Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes

Abstract

Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.KEYWORDS ferritin, FH, FOXM1, fumarate, HLRCC, NRF2 H ereditary leiomyomatosis and renal cell cancer (HLRCC) patients carry a germ line-inactivating mutation in one of the fumarate hydratase (FH) alleles and are prone to developing skin leiomyomas, uterine fibroids, and renal cell carcinoma of type 2 papillary morphology (1). HLRCC-associated tumors harbor a loss of heterozygosity at the FH locus, indicating biallelic FH inactivation as the tumor-initiating event (1). However, it remains unclear how FH inactivation drives carcinogenesis.The most direct consequence of FH inactivation is intracellular fumarate accumulation. Accumulated fumarate can covalently modify cysteine residues of proteins in an uncatalyzed process termed succination and cause many alterations in cellular signaling (2). Succination in HLRCC cells was first discovered on Kelch-like ECH-associated protein 1 (KEAP1), a negative regulator of the nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcription factor (3). Since HLRCC is driven by FH inactivation, chronic succination of KEAP1 results in constitutive NRF2 activation and increased expression of its target genes (3). Besides KEAP1, the Krebs cycle enzyme aconitase 2 (Aco2) was reported to be a succination target in Fh knockout mouse tissues, and succination inhibited its activity (2). Despite dramatic cellular changes induced by protein succina-

Fumarate hydratase inactivation in hereditary leiomyomatosis and renal cell cancer is synthetic lethal with ferroptosis induction

“…Future efforts to develop treatments for this deadly disease may focus on targeting this particular vulnerability. For example, we have previously shown that ferritin knockdown retards cell proliferation . Prolonged ferritin knockdown is actually lethal to HLRCC cells (result not shown), potentially through ferroptotic cell death.…”

“…Transfections were performed using Attractene (Qiagen, Hildebrand, Germany). UOK262‐ FH res and UOK262‐EV were generated previously …”

“…This accumulated fumarate can form adducts on cysteine residues of proteins in a process known as succination . Several proteins, including Kelch‐like ECH‐associated protein 1 (KEAP1), aconitase 2 (ACO2) and iron regulatory protein 2 (IRP2), have been reported to be succinated in FH‐inactivated cells, and the succination events have been implicated in altered cellular signaling. For example, succination of KEAP1 allows for the accumulation and activation of the nuclear factor (erythroid‐2)‐like 2 (NRF2) transcription factor, which orchestrates the dominant cellular transcriptional programming observed in HLRCC cells .…”

“…For example, succination of KEAP1 allows for the accumulation and activation of the nuclear factor (erythroid‐2)‐like 2 (NRF2) transcription factor, which orchestrates the dominant cellular transcriptional programming observed in HLRCC cells . More recently, we showed that NRF2 activation and IRP2 succination increase cellular ferritin level in a concerted manner, and the ferritin drives HLRCC cells’ proliferation . Despite the unique and distinguishing biology driven by the FH loss, a strategy to specifically target HLRCC cells has yet to come to fruition.…”

“…(IRP2), 6 have been reported to be succinated in FH-inactivated cells, and the succination events have been implicated in altered cellular signaling. For example, succination of KEAP1 allows for the accumulation and activation of the nuclear factor (erythroid-2)-like 2 (NRF2) transcription factor, which orchestrates the dominant cellular transcriptional programming observed in HLRCC cells.…”