The oncometabolite fumarate promotes pseudohypoxia through noncanonical activation of NF-κB signaling

Abstract

Inactivating mutations of the gene encoding the tricarboxylic acid cycle enzyme fumarate hydratase (FH) have been linked to an aggressive variant of hereditary kidney cancer (hereditary leiomyomatosis and renal cell cancer). These tumors accumulate markedly elevated levels of fumarate. Fumarate is among a growing list of oncometabolites identified in cancers with mutations of genes involved in intermediary metabolism. FH-deficient tumors are notable for their pronounced accumulation of the transcription factor hypoxia inducible factor-1α (HIF-1α) and aggressive behavior. To date, HIF-1α accumulation in hereditary leiomyomatosis and renal cell cancer tumors is thought to result from fumarate-dependent inhibition of prolyl hydroxylases and subsequent evasion from von Hippel-Lindau-dependent degradation. Here, we demonstrate a novel mechanism by which fumarate promotes HIF-1α mRNA and protein accumulation independent of the von Hippel-Lindau pathway. Here we demonstrate that fumarate promotes p65 phosphorylation and p65 accumulation at the HIF-1α promoter through non-canonical signaling via the upstream Tank binding kinase 1 (TBK1). Consistent with these data, inhibition of the TBK1/p65 axis blocks HIF-1α accumulation in cellular models of FH loss and markedly reduces cell invasion of FH-deficient RCC cancer cells. Collectively, our data demonstrate a novel mechanism by which pseudohypoxia is promoted in FH-deficient tumors and identifies TBK1 as a novel putative therapeutic target for the treatment of aggressive fumarate-driven tumors.

Keywords: Cancer Biology; Cell Metabolism; Hypoxia-inducible Factor (HIF); NF-κB (NF-KB); Small; Small Molecule.

FIGURE 1.

Cell models of high fumarate state demonstrate increased HIF-1α mRNA levels. A, HIF-1α mRNA expression was assessed in HK2- and FH-deficient UOK 262 cultured cells and normalized to 18 S RNA as described under “Experimental Procedures.” In parallel, HIF-1α protein expression was analyzed by Western blot. Tubulin was used as a loading control. The data were quantitated and the results are expressed as mean ± S.E., **, p < 0.01. B, HIF-1α mRNA levels were examined in FH-deficient UOK 262 stably transfected with FH FLAG or vector control (Vec). The data are expressed as mean ± S.E., *, p < 0.05. HIF-1α expression was analyzed in the nuclear extract and Lamin was used as a loading control. FLAG antibodies were used to detect overexpression of FH in whole cell lysate (WCE). Tubulin was used as a loading control.

FIGURE 2.

Effects of fumarate on HIF-1α expression in VHL-deficient cells. VHL-deficient RCC4 cells were treated with increasing concentrations of DEF or DMF in serum-free media for 8 h. A, upper panel, HIF-1α mRNA was assessed in DEF-treated cells as described in the legend to Fig. 1 and expressed relative to dimethyl sulfoxide-treated control. Lower panel, HIF-1α protein levels were examined by Western blot in parallel. Tubulin was used as a loading control. The data were quantitated and the results are expressed as mean ± S.E. *, p < 0.05 and **, p < 0.01. B, upper panel, HIF-1α mRNA was assessed in DMF-treated cells as described in the legend to Fig. 1 and expressed relative to dimethyl sulfoxide-treated control. Lower panel, HIF-1α protein levels were examined by Western blot in parallel. GAPDH was used as a loading control. The data were quantitated and the results are expressed as mean ± S.E. *, p < 0.05. C, HIF-1α mRNA expression was analyzed in RCC4 cells silenced of fumarate hydratase using siRNA (siFH) or scrambled control (scr). The data were quantitated and the results are expressed as mean ± S.E. ***, p < 0.001. D, HIF-1α and FH expression was assessed in total cell lysates prepared from RCC4 cells transfected with siFH or scrambled control from C. Actin was used as loading control.

FIGURE 3.

Fumarate-induces HIF-1α mRNA expression through an NF-κB-dependent mechanism. A, RCC4 cells were treated with increasing concentrations of DEF and DMF (left and right panels, respectively) in serum-free media for 8 h. Cell lysates were prepared and phospho-p65 Ser⁵³⁶ (p-p65 S⁵³⁶), total p65, and IκB expression were analyzed by Western blot. Actin was used as loading control. B, RCC4 cells were transiently transfected with siRNA oligos for fumarate hydratase (siFH) or scrambled control (scr). Total protein was analyzed for phospho-p65 Ser⁵³⁶, total p65, and FH. Actin was used as loading control. C, p65 was transiently down-regulated in RCC4 cells using small inhibitory RNA (siRNA). Scrambled oligos were transfected as a negative control. Cell lysates were prepared and expression of HIF-1α, phospho-p65 Ser^536, and total p65 were assessed. GAPDH was used as loading control. D, HIF-1α DNA associated with p65 was assessed in nuclear extracts of RCC4 cells treated with 100 μm DEF for 8 h using ChIP as described under “Experimental Procedures.” Input DNA from RCC4 cells treated or not with DEF was used as a positive control for HIF-1α PCR. Immunoprecipitation using nonspecific IgG was used as a negative control in RCC4 cells treated or not with DEF. HIF-1α is detected at 518 bp. Nonspecific (NS) bands and primer dimer bands (PD) are indicated by arrows. The data were quantitated from two independent experiments and the results are expressed as mean ± S.E. **, p < 0.01 (lower panel).

FIGURE 4.

Fumarate promotes p65 phosphorylation in an IKK-independent manner. A, HIF-1α phospho-p65 Ser⁵³⁶ (p-p65 S⁵³⁶) and total p65 were analyzed by Western blot analysis using cell lysates prepared from FH null MEFs or FH-deficient MEFs stably expressing FH (FH^−/− + FH). Tubulin expression was used as loading control. B, HIF-1α, phospho-p65 Ser⁵³⁶, and total p65 expression was examined in FH-null MEFs and wild-type (WT) MEFs treated with increasing concentrations of DMF in serum-free media for 6 h. Actin was used as loading control. C, DKO IKKα and IKKβ MEFs were treated with increasing concentrations of DEF in serum-free media for 6 h. Whole cell lysates were analyzed for phospho-p65 Ser⁵³⁶ and total p65, and IκB. Actin was used as loading control.

FIGURE 5.

Fumarate mediates HIF-1α mRNA expression through non-canonical NF-κB signaling. A, phospho-TBK Ser¹⁷² (p-TBK S¹⁷²) and total TBK expression was examined in DKO (IKKα and IKKβ) MEFs treated with increasing concentrations of DEF in serum-free media for 6 h. B, phospho-TBK Ser¹⁷² and total TBK expression was assessed in RCC4 cells treated with increasing concentrations of DEF (upper panel) and DMF (lower panel) for 8 h in serum-free media. C, RCC4 cells were transiently transfected with siFH or scrambled control and total protein was analyzed for phospho-TBK Ser¹⁷², and total TBK. Actin was used as loading control. D, total TBK and phospho-TBK Ser¹⁷² expression was assessed in wild-type (WT), FH-deficient (FH^−/−), or FH-deficient with FH stably added back (FH^−/− + FH) MEFs.

FIGURE 6.

Fumarate mediates HIF-1α mRNA expression through TBK1. A, FH or fumarate hydratase + Tank-binding kinase-1 (FH + TBK) were transiently silenced (siFH and siTBK/siFH, respectively) in DKO (IKKα and IKKβ) MEFs. Scrambled oligos were transfected as a negative control. After 48 h, cell lysates were prepared and analyzed for phospho-p65 Ser⁵³⁶ (p-p65 S⁵³⁶), total p65, total TBK and FH. Tubulin was used as loading control. B, HK2 cells were treated with DEF simultaneously with the Tank-binding kinase inhibitor BX795 at the indicated concentrations for 8 h. Total protein was used to assess HIF-1α, phospho-p65 Ser⁵³⁶ expression. Tubulin was used as loading control.

FIGURE 7.

Activated p65/TBK axis in FH-deficient tumor cells promotes HIF-1α expression. A, phospho-TBK Ser¹⁷² and total TBK expression was analyzed in cell extracts prepared from HK2, FH-deficient UOK 262 stably expressing vector control, or FH-FLAG. B, phospho-p65 Ser⁵³⁶ (p-p65 S⁵³⁶) and total p65 expression was assessed in FH-deficient UOK 262 stably expressing vector control or FH-FLAG. Tubulin was used as loading control. C, HIF-1α DNA associated with p65 was assessed in nuclear extracts of FH-deficient UOK 262 cells stably expressing vector control or FH using ChIP as outlined under “Experimental Procedures.” Input DNA was used as a positive control for HIF-1α PCR. Immunoprecipitation using nonspecific IgG was used as a negative control. HIF-1α is detected at 518 bp. The data were quantitated from two independent experiments and the results are expressed as mean ± S.E. *, p < 0.05 (lower panel). D, UOK 262 cells were transiently transfected with siTBK or scramble control. After 48 h nuclear and cytoplasmic extracts were prepared. HIF-1α, phospho-p65 Ser⁵³⁶ (p-p65 S⁵³⁶), and total p65 expression were analyzed in the nuclear fraction by Western blot analysis. CREB was used as loading control. Total TBK expression was assessed in the cytosolic lysate. Tubulin was used as loading control. E, UOK 262 cells was transiently transfected with siTBK or scramble control. HIF-1α mRNA expression was analyzed as described in the legend to Fig. 1. The data were quantitated and the results are expressed mean ± S.E., **, p < 0.01.

FIGURE 8.

TBK1 silencing reduces cell invasion in FH-deficient RCC cells. UOK 262 cells were transiently transfected with siTBK or scramble control (scr). A, left panel, transfected cells were seeded in a Matrigel invasion chamber. After 72 h of incubation, inserts were subjected for staining. Right panel, invaded cells were quantitated by counting and the results are expressed as mean ± S.E. ***, p < 0.0001. B, total protein was analyzed for total TBK and Tubulin as loading control.

FIGURE 9.

Model of fumarate-mediated HIF-1α mRNA expression through TBK1.