HER2 (neu) Signaling Increases the Rate of Hypoxia-Inducible Factor 1α (HIF-1α) Synthesis: Novel Mechanism for HIF-1-Mediated Vascular Endothelial Growth Factor Expression

Angiogenesis is essential for tumorigenesis as well as metastasis (11, 16, 64), and vascular density is an important prognostic factor in breast cancer (19, 27, 58, 59). Vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis (10), and its expression in breast cancer is inversely correlated with patient survival (29, 30). VEGF expression can be induced by exposure of tumor cells to hypoxia or growth factors and, in both cases, this expression is due in part to increased VEGF gene transcription that is mediated by hypoxia-inducible factor 1 (HIF-1) (6, 9, 12, 22, 44, 63, 65). HIF-1 is a heterodimer composed of HIF-1α and HIF-1β subunits (56, 57). Whereas HIF-1β is constitutively expressed, the expression and activity of the HIF-1α subunit are induced by exposure of cells to hypoxia or growth factors (reviewed in reference 49). HIF-1 activates the transcription of genes whose products are required for critical aspects of tumor progression including angiogenesis (plasminogen activator inhibitor 1 and VEGF), iron homeostasis (transferrin and transferrin receptor), and metabolic adaptation (glucose transporters and glycolytic enzymes), as well as several factors that affect tumor cell survival or proliferation (endothelin 1, inducible nitric oxide synthase, and insulin-like growth factor 2). HIF-1α is overexpressed in primary and metastatic human tumors (1, 4, 5, 53, 62, 66). In breast cancer, HIF-1α overexpression can be detected in ductal carcinoma in situ but not in benign ductal hyperplasia (5), i.e., in early-stage cancer prior to invasion but concomitant with increased angiogenesis (15). HIF-1 activity is increased both by intratumoral hypoxia and by genetic alterations, including loss-of-function mutations in the tumor suppressor genes encoding p53, PTEN, and VHL (von Hippel-Lindau protein) as well as gain-of-function mutations in oncogenes that activate the phosphatidylinositol 3-kinase (PI3K), SRC, and mitogen-activated protein (MAP) kinase signal-transduction pathways (24, 34, 40, 41, 47, 48, 65, 66, 68). Loss or gain of HIF-1 activity is negatively and positively correlated, respectively, with tumor growth and angiogenesis in xenograft assays (6, 24, 28, 33, 40, 44, 45). Among the genetic alterations identified in human breast cancer, one of the most important is the increased activity of the HER2 receptor tyrosine kinase encoded by the ERBB2 gene on chromosome 17q21, which occurs in approximately one-third of breast tumors and is associated with increased tumor grade, chemotherapy resistance, and decreased rates of patient survival (36, 43, 50, 51). Overexpression of HER2 transforms human mammary epithelial and mouse 3T3 cells and imparts resistance against the chemotherapeutic agents tamoxifen and Taxol (32, 39, 61). Treatment of breast cancer cells with a neutralizing antibody against HER2 results in a dose-dependent inhibition of VEGF expression (38). A humanized monoclonal antibody to HER2 inhibits breast cancer growth and has been approved for treatment of HER2-overexpressing tumors (35). Unlike other members of the epidermal growth factor receptor (EGFR) family, HER2 has tyrosine kinase activity in the absence of any known ligand. HER2 heterodimerizes with the EGFR family members HER3 and HER4, which bind the ligand heregulin (55). In breast cancer cells, heregulin activates AKT (also known as protein kinase B) via the PI3K pathway (31). HER2 overexpression is also associated with increased AKT activity (67). Recently, HER2 overexpression or heregulin stimulation has been shown to induce VEGF mRNA and protein expression in cancer cell lines (3, 60). Because HIF-1 has been shown to lie downstream of EGFR and PI3K-AKT signaling and upstream of VEGF expression in tumor cells (9, 65, 68), we have analyzed HIF-1 activity in HER2-overexpressing 3T3 cells and heregulin-stimulated MCF-7 cells. Our results indicate that HIF-1 contributes to the induction of VEGF expression in these cells. Because hypoxia (52) and mutations in the tumor suppressor genes VHL (7, 54) and p53 (40) induce HIF-1 activity by inhibiting the ubiquitination and proteasomal degradation of HIF-1α (20, 26, 46), it was assumed that receptor tyrosine kinase signaling induced HIF-1α expression by the same mechanism. However, our results demonstrate that HER2 signaling induces HIF-1α protein synthesis rather than inhibiting its degradation, thus representing a novel mechanism for the regulation of HIF-1 and VEGF expression.