Abstract 2498: Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer.
Authors: Cortez Maria A. , Byers Lauren Averett , Fan You Hong , Diao Lixia , Groth Philip , Paul Julianne , Wang Jing , Giri Uma , Gudikote Jayanthi , Tran Hai , Coombes Kevin , Minna John D. , Liu Ningshu , Heymach John V. 
Published: August 2014

The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. Here, we sought to identify new potential biomarkers and pathways that confer resistance to PI3K inhibitors in NSCLC. To this end, we used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. First, we evaluted the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. We found increased phosphorylation and inactivation of pro-apoptotic protein Bad and phospho-LKB1 in the resistant cell lines. An inverse correlation was found for phosphorylation and inactivation of c-Src. Interestingly, activation of EGFR and increased levels of PTCH and PKCα were found in the resistant cell lines. Next, paired t-test was applied to each marker comparing the difference between drug and control treated cells. As expected, treatment with BAY 80-6946, significantly downregulates components of PI3K/mTOR pathway, as illustrated by decreased levels of phospho-Akt (-4.28-fold relative expression) and phospho-S6 ribosomal protein (-9.64-fold), phospho-p70S6K (-3.47-fold) and phospho-mTOR (-1.42-fold). We observed elevated expression of negative regulators of mTOR pathway, LKB1 (1.11-fold), AMPKα (1.12-fold) and phospho-AMPKα (12-fold). On the other hand, our analysis identified new potential pathways of resistance that are activated upon PI3K inhibition. We found increased levels of phospho-EGFR (P&lt;0.0001), PTCH (P&lt;0.0001) and PKCα (P&lt;0.0001). We observed activation of c-Src pathway upon treatment with BAY 80-6946, total and phospho-Src (P&lt;0.0001 and P&lt;0.0001). Accordingly, key downstream survival cascades regulated by c-Src, FAK (P&lt;0.0001), Stat5 (P&lt;0.0001), phospho-Stat5 (P&lt;0.0001) and phospho-Stat3 (P&lt;0.0001) were upregulated in treated cells. Our results suggest that PI3K inhibition leads to EGFR/MAPK and c-SRC/Stat signaling activation in vitro, suggesting a compensatory pathway that may allow NSCLC survival. Taken together, these results suggest that activation of Src/Stat pathway as an important mechanism of resistance to PI3K inhibitors in NSCLC and may be clinically relevant targets for combination therapy.

Citation Format: Maria A. Cortez, Lauren Averett Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai Tran, Kevin Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2013-2498