Abstract 4499: The oncometabolite fumarate prevents hypoxia-induced ER stress by enhancing the pentose phosphate pathway
Authors: Schito Luana , Rey Sergio , Wouters Bradly G. , Koritzinsky Marianne 
Published: July 2017

Abstract
Cancer cells co-opt mechanisms of adaptation to hypoxia to minimize energy expenditure and halt proliferation thereby increasing hypoxic tolerance, a major factor contributing to chemo- and radioresistance in patients. The endoplasmic reticulum (ER) kinase PERK is one of the three signaling transducers of the unfolded protein response (UPR) that inhibits mRNA translation via eIF2α phosphorylation to alleviate hypoxia-induced ER stress. We have previously shown that accumulation of fumarate, resulting from loss-of-function (LOF) of the mitochondrial enzyme fumarate hydratase (FH), augments the antioxidant capacity of hypoxic cancer cells through upregulation of the pentose phosphate pathway (PPP). Here, we report for the first time that the fumarate-dependent increase of antioxidants generated by the PPP prevents hypoxic activation of the PERK→eIF2α axis enabling protein synthesis and proliferation leading to an impairment of hypoxic tolerance.
HeLa (cervix), HCT-116 and LS174T (colon) adenocarcinoma cells were transfected with lentiviral vectors encoding for shRNAs targeting FH. Immunoblot assays showed that FH LOF impaired PERK activity under hypoxia thus inactivating eIF2α. Moreover, the ability of fumarate to prevent PERK activation was specific to hypoxia since fumarate did not affect the pharmacological activation of the UPR by thapsigargin, DTT or tunicamycin. A comprehensive analysis utilizing a RT-qPCR array to profile the mRNA expression of 84 UPR genes, showed that FH LOF inhibited the UPR in HCT-116 cells. A similar response was observed in patient-derived UOK262 kidney cancer cells whereby bi-allelic FH mutations resulted in downregulation of 72% of the UPR genes whereas FH re-introduction restored UPR signaling. We employed LS174T mucinous adenocarcinoma cells as a model to assess protein synthesis. Mucin synthesis was not affected in hypoxic FH deficient cells whereas it was abrogated in wild-type cells. Consistent with these results, FH LOF increased 3D spheroid growth and decreased clonogenic potential in HCT-116 and HeLa cells. Pharmacological inhibition of the PPP by 6-aminonicotinamide activated the PERK→eIF2α axis in FH silenced cells thereby suggesting that PPP activation by fumarate is implicated in PERK inhibition. RNAseq data analysis in melanoma, prostate, colon, breast, lung and renal cancers from TCGA cohorts shows a negative correlation between PERK and the PPP enzyme PGLS suggesting that the mechanism hereby delineated occurs in cancer patients.
In conclusion, we show that fumarate impairs PERK→eIF2α UPR signaling in hypoxic cancer cells. Clinical and molecular data show that UPR activation is one of the mechanisms responsible for chemo- and radioresistance in hypoxic tumors. This study suggests that fumarate accumulation (either through extrinsic or FH LOF) represents a novel approach to target hypoxic cancer cells and improve patient prognosis.
Citation Format: Luana Schito, Sergio Rey, Bradly G. Wouters, Marianne Koritzinsky. The oncometabolite fumarate prevents hypoxia-induced ER stress by enhancing the pentose phosphate pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4499. doi:10.1158/1538-7445.AM2017-4499