HER2 (neu) signaling increases the rate of hypoxia-inducible factor 1alpha (HIF-1alpha) synthesis: novel mechanism for HIF-1-mediated vascular endothelial growth factor expression

Abstract

Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator composed of HIF-1alpha and HIF-1beta subunits. Several dozen HIF-1 targets are known, including the gene encoding vascular endothelial growth factor (VEGF). Under hypoxic conditions, HIF-1alpha expression increases as a result of decreased ubiquitination and degradation. The tumor suppressors VHL (von Hippel-Lindau protein) and p53 target HIF-1alpha for ubiquitination such that their inactivation in tumor cells increases the half-life of HIF-1alpha. Increased phosphatidylinositol 3-kinase (PI3K) and AKT or decreased PTEN activity in prostate cancer cells also increases HIF-1alpha expression by an undefined mechanism. In breast cancer, increased activity of the HER2 (also known as neu) receptor tyrosine kinase is associated with increased tumor grade, chemotherapy resistance, and decreased patient survival. HER2 has also been implicated as an inducer of VEGF expression. Here we demonstrate that HER2 signaling induced by overexpression in mouse 3T3 cells or heregulin stimulation of human MCF-7 breast cancer cells results in increased HIF-1alpha protein and VEGF mRNA expression that is dependent upon activity of PI3K, AKT (also known as protein kinase B), and the downstream kinase FRAP (FKBP-rapamycin-associated protein). In contrast to other inducers of HIF-1 expression, heregulin stimulation does not affect the half-life of HIF-1alpha but instead stimulates HIF-1alpha synthesis in a rapamycin-dependent manner. The 5'-untranslated region of HIF-1alpha mRNA directs heregulin-inducible expression of a heterologous protein. These data provide a molecular basis for VEGF induction and tumor angiogenesis by heregulin-HER2 signaling and establish a novel mechanism for the regulation of HIF-1alpha expression.

FIG. 1

Effect of HER2 overexpression on HIF-1 activity and VEGF mRNA levels. (A) Analysis of HIF-1 expression and activity. Mouse NIH 3T3 and 3T3/neu (NEU) cells were incubated in complete media under hypoxic (1% O₂) or nonhypoxic (20% O₂) conditions for 6 h prior to nuclear extract preparation. Aliquots were subjected to immunoblot assay using anti-HIF-1α (top) or anti-HIF-1β (middle) antibodies. An electrophoretic mobility shift assay (bottom) was also performed which detected HIF-1 and constitutively expressed (C) DNA-binding activities. (B) Analysis of VEGF and HIF-1α RNA expression. Cells were incubated under hypoxic or nonhypoxic conditions for 16 h prior to total RNA preparation. Aliquots were fractionated by agarose gel electrophoresis and subjected to serial blot hybridization using rat VEGF (top) and human HIF-1α (middle) cDNA probes. The agarose gel was stained with ethidium bromide prior to transfer to demonstrate equivalent quantity and quality of RNA (28S and 18S rRNA species are indicated) in each lane (bottom). (C) Analysis of HIF-1 transcriptional activity. 3T3 and NEU cells were cotransfected with control reporter pSV-Renilla, containing Renilla luciferase coding sequences under the control of an SV40 promoter, and p2.1, which contains a 68-bp HRE from the human ENO1 gene inserted upstream of a minimal SV40 promoter and firefly luciferase coding sequences, or pVEGF, which contains a 2.7-kb human VEGF promoter fragment inserted upstream of firefly luciferase coding sequences. Luciferase activities were measured 48 h after transfection. For each condition, the ratio of firefly to Renilla luciferase was determined and normalized to the value obtained for 3T3 cells transfected with p2.1 (Relative Expression).

FIG. 2

Effect of heregulin stimulation on HIF-1α expression. Human MCF-7 breast cancer cells were serum starved for 20 h and then exposed to no treatment, 10% FBS (serum), or 25 to 100 ng of heregulin/ml for 6 h, prior to nuclear extract preparation and immunoblot assay using anti-HIF-1α (top) or anti-HIF-1β (bottom) antibodies.

FIG. 3

Effect of heregulin stimulation on HIF-1 transcriptional activity. Transfected cells were serum starved and exposed to no growth factor (−), 10% FBS, heregulin (HRG) or FBS and HRG for 24 h, under either nonhypoxic (20% O₂) (top panels) or hypoxic (1% O₂) (bottom panels) culture conditions, prior to preparation of cell lysates for dual luciferase assays. (A) Analysis of HIF-1-mediated reporter gene transcription. MCF-7 cells were cotransfected with pSV-Renilla and p2.1 or p2.4, which contain a wild-type and mutated HRE, respectively. The ratio of firefly to Renilla luciferase expression was determined and normalized to the value obtained from nonhypoxic cells transfected with p2.1 (Relative Expression). (B) Analysis of HIF-1α transactivation domain function. MCF-7 cells were transfected with: pSV-Renilla; pG5E1bLuc, which contains five copies of a GAL4 DNA-binding site upstream of the Elb promoter and firefly luciferase coding sequences; and either pGal0 or pGalA, which encodes the GAL4 DNA-binding domain either alone or fused to HIF-1α amino acids 531 to 826, respectively. The ratio of firefly to Renilla luciferase expression was determined and normalized to the value obtained from nonhypoxic cells transfected with pGal0 (Relative Expression).

FIG. 4

Involvement of PI3K and AKT in signaling from HER2 to HIF-1α. (A) Analysis of AKT activity. 3T3 and 3T3/NEU (NEU) cells were incubated under nonhypoxic or hypoxic conditions, and cell lysates were prepared. Aliquots were subjected to immunoblot assay with antibodies that recognize only phospho-AKT (top) or total AKT (bottom). (B) Effect of kinase inhibitors on HIF-1α expression. 3T3 and NEU cells were pretreated with no drug, 100 μM AG825, 100 μM LY294002, 100 μM PD098059, or 100 nM rapamycin for 30 min and incubated under nonhypoxic or hypoxic conditions for 6 h prior to HIF-1α immunoblot assay. (C) Effect of dominant-negative AKT on HER2-induced HIF-1 transcriptional activity. 3T3 cells were cotransfected with pSV-Renilla; wild-type p2.1 or mutant p2.4 reporter gene; and either empty vector (E) or expression vector encoding HER2 (N) in the presence or absence of expression vector encoding kinase-dead AKT (AKT-KD). Cell lysates were subjected to dual luciferase assays.

FIG. 4

Involvement of PI3K and AKT in signaling from HER2 to HIF-1α. (A) Analysis of AKT activity. 3T3 and 3T3/NEU (NEU) cells were incubated under nonhypoxic or hypoxic conditions, and cell lysates were prepared. Aliquots were subjected to immunoblot assay with antibodies that recognize only phospho-AKT (top) or total AKT (bottom). (B) Effect of kinase inhibitors on HIF-1α expression. 3T3 and NEU cells were pretreated with no drug, 100 μM AG825, 100 μM LY294002, 100 μM PD098059, or 100 nM rapamycin for 30 min and incubated under nonhypoxic or hypoxic conditions for 6 h prior to HIF-1α immunoblot assay. (C) Effect of dominant-negative AKT on HER2-induced HIF-1 transcriptional activity. 3T3 cells were cotransfected with pSV-Renilla; wild-type p2.1 or mutant p2.4 reporter gene; and either empty vector (E) or expression vector encoding HER2 (N) in the presence or absence of expression vector encoding kinase-dead AKT (AKT-KD). Cell lysates were subjected to dual luciferase assays.

FIG. 5

Involvement of the PI3K-AKT-FRAP pathway in heregulin-induced HIF-1α expression. (A) Stimulation of AKT activity and HIF-1α expression by heregulin. Serum-starved MCF-7 cells were exposed to vehicle or heregulin for 6 h and analyzed for phospho-AKT (top), total AKT (middle), or HIF-1α protein (bottom) by immunoblot assay. (B) Effect of serum and heregulin stimulation on S6 kinase activity. Serum-starved cells were treated with serum or heregulin for 6 h prior to immunoblot assay with antibodies specific for phosphorylated p70^s6k and its p85 isoform (top) or total p70^s6k protein (bottom). (C) Effect of kinase inhibitors on heregulin-stimulated HIF-1α expression. Serum-starved cells were pretreated for 30 min with vehicle or inhibitor (AG825, PD98059, rapamycin, or LY294002) and then exposed to no treatment, 10% FBS (Serum), or 100 ng of heregulin/ml for 6 h prior to HIF-1α immunoblot assay of whole-cell lysates. (D) Analysis of HIF-1α mRNA expression. MCF-7 cells were serum starved and exposed to heregulin in the absence or presence of rapamycin for 6 h. Total RNA was isolated, and blot hybridization was performed with a HIF-1α cDNA probe (top). A photograph of the ethidium bromide-stained gel demonstrates equal loading of RNA as determined by the intensity of the 18S and 28S rRNA bands (bottom).

FIG. 6

Effect of hypoxia, cobalt, and heregulin stimulation on HIF-1α stability. HIF-1α expression was induced by exposure of MCF-7 cells to 1% O₂ (Hypoxia), 100 μM CoCl₂ (Cobalt), or 100 ng of heregulin/ml for 6 h. Cycloheximide (CHX) was added to a final concentration of 100 μM, and the cells were harvested after being incubated for the indicated time in the presence of CHX and the inducer. Nuclear extracts were analyzed for the expression of HIF-1α (top) and HIF-1β (bottom) by immunoblot assay.

FIG. 7

Metabolic labeling experiments. (A) Pulse-labeling of MCF-7 cells. Serum-starved cells were pretreated with no drug, heregulin, or heregulin plus rapamycin for 30 min in Met-free medium. [³⁵S]Met-Cys was added, and the cells were incubated for 20 or 40 min prior to preparation of cell lysates and immunoprecipitation of HIF-1α. (B) Pulse-chase. Serum-starved MCF-7 cells were pretreated with 100 μM cobalt chloride (C) or 100 ng of heregulin/ml (H) for 30 min in Met-free medium. [³⁵S]Met-Cys was added, and then the cells were incubated for 40 min and either harvested directly for analysis of pulse-labeling (P) or rinsed and incubated in medium containing unlabeled Met-Cys for 25 min in the presence or absence of heregulin for pulse-chase analysis (P/C) of immunoprecipitated HIF-1α.

FIG. 8

Effect of HIF1A 5′-UTR on luciferase expression. (A) MCF-7 cells were cotransfected with pSV-Renilla and firefly luciferase (LUC) reporter genes containing 572 bp of 5′-FS from the human HIF1A gene, followed by either 284 or 32 bp of the 5′-UTR. (B) Transfected cells were exposed to heregulin or vehicle for 24 h, and the ratio of firefly luciferase to Renilla (Relative Luciferase) activity was determined. The mean and standard deviation for each condition are shown based upon three independent transfections. (C) Expression of Renilla (Ren) and firefly luciferase (Luc) mRNA in transfected cells was determined by RT-PCR. The ratio of Renilla to firefly luciferase expression was determined by densitometry for the 20- and 24-cycle PCR but was not determined (N/D) for the 16-cycle PCR due to the low levels of product.

FIG. 9

Dual mechanisms for induction of HIF-1α protein and VEGF mRNA expression. Signal transduction from HER2 (and possibly other tyrosine kinases such as EGFR and V-SRC) to PI3K, AKT, and FRAP increases the rate of HIF-1α synthesis, whereas hypoxia and loss of VHL or p53 activity decrease the rate of HIF-1α degradation by reducing its ubiquitination (VHL and p53-recruited MDM2 are ubiquitin-protein ligases).