Mechanism of von Hippel-Lindau protein-mediated suppression of nuclear factor kappa B activity

Abstract

Biallelic inactivating mutations of the von Hippel-Lindau tumor suppressor gene (VHL) are a hallmark of clear cell renal cell carcinoma (CCRCC), the most common histologic subtype of RCC. Biallelic VHL loss results in accumulation of hypoxia-inducible factor alpha (HIFalpha). Restoring expression of the wild-type protein encoded by VHL (pVHL) in tumors with biallelic VHL inactivation (VHL(-)(/)(-)) suppresses tumorigenesis, and pVHL-mediated degradation of HIFalpha is necessary and sufficient for VHL-mediated tumor suppression. The downstream targets of HIFalpha that promote renal carcinogenesis have not been completely elucidated. Recently, VHL loss was shown to activate nuclear factor kappa B (NF-kappaB), a family of transcription factors that promotes tumor growth. Here we show that VHL loss drives NF-kappaB activation by resulting in HIFalpha accumulation, which induces expression of transforming growth factor alpha, with consequent activation of an epidermal growth factor receptor/phosphatidylinositol-3-OH kinase/protein kinase B (AKT)/IkappaB-kinase alpha/NF-kappaB signaling cascade. We also show that components of this signaling pathway promote the growth of VHL(-)(/)(-) tumor cells. Members of this pathway represent viable drug targets in VHL(-)(/)(-) tumors, such as those associated with CCRCC.

FIG. 1.

HIFα is necessary and sufficient for pVHL-mediated modulation of NF-κB activity. (A) Immunoblots for pVHL and HIF2α (nuclear protein). 786-0-v (v) represents stable transfection of empty control vector; 786-0-VHL (VHL) represents stable transfection of wild-type VHL; 786-0-v/v (v/v) represents stable transfection of two empty control vectors; 786-0-VHL/v (VHL/v) represents stable transfection of wild-type VHL and one control vector; and 786-0-VHL/HIF2α^M (VHL/HIF2α^M) represents stable transfection of VHL and a HIF2α mutant that is resistant to pVHL-mediated ubiquitination. Western blots for actin and γ-tubulin (nuclear protein) are protein loading controls. (B) Effects of gene silencing of HIF2α on NF-κB activity. (Top panel) Immunoblots demonstrate that HIF-2α siRNA selectively silences HIF2α protein expression. (Bottom panel) HIF2α siRNA reduces NF-κB activity as measured by EMSA. Oct-1 EMSA is a negative control (cont) (C) Transient cotransfection of HIF2α siRNA and an NF-κB-driven reporter. Transient transfection experiments were performed in triplicate, and data represent the means of the results and are reported as relative luciferase units (RLU) ± standard deviation. (D) Effects of restoration of pVHL and HIF2α expression on NF-κB activity. (Top and bottom panels) EMSA for NF-κB activity (top) and Oct-1 (bottom) as a negative control. (E) Effects of HIF1α siRNA on HIF1α protein expression and NF-κB activity in UMRC6 cells. (Top and middle panels) HIF1α siRNA reduces HIF1α but not γ-tubulin expression. (Bottom panel) EMSA for NF-κB activity shows that silencing of HIF1α expression reduces constitutive NF-κB activity. At the far right of the EMSAs are cold competition experiments with molar excess of cold wild-type (WT) and mutant (M) κB and Oct-1 probes, respectively.

FIG. 2.

EGFR-mediated activation of NF-κB. (A) Effects of restoration of pVHL and HIF2α expression on EGFR activity. Whole-cell extracts were immunoprecipitated (IP) with an anti-EGFR antibody followed by immunoblotting (Western blotting [WB]) with phospho-specific and total EGFR antibodies. (B and C) Inhibition of EGFR activity by PD153035 (top panels) results in reduced NF-κB activity as measured by EMSA (bottom) and NF-κB-driven reporter gene expression (C) (mean ± standard deviation). RLU, relative luciferase units.

FIG. 3.

VHL- and HIF2α-dependent differential activation of the Ras/Raf/ERK and AKT but not JAK/STAT or JNK pathways. (A) Activation status of STAT1, -3, and -5. There was no detectable phosphorylation of STAT1 or -5 (top and bottom panels, respectively). STAT3 is constitutively but not differentially activated in all 786-0 clones (middle panels). Treatment of HeLa cells with alpha interferon (IFN-α) for 30 min was a positive control for STAT activation. (B) Immunoblotting with a phospho-specific JNK antibody. Treatment of human embryonic kidney 293 cells with tetradecanoyl phorbol acetate (TPA ester; 20 ng/ml for 30 min) served as a positive control for JNK activation. (C) Immunoblotting for phospho-ERK as a measurement of Ras/Raf/ERK pathway activation (top panels). Gene silencing of HIF2α reduces ERK phosphorylation (bottom panels). (D) Assessment of constitutive AKT activation. (Top panels) Immunoblotting (Western blotting) [WB] with phospho-specific AKT antibodies. (Bottom panels) In vitro kinase assays for AKT activation. Densitometry readings are provided above the autoradiograph; densitometric readings from 786-0-VHL/v cells were assigned a value of 1.0. IP, immunoprecipitation. (E) Suppression of HIF2α by siRNA reduces AKT activation as measured by immunoblotting with phospho-specific AKT antibodies. (F) Inhibition of EGFR activation with PD153035 reduces phosphorylation of AKT and ERK in 786-0-v and 786-0-VHL cells.

FIG. 4.

The Ras/Raf/ERK pathway does not regulate NF-κB activity in 786-0 cells. (A) A Raf1 inhibitor has no effect on an NF-κB-driven reporter (top panel) but does inhibit ERK phosphorylation in 786-0-v and 786-0-VHL cells (bottom panels). RLU, relative luciferase units. (B) Raf1 siRNA does not affect NF-κB activity. (Top panels) Western blots for Raf1 and actin (as a specificity/loading control). (Bottom panels) EMSAs for NF-κB and Oct-1. Cold competition experiments are shown in the rightmost two lanes of the EMSAs.

FIG. 5.

PI3K-dependent regulation of NF-κB activity. (A) The PI3K inhibitor wortmannin inhibits NF-κB activity. (Top panels) Wortmannin reduces phosphorylation of AKT in 786-0-v and 786-0-VHL cells as measured by immunoblotting with phospho-specific AKT antibodies. (Middle panel) EMSA demonstrating that wortmannin inhibits NF-κB. In the far right two lanes are cold competition experiments with cold wild-type (WT) and mutant (M) NF-κB probes. (Bottom panel) Wortmannin inhibits NF-κB-driven reporter gene expression. (B) Same as panel A, but with the PI3K inhibitor Ly294002. Relative luciferase units (RLU) are means of three experiments ± standard deviation.

FIG. 6.

AKT regulates NF-κB activity. (A) Effects of a chemical AKT inhibitor on NF-κB activity. The AKT chemical inhibitor blocks phosphorylation of AKT (top panels) and NF-κB activity as measured by reporter gene expression (middle panel) and EMSA (bottom panel). (B) Transient transfection of a kinase-dead AKT-DN inhibits NF-κB-driven reporter gene expression in 786-0-v and 786-0-VHL cells. RLU, relative luciferase units. (C) Selective activation of AKT by transient transfection of an AKT-DA plasmid into 786-0-VHL cells is sufficient to increase NF-κB-driven reporter gene expression. Data were normalized to those of 786-0-v cells transfected with an empty control vector. (D) AKT phosphorylates IKKα. (Top panels) In vitro kinase assay of immunoprecipitated (IP) AKT with recombinant GST-IKKα as a substrate. Densitometry readings are provided above the autoradiograph; densitometric readings from 786-0-VHL cells were assigned a value of 1.0. WB, Western blotting. (Bottom panels) Inhibition of AKT phosphorylation by the AKT inhibitor (10 μM) was confirmed in the cellular extracts used for the in vitro kinase assays. All reporter gene data represent means of three experiments ± standard deviation. For all reporter assays, total DNA was held constant with a control vector.

FIG. 7.

Effects of modulating components of the PI3K/AKT/NF-κB pathway on RCC cell viability. (A) 786-0 cells. Cells were incubated in ITS medium overnight prior to the addition of either 10% FBS or ITS ± the indicated inhibitor/activator (wortmannin [Wort], 100 nM; AKT inhibitor, 10 μM; and Raf1 inhibitor, 10 nM) for 48 h prior to cell counting by trypan blue exclusion. (Left panel) Relative cell number. (Right panel) Absolute number of dead (trypan blue positive) cells. (B) UMRC6 cells. Cells were treated and analyzed as in panel A, but UMRC6 cells were maintained in serum-replete medium (10% FBS) throughout the experiment. Relative cell number values represent means of three experiments ± standard deviation.