Abstract 4440: The functional role of insulin-like growth factor binding protein-2 in esophageal adenocarcinoma chemoresistance
Authors: Myers Amy L. , Lin Lin , Nancarrow Derek J. , Wang Zhuwen , Ferrer-Torres Daysha , Beer David G. , Chang Andrew C. 
Published: August 2015

Abstract
Despite improved understanding of esophageal adenocarcinoma (EAC) progression and advances in endoscopic surveillance and multimodality treatment, patients commonly present with advanced stage disease and demonstrate resistance to therapy, with complete response rates below 40%. Understanding the molecular mechanisms of resistance is crucial for predicting and overcoming this resistance.
IGFBP2 is a member of the IGFBP family of proteins that has been shown to modulate both insulin growth factor (IGF) and integrin signaling and is a mediator of cell growth, invasion and resistance in other tumor types. Because these pathways have been implicated in esophageal cancer growth and progression, we evaluated the functional role of IGFBP2 in EAC tumorigenesis and chemoresistance. High IGFBP2 expression was observed in a subset of EAC patients and was found to be significantly higher in patients with shorter disease-free intervals as well as in treatment-resistant EACs as compared to chemonaive EACs.
To evaluate the role of IGFBP2 as a potential modulator of chemoresistance in vitro, IGFBP2-expressing Flo-1 EAC cells were pretreated with siRNA to IGFBP2 while non-expressing OE33 EAC cells were stably-transfected with an IGFBP2 expression construct or pre-treated with exogenous recombinant IGFBP2 followed by 3-day treatment with chemotherapy. All experiments were performed in serum-deprived and serum-replete conditions and were assessed using WST-1 cell proliferation reagent.
IGFBP2 siRNA inhibition sensitized Flo-1 cells to cisplatin in the presence of serum but exogenous treatment or ectopic expression of IGFBP2 in OE33 cells did not alter their chemosensitivity. Immunoblot analyses of Flo-1 cells treated with cisplatin showed a dose-dependent reduction in IGFBP2 expression but a dose-dependent induction of ERK and AKT phosphorylation at 24 hours post-treatment. Knockdown of IGFBP2 blocked IGF1 and CDDP-induced ERK activation at 15 minutes and 3-days post-treatment, respectively. In contrast, IGFBP2 knockdown resulted in the activation of AKT and was further enhanced by the addition of IGF1 up to 24 hours post-treatment. In an attempt to augment siIGFBP2-induced CDDP chemosensitization, we pretreated Flo-1 cells with either a MEK1/2 inhibitor, selumetinib or trametinib, or the AKT inhibitor, AKT Inhibitor VIII, in repeat cytotoxicity assays. The MEK1/2 inhibitors increased sensitivity in serum-replete conditions while AKT Inhibitor VIII increased sensitivity in serum-deprived conditions as compared to CDDP alone, IGFBP2 knockdown alone, and the combination of IGFBP2 knockdown and CDDP. These results suggest that targeted modulation of IGFBP2 and either the MAPK or PI3K/AKT signaling pathway in IGFBP2-overexpressing EAC tumors may be an effective approach for sensitizing resistant tumors to standard-of-care chemotherapy treatment options.
Citation Format: Amy L. Myers, Lin Lin, Derek J. Nancarrow, Zhuwen Wang, Daysha Ferrer-Torres, David G. Beer, Andrew C. Chang. The functional role of insulin-like growth factor binding protein-2 in esophageal adenocarcinoma chemoresistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4440. doi:10.1158/1538-7445.AM2015-4440