Abstract 3260: Antiproliferative effects of PI3K-mTOR pathway inhibitors alone or in combination with MEK inhibitor GSK1120212 in pancreatic cancer cell lines are not correlated with molecular effects on ERK and S6 phosphorylation.
Authors: Neuzillet Cindy , Serova Maria , Tijeras-Raballand Annemilai , Astorgues-Xerri Lucile , Riveiro Maria E. , de Gramont Armand , Ruszniewski Philippe , Hammel Pascal , Faivre Sandrine , Raymond Eric 
Published: August 2014

Background: Activating K-RAS mutations are frequent (&gt;70%) in pancreatic adenocarcinoma (PAC) and drive downstream deregulation of both Raf-MEK-ERK and PI3K-mTOR pathways. Moreover, many cross-talks exist between these two pathways, so that inhibition of one leads to activation of the other. MEK inhibitors (MEKi) are currently under clinical evaluation in PAC, in combination with chemotherapy or targeted agents including PI3K-mTOR pathway inhibitors. To date, no predictive marker has been validated for such combinations in PAC. This study aimed to explore the effects of various PI3K-mTOR pathway inhibitors alone or in combination with MEKi GSK1120212, in MIAPACA-2 and PANC-1 PAC cell lines.

Materials and Methods: Everolimus is a mTORC1 inhibitor, AZD8055 a dual mTORC1/mTORC2 inhibitor, BEZ235 a dual PI3K/mTOR inhibitor, and BKM120 a pan-class PI3K inhibitor. GSK1120212 is an allosteric non-ATP competitive MEKi. Effects on proliferation were evaluated by MTT assay. Protein expression was assessed by Western blot. Combinations were analyzed using the Chou-Talalay method.

Results: We had previously shown that MIAPaCa-2 and PANC-1 were two mesenchymal K-RAS mutated PAC cell lines with different response to GSK1120212, MIAPaCa-2 being the most sensitive (72h-IC50=0.009μM) and PANC-1 the most resistant (72h-IC50=33μM). We additionally screened these cell lines for predictive markers of response to mTOR pathway inhibitors: no mutation was found in B-RAF, EGFR and PI3KCA genes; MIAPaCa-2 exhibited lower p27, PTEN and higher Bcl-2 levels than PANC-1. PANC-1 exhibited high basal level of Akt associated with high level of pAkt and downstream pP70S6K and pS6, suggesting basal activation of the pathway. PI3K-mTOR pathway inhibitors exerted antiproliferative effects on both cell lines. Unexpectedly, MIAPaCa-2 was more sensitive than PANC-1 to everolimus (IC50=23.3μM vs 47.0μM) and other PI3K-mTOR inhibitors (IC50 range=0.385-4.19μM vs 6.31-31.6μM). Combination of GSK1120212 and the PI3K-mTOR inhibitors for 72h resulted in synergistic effects in MIAPaCa-2 sensitive cell line but not in PANC-1. We further studied cell signaling in PANC-1. MEK inhibition abolished pERK expression (-70%) and increased pAkt expression (+60%). mTOR pathway inhibition by everolimus or BKM120 (0.1μM, 1μM) decreased pS6 expression (-90% and -80%, respectively) and induced a slight increase in pERK (+10%). Combination therapy resulted in extinction of both pERK and pS6 expression.

Conclusion: Response to combined MEK/mTOR pathway inhibition was not correlated with usually reported biomarkers of response to MEKi or mTOR pathway inhibitors. PANC-1 cell line was resistant despite effective inhibition of both pERK and pS6, suggesting that resistance may be due to activation of other alternative pathway(s).

Citation Format: Cindy Neuzillet, Maria Serova, Annemilai Tijeras-Raballand, Lucile Astorgues-Xerri, Maria E. Riveiro, Armand de Gramont, Philippe Ruszniewski, Pascal Hammel, Sandrine Faivre, Eric Raymond. Antiproliferative effects of PI3K-mTOR pathway inhibitors alone or in combination with MEK inhibitor GSK1120212 in pancreatic cancer cell lines are not correlated with molecular effects on ERK and S6 phosphorylation. [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 3260. doi:10.1158/1538-7445.AM2013-3260