Abstract B158: Targeting APE1/Ref-1 results in inhibition of hypoxia signaling genes
Authors: Logsdon Derek P. , Cheng Huiwen , Luo Meihua , Shahda Safi , Hao Yangyang , Tong Yan , Yu Zhangsheng , Zyromski Nicholas , Schipani Ernestina , Liu Yunlong , Supuran Claudiu T. , Ivan Mircea , Kelley Mark R. , Fishel Melissa L. 
Published: May 2016

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease, and ∼95% die within five years. Treatment with chemotherapy has not changed the natural course of this disease, and just recently, with combination of chemotherapeutic agents, the median survival reached a year. Several mechanisms are proposed to play a role in the aggressive, treatment-resistant phenotype of PDAC, including adaptation to hypoxia, which leads to increased potential for metastasis and impairs the efficacy of chemotherapy and radiotherapy. Hypoxia-Inducible Factor-1α (HIF1α), a major oxygen sensor in cells, is a transcription factor that is rapidly degraded under normoxic conditions but upregulates a number of genes under hypoxic conditions that contribute to survival, metastasis, and angiogenic signaling in the tumor microenvironment. One of the most notable HIF targets is Carbonic Anhydrase IX (CA9), which promotes tumor cell survival and metastasis by maintaining a steady intracellular pH while acidifying the microenvironment, thereby encouraging epithelial-mesenchymal transition and contributing to extracellular matrix degradation. AP Endonuclease1/ Redox Effector Factor 1 (APE1/Ref-1) is a dual function protein that possesses a DNA repair function as well as the ability to reduce transcription factors and enable them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in preventing apoptosis, survival mechanisms, and hypoxia signaling, including HIF-1α. Therefore, we hypothesized that APE1/Ref-1 inhibition impairs HIF-1α-mediated signaling, and this leads to decreased survival and invasion of tumor cells exposed to hypoxic conditions.
Methods: We performed co-immunoprecipitation (co-IP) studies to look at the interaction of APE1/Ref-1 with transcriptional targets, HIF-1α, STAT3, and NFκB along with RT-PCR and Western blotting to confirm expression of hypoxia signaling genes. Luciferase reporter assays were used to quantitate transcriptional activation under hypoxia. Boyden chamber was used to look at migration and invasion as well as proliferation based assays following manipulation of APE1/Ref-1 and hypoxia.
Results: HIF-1α and STAT3, but not NFκB associate with APE1/Ref-1 under hypoxia. Moreover, we found that knockdown of APE1/Ref-1 protein diminishes HIF-mediated transcription in hypoxia, as shown by analysis of luciferase reporter assays. Next, we showed that, in hypoxia, APE1/Ref-1 inhibition diminishes HIF-1α-induced downstream targets including CA9 and ANGPTL4 further indicating that APE1/Ref-1 redox activity regulates HIF signaling. Importantly, we found that hypoxia, in the presence or absence of APE1/Ref-1, no longer induced CA9 mRNA levels in HIF-deficient MEFs, proving that hypoxia-dependent regulation of CA9 expression is fully mediated by HIF-1α. A blockade of both CA9 activity via small molecule and CA9 transcription via APE1/Ref-1 leads to decreased PDAC cell proliferation under hypoxia. These data indicate that APE1/Ref-1 inhibition interferes with ηψπoξια-mediated signaling and can further sensitize PDAC cells to CA9/12 inhibition even under the conditions of extreme oxygen deprivation. Ongoing experiments will determine the role APE1/Ref-1 plays in the survival and invasion of tumor cells exposed to hypoxia.
Citation Format: Derek P. Logsdon, Huiwen Cheng, Meihua Luo, Safi Shahda, Yangyang Hao, Yan Tong, Zhangsheng Yu, Nicholas Zyromski, Ernestina Schipani, Yunlong Liu, Claudiu T. Supuran, Mircea Ivan, Mark R. Kelley, Melissa L. Fishel. Targeting APE1/Ref-1 results in inhibition of hypoxia signaling genes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B158.