Abstract 521: Downregulation of type I IFN response in hypoxia as a possible mechanism of immune escape

Abstract

Introduction: Hypoxia is a common phenomenon in solid tumors and is considered a hallmark of cancer. Increasing evidence shows that tumor hypoxia promotes local immune suppression and inhibits immune cell killing functions. However, there is little information about the relationship between hypoxia and the type I interferon (IFN) pathway, which comprises the sensing double-stranded RNA and DNA (dsRNA/dsDNA) in the tumor microenvironment, followed by IFNα/β secretion. The aim of this study is to determine the effect of hypoxia on the type I IFN pathway in breast cancer and the consequences for tumor survival. Methods: A panel of breast cancer cell lines [MCF7, T47D, BT474, SKBR3, MDA231, MDA453, MDA468] were analyzed by Western blot and RT-qPCR in normoxia and acute hypoxia (0.1% O 2 ) for 24 hours, using medium containing 5mM glucose. Cells were stimulated with the dsRNA mimic poly I:C [10ng/ml to 20ug/ml] to activate the IFN type I pathway. Results: An overall 50% decrease in expression of the dsRNA-sensing branch (sensors: RIG-I, MDA5; adaptor: MAVS; effectors: IRF3, IRF7, STAT1, STAT2; interferon stimulated genes (ISGs): PKR, ADAR1) was observed in all breast cancer cell lines studied when they were treated with 0.1% O 2 for 24h both at RNA and protein levels. Moreover, when the pathway was stimulated using poly I:C, hypoxia also showed a decrease in the stimulation and in the activation of pIRF3 and pSTAT1. Different IFN bioassays were performed using HEK293 cells expressing (i) the IFN stimulated response element (ISRE), which were cultured for 24h with supernatant from MCF7 previously treated with normoxia/hypoxia and different concentration of poly I:C, and (ii) the IFNβ promoter, which were transfected with total RNA from normoxic/hypoxic cells. Consistent with reduced expression of factors involved in the IFN pathway, there was lower activation of the ISRE by supernatants from hypoxic cells compared to samples from normoxic cells, indicating lower IFN production in hypoxia. In addition, RNA from hypoxic cells showed much lower activation of the IFNβ promoter pointing to lower IFNβ transcription in hypoxic cells. HIF1α and/or HIF2α downregulation using siRNAs partially upregulated the expression of some ISGs (i.e. RIG-I, ADAR1), but this treatment did not completely reverse the effect of hypoxia, suggesting that the effect observed in hypoxia does not fully depend on HIF1α and HIF2α. Further studies on how chromatin conformation under hypoxia could affect the expression of genes in the type I IFN pathway are on going. Conclusions: Hypoxia downregulates the dsRNA-sensing branch of the type I IFN system in breast cancer, thus favoring an immunosuppressive status that could help immune evasiveness. Note: This abstract was not presented at the meeting. Citation Format: Ana Miar, Jan Rehwinkel, Christos Zois, Adrian L. Harris. Downregulation of type I IFN response in hypoxia as a possible mechanism of immune escape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 521.