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.

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

Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1α, a basic helix–loop–helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1α for hypoxia-induced growth arrest and activation of p21cip1, a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21cip1 activation has been elusive. Here we demonstrate that HIF-1α, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21cip1. The HIF-1α antagonism is mediated by displacing Myc binding from p21cip1 promoter. Neither HIF-1α transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1α. In keeping with its antagonism of Myc, HIF-1α also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF-1α pathway, which regulates a distinct group of Myc target genes in response to hypoxia.

/pdf/hif-1a-induces-cell-cycle-arrest-by-functionally-1grvhux8e1.pdf

HIF‐1α induces cell cycle arrest by functionally counteracting Myc

A classical cellular response to hypoxia is a cessation of growth. Hypoxia-induced growth arrest differs in different cell types but is likely an essential aspect of the response to wounding and injury. An important component of the hypoxic response is the activation of the hypoxia-inducible factor 1 (HIF-1) transcription factor. Although this transcription factor is essential for adaptation to low oxygen levels, the mechanisms through which it influences cell cycle arrest, including the degree to which it cooperates with the tumor suppressor protein p53, remain poorly understood. To determine broadly relevant aspects of HIF-1 function in primary cell growth arrest, we examined two different primary differentiated cell types which contained a deletable allele of the oxygen-sensitive component of HIF-1, the HIF-1alpha gene product. The two cell types were murine embryonic fibroblasts and splenic B lymphocytes; to determine how the function of HIF-1alpha influenced p53, we also created double-knockout (HIF-1alpha null, p53 null) strains and cells. In both cell types, loss of HIF-1alpha abolished hypoxia-induced growth arrest and did this in a p53-independent fashion. Surprisingly, in all cases, cells lacking both p53 and HIF-1alpha genes have completely lost the ability to alter the cell cycle in response to hypoxia. In addition, we have found that the loss of HIF-1alpha causes an increased progression into S phase during hypoxia, rather than a growth arrest. We show that hypoxia causes a HIF-1alpha-dependent increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27; we also find that hypophosphorylation of retinoblastoma protein in hypoxia is HIF-1alpha dependent. These data demonstrate that the transcription factor HIF-1 is a major regulator of cell cycle arrest in primary cells during hypoxia.

Hypoxia-Inducible Factor 1α Is Essential for Cell Cycle Arrest during Hypoxia

As formulated in 1974, the concept of the restriction point of the cell cycle was based on cell biological experiments, yet allowing accurate molecular predictions and spurring a search for the restriction factor. Although cyclin D meets the criteria of the R-factor, the picture as outlined here is more interesting and far more complex. We discuss the relationship between the restriction knot and DNA damage-checkpoints. Finally, we discuss how loss of the restriction point in cancer leads to loss of checkpoint control and to insensitivity to antimitogens including some mechanism-based anticancer therapeutics.

The Restriction Point of the Cell Cycle

Activation of the HIF pathway in cancer

Hypoxia limits tumor growth but selects for higher metastatic potential. We tested the functional activity of hypoxia-inducible factor-1 (HIF-1) in prostate cell lines ranging from normal epithelial cells (PrEC), hormone-dependent LNCaP, hormone-independent DU145, PC-3 to highly metastatic PC-3M cancer cell lines. We found that HIF-1-stimulated transcription was the lowest in PrEC and LNCaP cells and the highest in PC-3M cells. The induction by hypoxia of the HIF-1 dependent genes Cap43 and GAPDH was the highest in the most aggressive PC-3M cancer cells. Because these advanced prostate cancer cell lines have lost p53 function, this further shifts a balance from p53 to HIF-1 transcriptional regulation, and a high ratio of HIF-1-dependent:p53-dependent transcription was a marker of the advanced malignant phenotype. Transient transfection of HIF-1alpha expression vector induced transcription from p21 promoter construct in prostate cancer cell lines. Furthermore, hypoxia slightly induced p21 mRNA in these cells. However, neither expression of p21 nor hypoxia caused growth arrest in PC-3M cells. Therefore, high inducibility of HIF-1-dependent genes, loss of p53 functions with high ratio of HIF-1-dependent:p53-dependent transcription, and loss of sensitivity to p21 inhibition is a part of hypoxic phenotype associated with aggressive cancer behavior.

https://cancerres.aacrjournals.org/content/canres/60/20/5630.full.pdf

Hyperinducibility of Hypoxia-responsive Genes without p53/p21-dependent Checkpoint in Aggressive Prostate Cancer

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Hyperinducibility of Hypoxia-responsive Genes without p53/p21-dependent Checkpoint in Aggressive Prostate Cancer