network may be necessary to treat this group of patients. In addition, for patients with KRAS MT tumors and activation of the ER-α, anti-estrogen therapy may have important clinical implications.

Keywords: KRAS mutation; laser capture microdissection; non-small cell lung cancers; reverse phase protein microarray; signaling networks.

Figure 1. Correlation analysis of KRAS downstream substrates (activated c-Raf, Mek 1/2 and ERK 1/2) and expression/activation levels of 145 endpoints analyzed

Only correlations with p < 0.0003 are shown.

Figure 2

Panel A: Representation of selected proteins that were significantly higher in KRAS MT tumors Panel B: Scatter plots of RPPA intensity values with mean and standard error of mean.

Figure 3. Example of ER-α S118 staining by IHC (x400 magnification)

Panel A, B, C, and D show no activation, weak activation (1+), moderate activation (2+), and strong activation (3+) respectively. Panel E shows activation of ER-α S118 in the adenocarcinoma component of the sample with mixed adeno-squamous histology.