Abstract 5632: MEK inhibition leads to elevated HER3:PI3K in EGFR or HER2 driven cancer through feedback induction of HER1:3 and HER2:3 dimers.
Authors: Hoe Nicholas , Mateling Michael , Ma Yating , Jin Kelly , Zhou JinYao , Kirkland Richard , Kuy Crystal , Liu Xinjun , Kim Phillip , Singh Sharat 
Published: August 2014

Abstract
In wide range of human cancer, the phosphoinositide 3-kinase (PI3K)/AKT and RAF/MEK/ERK signaling pathways are activated, regulating cell growth, metabolism, survival, and proliferation. Inhibition of either PI3K/AKT or MEK/ERK signaling pathway as a single-agent targeted therapy is often ineffective due to feedback mechanisms in which inhibition of one signaling pathway leads to activation of another. Treatment with a single-agent MEK inhibitor substantially increases ERBB3/PI3K/AKT phospho levels by relieving an ERK mediated negative feedback on threonine phosphorylation on the juxtamembrane domains of EGFR (T669) and HER2 (T677). Loss of inhibitory threonine phosphorylation in EGFR and HER2 suggests that increase in phospho HER3 levels may be due to formation of heterodimers (EGFR:HER3 and HER2:HER3) driving trans-phosphorylation of ERBB3.
Herein we report, utilizing Collaborative Enzyme Enhanced Reactive (CEERTM) immunoassay, comprehensive analysis of key receptor tyrosine kinases (HER1, HER2, HER3, cMET, IGF1R, and others) and their downstream signal proteins (PI3K, Shc, AKT, MEK, ERK, PRAS40, RPS6, P70S6K, RSK), and dimerization partners (HER1:HER2, HER2:HER3, HER1:HER3, and HER3:PI3K) in MDA-MB-468, and BT474 treated with either MEK inhibitor alone (AZD6244) or in combination with PI3K inhibitor (GDC0941). CEER platform utilizes the formation of an unique “triple-antibody-enzyme-channeling” immuno-complex, requiring minimal sample amount.
At MEK inhibitor concentration of 1μmol/L, both MDA-MB-468 and BT474 cells sufficiently inhibited ERK and RSK phosphorylation. In each cell line, we observed increase in phospho-HER3 and activated HER3:PI3K complex, a phospho-tyrosine signaling cascades that directly activate AKT. Levels of HER1:3 and HER2:3 dimers were elevated in MDA-MB-468 and BT474, respectively post MEK inhibitor treatment. Feedback activation of AKT with AZD6244 was suppressed when combined with GDC0941 at concentration of 1μmol/L. With combination of MEK and PI3K inhibitors, levels of HER1:3 and HER2:3 dimers were unaffected in their respective cell line, but HER3:PI3K complex was completely suppressed in both cell lines. The data suggests that loss of negative feedback with MEK inhibition alone promoted alternate oncogenic AKT signaling. As often the case, single-targeted therapies are ineffective, since the pathway it is targeted to inhibit, leads to feedback contributing to therapeutic resistance. Utilizing CEERTM, clinical specimens with limited availability can be profiled in a comprehensive manner for potential feedback mechanisms and selection of combinational therapies suppressing both the oncoprotein and the feedback program.
Citation Format: Nicholas Hoe, Michael Mateling, Yating Ma, Kelly Jin, JinYao Zhou, Richard Kirkland, Crystal Kuy, Xinjun Liu, Phillip Kim, Sharat Singh. MEK inhibition leads to elevated HER3:PI3K in EGFR or HER2 driven cancer through feedback induction of HER1:3 and HER2:3 dimers. [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 5632. doi:10.1158/1538-7445.AM2013-5632