Abstract B71: Resistance to MEK inhibition in pancreatic cancer is associated with amphiregulin mediated EGFR-STAT3 activation
Authors: Nagathihalli Nagaraj , Castellanos Jason , Shi Chanjuan , Roberts Casey , VanSaun Michael , Merchant Nipun 
Published: December 2016

Abstract
Introduction: Mutations in the KRAS oncogene occur in the majority of pancreatic ductal adenocarcinomas (PDAC), resulting in aberrant activation of the MAPK (RAS-RAF-MEK-ERK) pathway, driving malignant progression. Targeting KRAS has remained an elusive goal. Therefore, efforts have focused on targeting downstream effectors of RAS. The clinical efficacy of MEK inhibitors in other malignancies confirms that targeting the MAPK pathway has therapeutic potential. Unfortunately, clinical trials of MAPK-directed therapies have been unsuccessful in PDAC. Here, we report a novel mechanism of resistance to MAPK-directed therapies, which is associated with amphiregulin (AREG)-mediated activation of EGFR-STAT3 signaling.
Experimental procedure: The effects of MEK inhibition on the phosphorylation of multiple signaling proteins and EGF family ligands was assessed. Activation of MAPK and STAT3 was quantified in human pancreatic tissues, and then correlated with overall survival (OS). Cell lysates from mouse PanIN lines derived from the LSL-KrasG12D/+;Pdx1Cre/+ and PDA line from LSL-KrasG12D/+; Trp53R172H/+;Pdx1Cre/+ mouse models of PDAC were immunoblotted for activated MAPK and STAT3 expression. AREG release was measured in the conditioned media of PDAC cells treated with MEK, EGFR, STAT3, and/or TACE inhibitors and TACE siRNA knock down cells treated with MEK, EGFR, or STAT3 inhibitors. Cell invasion, colony formation, spheroid formation, metabolic activity, cell cycle analysis, and apoptosis assays were performed with human PDAC cells treated with inhibitors for TACE, MEK, EGFR and STAT3 in combinations. PDAC xenografts and patient derived xenografts (PDXs) were treated either with vehicle, the MEK inhibitor AZD6244, the STAT3 inhibitor AZD1480 or both drugs and analyzed for spheroid growth. PKT mice (Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl) were treated with AZD6244 and AZD1480 and assessed for OS. Tissues from the xenografts and PKT mice were analyzed for cell proliferation and apoptosis markers. Plasma from the PKT mice treated with the drugs and their combinations was analyzed released AREG by ELISA.
Results: Targeting the MAPK pathway is a novel approach to inhibit KRAS mutant tumor progression and improve therapeutic response in PDAC. However, our results show that MAPK inhibition leads to activation of TACE and EGFR and subsequent activation of STAT3 signaling. Combined inhibition of MEK/STAT3 or MEK/EGFR resulted in sustained blockade of MEK, EGFR and STAT3 signaling, decreased cell invasion, colony formation, reduced spheroid size, and metabolic activity in vitro. Growth of flank PDAC xenografts and human PDX tumors in vivo were significantly decreased with combined MEK and STAT3 inhibition when compared to vehicle or monotherapy treatment. OS in PKT mice was extended to a median of 85 days with combined MEK and STAT3 inhibition vs. 52 days for vehicle treated mice (p &lt; 0.001). AREG release was significantly reduced with combined MEK/STAT3 inhibition. TACE siRNA in PDAC cells confirms the role of AREG release in mediating EGFR signaling and overcoming MEK inhibitor resistance. Evaluation of TACE activation and AREG shedding/release in response to MEK inhibition demonstrated that resistance to MEK inhibition in PDAC is mediated by reactivation of the STAT3 pathway, which is strongly influenced by increased AREG production.
Conclusions: Our study provides insights into the molecular mechanisms that help explain the heterogeneous response and therapeutic resistance of PDAC to MAPK pathway inhibition and provide a strong rationale that AREG mediated EGFR-STAT3 pathway activation is a major resistance mechanism that impairs the efficacy of MEK inhibitors.
Citation Format: Nagaraj Nagathihalli, Jason Castellanos, Chanjuan Shi, Casey Roberts, Michael VanSaun, Nipun Merchant.{Authors}. Resistance to MEK inhibition in pancreatic cancer is associated with amphiregulin mediated EGFR-STAT3 activation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B71.