Abstract 4810: HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma

Abstract

Abstract Purpose/Objective(s): Pancreatic ductal adenocarcinoma (PDAC) is characterized by an unusually high degree of hypoxia in the tumor microenvironment, which activates hypoxic signaling pathways orchestrated by hypoxia-inducible factor 1α (HIF1α). It has been shown previously that HIF1α significantly increases PDAC radioresistance and growth, though its mechanism of action is unknown. Here, we test the hypothesis that HIF1α confers these traits in PDAC through involvement in DNA damage repair and KRAS signaling in vitro. Materials/Methods: We performed studies with the KPC cell line (KrasLSL-G12D/+;TP53LSL-R172H/+). We used CRISPR-Cas9, a genome editing tool, to generate a HIF1α knockout (KO) cell line. Hypoxic microenvironments were induced using 100uM of the oxygen scavenger CoCl2. Radioresistance and double-strand DNA damage was evaluated and compared between wild-type (WT) and KO cells after treatment with increasing doses of ionizing radiation using neutral comet assays. Tumor signaling pathways were mapped and analyzed using western blot and co-immunoprecipitation. The KRASG12D inhibitor, BI-2852, was obtained from Boehringer-Ingelheim. Cancer cells were evaluated for growth and migration using cell proliferation assays and scratch wound assays following treatment with 50 uM BI-2852. Bioinformatic analysis was conducted using TCGA (The Cancer Genome Atlas) data; high and low expression were defined as samples with mRNA expression z-scores ≥ 1 and ≤ -1, respectively, Results: HIF1α KO cells demonstrated significantly increased radioresistance compared to WT cells following 8 and 10 Gy of radiation in hypoxic conditions (t-test, P<0.05). The olive moment of KO cells treated with the IC50 radiation dose (4 Gy) was significantly higher than that of WT cells (t-test, P<0.0001), suggesting that the DNA damage repair was upregulated by HIF1α. In addition, WT cells demonstrated higher tumor proliferation (t-test, P<0.01) and migration (t-test, P<0.05) compared to KO cells under hypoxia, suggesting HIF1α promoted tumor growth. WT cells treated with BI-2852 demonstrated significantly lower levels of TP53; using coimmunoprecipitation, we show that TP53 was likely degraded through Mdm2. HIF1α KO was necessary to induce high levels of apoptosis in cells treated with BI-2852. Our results suggest that HIF1α may act synergistically with KRAS to promote tumor survival through anti-apoptotic pathways that target TP53 for degradation. TCGA data also suggest that tumors with high expression of HIF1α is are associated with significantly poorer prognosis coimpared to tumors with low expression of HIF1α (Logrank test, P=0.003). Conclusion: Overall, this study demonstrates that HIF1α promotes DNA damage repair in PDAC and reveals a HIF1α/KRAS/p53 signaling axis. Future studies will test anti-HIF1α and anti-KRAS combination therapies in PDAC mouse models. Citation Format: Kevin J. Tu, Sanjit K. Roy, Amit Sawant, Hem D. Shukla. HIF1α is involved in radiation-induced DNA damage repair and anti-apoptotic pathways in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4810.