Hypoxia-Inducible Factor in Prostate Cancer Progression
January 1, 2001 9 minutes read
Hypoxia-Inducible Factor in Prostate Cancer Progression
By Mikhail V. Blagosklonny, PhD and Konstantin Salnikow, PhD
Prostate cancer is the second leading cause of all cancer deaths in men in the United States. Between 1973 and 1992, the incidence rate in the United States increased more than 250%.1 These numbers demonstrate the need for continued exploration of the molecular mechanisms of prostate carcinogenesis in order to estimate prognosis and develop new targets for treatment and prevention.
Role of Hypoxia in Prostate Tumor Progression
Fatal outcome in prostate cancer is linked to the development of hormone independency, and to the development of prostate cancer metastasis to bone and other tissues. Tumor progression toward aggressive and metastatic potential is a fundamental process in neoplasia, but stimuli that drive this progression are poorly understood. Although hypoxia limits tumor growth, and tumors with poor vascularization fail to grow and form metastases, hypoxia eventually selects a more aggressive, metastatic cancer phenotype that is associated with poor prognosis. We proposed that hypoxia is indeed a driving force for selection of aggressive, autonomous, and metastatic cancer cells.2 Hypoxia already exists in primary prostate carcinomas, and highly metastatic human prostate cancers growing within the prostate of athymic mice overexpress vascular endothelial growth factor (VEGF).3 Although hypoxia limits tumor growth, it is inevitably associated with tumor progression. We envision the ability of prostate cancer cells to survive hypoxia as a natural test, allowing further tumor progression.
Hypoxia, Cancer, and HIF-1
Hypoxia is an important pathological condition that accompanies tumor growth. Hypoxia develops early in tumors because of inadequate vascularization of the tumor. In response to hypoxia, cells increase synthesis of enzymes that metabolize glucose. Since hypoxia often is a result of inadequate angiogenesis, cells secrete VEGF that in turn stimulates endothelial cell proliferation and forms new blood vessels. Angiogenesis (formation of new blood vessels) is absolutely required for tumor growth. Numerous genes are important for cell survival under hypoxic conditions, including glucose transporters, glycolytic enzymes, VEGF, and nitric oxide (NO). Cells adapt to hypoxia by induction of hypoxia-inducible factor 1a (HIF-1a). HIF-1 is a transcription factor that stimulates expression of VEGF, glucose transporters, glycolytic enzymes, transferrin, NO, and other hypoxia-inducible genes.4 In kidney, HIF-1 also transactivates the gene that increases whole body oxygen supply, namely erythropoietin, a growth and differentiation factor for red blood cells. The HIF-1 transcription factor activity is regulated by the stability of the HIF-1a protein, which is the limiting, hypoxia-inducible subunit of the HIF-1 transactivator. Under normal oxygen conditions, the HIF-1a protein is rapidly degraded, whereas under hypoxic conditions this degradation is prevented. HIF-1a accumulates, binds the HIF-1b subunit, and transactivates target genes.4
Cells deficient in either subunit of HIF-1 have impaired ability to form tumors.5 Lack of HIF-1 retards solid tumor growth and vascularity because of the reduced capacity to produce VEGF during hypoxia. The HIF-1-dependent production of VEGF resolves hypoxia due to new blood vessel formation, although VEGF does not provide immediate protection against hypoxia. A switch from cellular metabolism to glycolysis may provide immediate protection to cells from hypoxia, and most glycolytic enzymes are HIF-1 inducible.
Hypoxia-Mimicking Metals Induce Malignant Transformation
Additional evidence that hypoxia and HIF-1 may play a role in tumor progression came from studies on nickel-induced carcinogenesis. Nickel is a potent non-mutagenic carcinogen. In vitro nickel compounds displayed incredible transforming capability in human and rodent cell systems.6 In addition to high transforming potential, nickel compounds display tumor-promoting properties when added to polycyclic hydrocarbons.
Hypoxia and carcinogenic nickel exert almost identical effects on gene expression; furthermore, nickel induces gene expression, in part through the HIF-1 transcription factor.7 Levels of HIF-1a, HIF-1-driven transcription, and ratio of HIF-driven transcription to p53-driven transcription are increased in the nickel-transformed cells.8
HIF Is Overexpressed in Highly Aggressive Prostate Cancer
Metastatic human prostate cancer cells exhibited enhanced VEGF production and tumor vascularity compared with prostate cancer cells of lower metastatic potential. HIF-1a protein was detected in PC-3 prostate cancer cells under normoxic conditions.9,10 This prostate cancer cell line has lost dependence to testosterone and developed the ability to form metastases in nude mice. Metastasis-derived PC-3 cells were obtained and these cells display much higher metastatic potential than PC-3 cells. We observed that under hypoxic conditions, PC3-M cells produced lactate, indicating high levels of glycolysis. This may reflect high levels of HIF-1a and HIF-1-dependent transcription in PC-3-M cells. We found that levels of HIF-1 and its inducibility were higher in PC-3-M cells than in PC-3 cells.2 In a panel of prostate cell lines ranging from normal prostate epithelial cells to the most aggressive PC-3-M cells, HIF-1-dependent transcription correlated with tumor progression.2 The comparison of PC-3-M cells with normal prostate epithelial cells and LNCaP cells (lowly aggressive, testosterone-dependent, and non-metastatic prostate cancer cells) showed higher inducibility of such HIF-1-dependent genes as VEGF and Cap43, and higher levels of hypoxia-dependent transcription. Our data suggest that an increased inducibility of HIF-1-dependent genes may be a hallmark of hypoxia-driven selection.
NDRG-1/Cap43 in Prostate Cancer
NDRG-1/Cap43 is a new human gene recently cloned in a few laboratories, including ours, based on its high inducibility by nickel compounds.11 The gene had different names, including RTP, DRG, and NDR; the latest nomenclature uses the name NDRG-1. The gene is highly induced by hypoxia and may be involved in cellular survival under hypoxic conditions. NDRG-1 was mapped on human chromosome 8q24. Although, the direct involvement of this gene in prostate cancer development was not shown, it is interesting to note that numerous studies reported 8q gain in advanced prostatic cancers. This gene is expressed at low levels in different tissues; however, because it is regulated by androgens, this gene is expressed at relatively high levels in normal prostate tissue.12 In LNCaP cells that have androgen receptors, NDRG-1 is up-regulated markedly by testosterone and to a much lesser extent by hypoxia.2,12 Other prostate cancer cells (e.g., PC-3, PC-3M, and DU145) have lost their androgen receptors and consequently are no longer regulated by testosterone. In these cells, hypoxic up-regulation of Cap43 was much stronger than in LNCaP or normal prostate epithelial cells.2
Is HIF-1 an Oncoprotein?
A tumor suppressor is a gene whose loss or inactivation increases cancer incidence. In von Hippel-Linday (VHL) syndrome, a familial cancer caused by germline mutations of the VHL tumor suppressor gene, inactivation of the pVHL protein was found in highly vascular tumors that overproduce angiogenic factors such as VEGF.13 VHL-associated tumor cells express high levels of mRNA for all hypoxia-inducible genes in both normoxic and hypoxic conditions.13
VHL functions have recently been elucidated: VHL targets HIF-1a for degradation under normal oxygen conditions.14 In the absence of VHL, HIF-1a is not degraded and, therefore, it is overexpressed in a cell. Such cells live under normal oxygen conditions as they do in deep hypoxia. This explains why all HIF-1-dependent genes are up-regulated when VHL is mutated or lost. Since the loss of the VHL tumor suppressor results in HIF-1 overexpression and that leads to cancerous phenotype, HIF-1 may be defined as an oncoprotein.
HIF, p53, and p21
Hypoxia represents severe stress that inhibits cell proliferation and induces apoptosis.15 For example, rodent fibroblasts cease proliferation and eventually die following hypoxia.16 We have shown that human metastatic prostate cancer cells neither arrest growth nor die under hypoxic conditions.2 Such high tolerance of hypoxia in advanced prostate cancers is characterized by high basal and hypoxia-induced levels of HIF-1-dependent transcription, loss of p53 function, and inability of HIF-1 and p21WAF1/CIP1 to induce growth arrest. Hypoxia induces the p53 tumor suppressor, which in turn leads to growth arrest or cell death.15,17 Growth arrest is largely mediated by the induction of several proteins, including p21, an inhibitor of cyclin-dependent kinases.
p53 mutations in primary prostate cancer are relatively infrequent. In contrast, they occur at high rates in metastatic disease, suggesting that prostate cancer progression involves p53 inactivation.18 Hypoxia may select for the loss of p53, thus facilitating selection of a more malignant phenotype. Therefore, highly malignant cells can tolerate hypoxia and are characterized by a loss of p53 function and an increase in HIF-1 function. Literally, HIF-1 substitutes for p53 as a stress regulator in highly metastatic cells. Tumor-associated nitric oxide production that is under the control of HIF-1 may promote cancer progression by providing a selective growth advantage to tumor cells with mutant p53.
In contrast to apoptosis, hypoxia-induced growth arrest is p53-independent. We showed that under hypoxic conditions, growth arrest may be mediated directly by HIF-1, which induces p21. It is noteworthy that HIF-1-null fibroblasts grow faster than normal fibroblasts, indicating that HIF-1 may inhibit proliferation. Additionally, it has been shown that hypoxia failed to induce p21 in cells lacking HIF-1, but induced p21 in normal cells,19making it plausible to link HIF-1 with p21 induction in normal cells. It has been proposed that p21 is regulated by an alternative signaling system in prostate tumors with p53 inactivation.20 We demonstrated that HIF-1 transactivates the p21 promoter in cells that lack wild type p53. Furthermore, the p21 promoter contains the ACGTG sequence, which has been implicated in the regulation of lactate dehydrogenase A by hypoxia. Hypoxia slightly up-regulated the p21 mRNA in DU145 and PC-3-M prostate cancer cells. However, neither HIF-1a nor hypoxia induced growth arrest in these prostate cancer cells. Thus, induction of p21 is dissociated from growth arrest in the advanced prostate cancer cells. This situation results in uncontrolled growth without apoptosis that is associated with the most malignant phenotype.21 In summary, increased HIF-1a expression, loss or mutation of p53, and resistance to p21-dependent growth arrest contribute to the aggressive metastatic phenotype in prostate cancer. (Dr. Blagosklonny is an Investigator/Contractor, Medicine Branch, Division of Clinical Sciences, National Cancer Institute, National Institute of Health, Bethesda, MD; Dr. Salnikow is a Research Assistant Professor of Environmental Medicine, Nelson Institute of Environmental Medicine, and Kaplan Comprehensive Cancer Center, New York University School of Medicine, New York, NY.)
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