Abstract C34: Signaling regulation of epithelial-mesenchymal transition in pancreas cancer cells cultured under hypoxic conditions
Authors: McGirr Brooke A. , Seyler Nicholas M. , Lazzara Matthew J. 
Published: March 2020

Abstract
Pancreatic ductal adenocarcinoma (PDAC) tumors exhibit hypoxia due to high oxygen demand and oxygen diffusional limitations (1). Hypoxia has been reported as a driver of epithelial-mesenchymal transition (EMT) (2), which occurs early in pancreas cancer dissemination and promotes resistance to chemotherapy (3,4). Druggable signaling pathways that promote EMT in the hypoxic PDAC microenvironment may therefore represent useful targets for combination therapy. Our lab previously studied the roles of ERK and SHP2 in driving EMT in response to growth factors (5,6). More recently, our lab employed an unbiased data-driven modeling approach and found that the ERK, JNK, and NF-kappaB pathways coordinate to drive robust EMT in PDAC cells treated with combinations of growth factors (unpublished). In the present study, we sought to determine the roles of those pathways in promoting EMT in PDAC cells in response to a shift from normoxic to hypoxic conditions. The immortalized human PDAC cell line HPAF-II, which exhibits baseline epithelial characteristics, was used for these studies. In simple two-dimensional cell culture, a shift from 20% to 1% oxygen for ≤120 hr led to several indications of EMT, including decreased epithelial cell cluster circularity, reduced junctional E-cadherin expression and an increased fraction of vimentin-positive cells (determined by an automated image analysis pipeline), and increased expression of the mesenchymal transcripts VIM, SNAI1, and SNAI2 (determined by RT-qPCR). A hypoxic cellular response was confirmed by immunoblotting, which showed an early (4 hr) peak in HIF-1alpha expression and a relatively late (48 hr) peak in HIF-2alpha expression, as well as increased expression of the HIF target genes PGK1 and SLC2A1. In three-dimensional cultures of HPAF-II cell spheroids in a collagen I matrix, a shift to hypoxia promoted cell invasion into the matrix, a behavior also observed when HPAF-II cells were treated with exogenous growth factors that drive EMT. To probe for the activation of the ERK, JNK, and NF-kappaB pathways, we used a combination of immunoblotting and immunofluorescence microscopy. A shift from 20% to 1% oxygen increased the phosphorylation of ERK and promoted the nuclear localization of c-Jun, a substrate of JNK. Inhibition of MEK or JNK at the same time as the shift to 1% oxygen prevented the loss of junctional E-cadherin that otherwise occurred, with JNK inhibition having the greater effect. JNK inhibition also abrogated loss of CDH1 transcripts and the increase of VIM transcripts otherwise observed in response to hypoxia. Thus, activation of the ERK and JNK pathways in PDAC cells in response to a shift to low oxygen conditions promoted EMT. These pathways may therefore represent useful targets for antagonizing EMT in the hypoxic microenvironment characteristic of PDAC tumors.
References: 1. Bertout et al., Nat Rev Cancer 2008. 2. Marie-Egyptienne et al., Cancer Lett 2013. 3. Rhim et al., Cell 2012. 4. Zheng et al., Nature 2015. 5. Buonato et al., Cancer Res 2014. 6. Buonato et al., J Cell Sci 2015.
Citation Format: Brooke A. McGirr, Nicholas M. Seyler, Matthew J. Lazzara. Signaling regulation of epithelial-mesenchymal transition in pancreas cancer cells cultured under hypoxic conditions [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C34.