P3-18-04: Pathway Guided Selection of Targeted Inhibitors for Breast Cancer Treatment.
Authors: von Ahsen O , Kim P , Langley E , Hazra S , Lee T , Leesman G , Schneider C , Singh S , Krahn T 
Published: June 2012

Background: The ability to functionally profile a whole spectrum of pathway proteins in tumor may provide valuable information about the likelihood of drug response and potential mechanisms for drug resistance in breast cancer (BCA) patients. Here we report a comprehensive pathway analysis of membrane associated kinases such as HER1, HER2, HER3, cMET, IGF1R, PI3K and downstream signal transduction proteins including Shc, AKT, ERK, MEK, PDK1, PRAS40, p70S6K and eIF4e in breast cancer cell lines and xenografts as in response to inhibitors targeting the Her1/Her2 and PI3K/AKT and MAPK pathways. For comparison we also analyzed NSCLC lines driven by other receptor tyrosine kinases like EGFR and MET. Methods: Lysates prepared from KPL4 cells and MCF7 cells both harboring PIK3CA mutations (with HER2−amplification and low level HER2 expression, respectively) treated with 6 inhibitors (an allosteric AKT inhibitor, Lapatinib, Regorafenib, MET inhibitor, an allosteric MEK inhibitor or PI3K inhibitor) targeting PI3K/AKT and MAPK pathways were analyzed for modulations of pathway protein phosphorylation using a multiplexed immuno-micorarray.

Tissue lysates prepared from KPL4 xenografts treated with AKT inhibitor were analyzed for pathway modulation in response to the drug treatment. The pathway activation profile shift in response to drug treatments in BCA models are compared to other tumor type models with diverse oncogenic backgrounds.

Results: The level of phosphorylated AKT (pAKT) was reduced when MCF7 cells were treated with AKT inhibitor as well as PI3K inhibitor. However, compensatory AKT activation was observed when these cells were treated with BRAF or MEK inhibitor. KPL4 cells also showed reduction of pAKT when treated with Lapatinib, AKT inhibitor or PI3K inhibitor. Compensatory hyper-phosphorylation of ERK (pERK) was observed in both cell lines and KPL4 xenografts with AKT inhibitor treatment while PI3K inhibition did not induce hyper-ERK phosphorylation. Reduction of pERK level was observed when both cell lines were treated with MEK inhibitor. Downstream analytes like PRAS40 and RPS6 summarize PI3K/AKT pathway activity and correlate well with response to treatment whereas eIF4e is a good final readout for activity in the RAS/RAF/MEK/ERK pathway. Treatment with single agents often even shows adverse profile shift through feedback or pathway cross-talk. The comparative analysis of pathway modulation in BCA models to other tumor types in response to drug treatments revealed several adaptive drug resistance mechanism with different oncogenic backgrounds.

Discussion: Evaluation of drug specific pathway modulations in cancer cells provided comprehensive information on efficacy of specific agents on target protein and pathway inhibition. Multiplexed pathway analysis provides valuable information for drug resistance mechanisms due to either redundant pathway activation, cross-talk or through feedback mechanism and may guide appropriate selection of targeted drug-combinations or drug-sequencing in clinical setting. For example, the observed increase in ERK phosphorylation due to AKT inhibition could be blocked by combination therapy with a MEK inhibitor.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-04.