A major feature of solid tumours is hypoxia, decreased availability of oxygen, which increases patient treatment resistance and favours tumour progression. How hypoxic conditions are generated in tumour tissues and how cells respond to hypoxia are essential questions in understanding tumour progression and metastasis. Massive tumour-cell proliferation distances cells from the vasculature, leading to a deficiency in the local environment of blood carrying oxygen and nutrients. Such hypoxic conditions induce a molecular response, in both normal and neoplastic cells, that drives the activation of a key transcription factor; the hypoxia-inducible factor. This transcription factor regulates a large panel of genes that are exploited by tumour cells for survival, resistance to treatment and escape from a nutrient-deprived environment. Although now recognized as a major contributor to cancer progression and to treatment failure, the precise role of hypoxia signalling in cancer and in prognosis still needs to be further defined. It is hoped that a better understanding of the mechanisms implicated will lead to alternative and more efficient therapeutic approaches.
KeywordsAngiogenesis Autophagy Bcl-2/adenovirus EIB 19 kDa-interacting protein 3 Cancer Carbonic anhydrase Hypoxia Hypoxia-inducible factor Oxygen-sensor Tumour metabolism pH regulation
Cancer is presently a major cause of mortality in developed countries and will become even more so in low-income countries as the global population increases and ages and as improvements in detection are implemented . Although some cancers occur in the young, most are associated with the elderly, and both events represent the accumulation of genetic and epigenetic cell damage . Cancer includes a diverse collection of diseases, from a cellular origin point of view, rather than a single disease, the causes of which are equally as diverse . Aberrant cell-cycle checkpoint control, overactivation of oncogenes and inhibition of tumour-suppressor genes are considered to be primordial in the initiation of tumourigenesis. However, other factors related to the tumour microenvironment are now being recognized as fundamental in tumour progression, increased resistance and metastasis. Hypoxia is one of these factors, the repercussions of which are shared by all cancer types including haematological cancers .
The hypoxic tumour phenotype
Hypoxia-inducible factor, the molecular key to hypoxia
Hypoxia activates an alpha/beta heterodimeric transcription factor termed appropriately the hypoxia-inducible factor (HIF). Activation resides in the inhibition of posttranslational hydroxylation of the alpha subunit that permits stabilization, heterodimerisation and binding to hypoxia-response elements (HRE) in target genes. The details of the mechanisms of regulation of the stability and activity of HIF-α have been extensively reviewed by us [10, 11, 12] and others [13, 14, 15, 16]. Suffice it to say that posttranslational hydroxylation by oxygen-dependent oxygenases, prolyl hydroxylase domain proteins and factor inhibiting HIF (FIH) destabilize and inactivate, respectively, HIF-α. The former, by favouring von Hippel-Lindau (VHL) E3 ubiquitin ligase-mediated proteasomal degradation, and the latter, by inhibiting interaction with co-activators such as p300/CBP.
The HIF-mediated cellular response
HIF-mediated expression of gene products including the vascular endothelial growth factor-A (VEGF-A) and angiopoïetin-2 (Ang-2) allow tumour cells to turn around the hypoxic situation by inducing regrowth of the vascular network, a phenomenon termed angiogenesis . Thereby an oxygenated and nutritional environment is reestablished for maintenance of growth. However, the neo-vessels formed are often distorted and irregular and thus less efficient in oxygen, nutrient transport and drug delivery.
Cell survival or death
Thus, hypoxia initiates a cascade of events that allows tumour cells to continue to proliferate; however, if too severe, hypoxia can also lead to cell death as shown by the presence in tumours of a central necrotic zone. In fact, it can be envisaged that highly variable levels of hypoxia accompany the dynamics of spatiotemporal development of the tumour mass so that a multitude of tumour cell responses are manifested (Fig. 1). Interplay between FIH and the transcriptional activation domains of HIF-1α, based on the degree of oxygen dependence of FIH for activity, has been proposed to select for different gene profiles that determine cell fate . Gene-profile selectivity may also arise from differential action of the three HIF-α subunits and, within the context, may promote cell proliferation or death [21, 22]. The genes bnip3, Bcl-2/adenovirus EIB 19 kDa-interacting protein 3, and bnip3L (bnip3-like), the products of which are members of the BH3-only protein family of cell death factors, are highly induced in hypoxia. Although many studies have pointed at the pro-apoptotic features of these two gene products, these findings are largely controversial. We propose instead that the BH3 domains of BNIP3 and BNIP3L belong to another class, like the BH3 domain of Beclin1, that do not induce cell death but survival by triggering autophagy [12, 23, 24]. Macroautophagy is a process that allows cells to recycle intracellular organelles such as ribosomes and mitochondria for nutritional and protective purposes . Catabolism of organelle components provides nutrient-depleted cells with a source of lipids, amino acids and sugars, and autophagy of mitochondria may protect cells from harmful reactive oxygen species.
A substantial number of genes involved in cellular metabolism, in particular those of glucose, are HIF-mediated. It has been known for many years that cancer cells divert pyruvate metabolism away from mitochondrial oxidative phosphorylation (OXPHOS) toward cytoplasmic conversion of pyruvate to lactic acid . Although this latter simplified pathway produces less adenosine triphosphate (ATP) per molecule of glucose, cells compensate for a reduced yield in ATP production by increasing both the uptake of glucose and the flux in conversion of glucose to pyruvate, i.e. glycolysis. This is made possible through an increase in HIF-mediated expression of both glucose transporters and enzymes of the glycolytic pathway, giving tumours a “glycolytic” phenotype. Diversion of pyruvate toward lactate and away from OXPHOS is also promoted through increased HIF-mediated expression of two key enzymes; lactate dehydrogenase A (LDH-A)  and pyruvate dehydrogenase kinase 1 (PDK1) [27, 28]. LDH-A is the enzyme responsible for conversion of pyruvate to lactate, and PDK1 is an inhibitor of pyruvate dehydrogenase that feeds pyruvate into the tricarboxylic acid cycle and thus toward OXPHOS. Thereby, HIF not only channels glucose towards glycolysis by repressing mitochondrial respiration but it also optimizes low levels of respiration by regulating the ratio of isoforms of cytochrome c oxidase, components of the electron transport chain . This strategy not only makes respiration more efficient but may also protect cells from oxidative damage under hypoxic conditions. Metabolic regulation via HIF also brings into play products of tumour suppressors and oncogenes such as p53, c-Myc, Ras and Akt [11, 21, 30].
Another pathway related to nutrient availability, which is modified by HIF, is that of mammalian target of rapamycin (mTOR). On the one hand, growth factors and nutrients potentiate the mTOR pathway in conveying signals of growth and survival through increased protein synthesis, and on the other hand, energy depletion and hypoxia suppress mTOR, saving on energy-consuming protein synthesis, allowing for cellular adaptation and subsequent survival .
Regulation of pH
Substantial data points toward hypoxic promotion of the invasive potential of tumour cells. HIF activation is associated with loss of E-cadherin, a component of adherens junctions that acts as a suppressor of invasion and metastasis . In this context, it is interesting that TWIST1, a regulator of epithelial-mesenchymal transition, is induced in hypoxia . In addition, cells that survive acidosis not only develop a growth advantage but also become more aggressive and invasive [6, 44]. This occurs in part through the activation of HIF-up-regulated proteins implicated in matrix remodeling, such as lysyl oxydase (LOX) [12, 45], metalloproteases that disrupt cell–cell and cell–matrix (ECM) interactions . HIF also activates other genes known to be involved in metastasis and invasion such as the c-met proto-oncogene, the chemokine receptor CXCR4 and the autocrine motility factor (AMF) [41, 47].
Clinical significance of hypoxia, HIF and HIF downstream gene products in prognosis
Since HIF-α and HIF-induced proteins such as CA IX, CA XII and Glut1 are highly expressed in renal cell carcinomas (RCC) and in multiple human cancers, their expression has been investigated as markers of tumour aggressiveness and in determining prognosis . RCC is a prototype cancer for understanding the role of HIF in cancer progression since it carries loss-of-function mutations in the VHL gene, the product of which is responsible for targeting HIF-α for proteasomal degradation . Thus, in these cancers, HIF-α is stable, and downstream gene products are induced. To better appreciate the implication of HIF in tumour progression and prognosis, immunohistochemical studies have been performed in several other cancer types to detect for both HIF-α and HIF-downstream gene products such as BNIP3, CA IX and XII, Glut1 and VEGF (Figs. 3 and 4). The inherent problem related to the detection of HIF-α in tissue specimens is the short half-life of HIF-α not only in vivo but also when the specimen comes in contact with atmospheric oxygen during surgical removal. In fact, recent studies have established that this was not the case and make these studies reporting levels of HIF-α relevant . It was shown that HIF-1α and CA IX expression correlate with poor prognosis in breast cancer . The longer half-life of the other potential marker proteins may make interpretation difficult as detection may reflect only past events. These markers were shown to correlate for both primary breast tumours and lymph node metastases , and further studies have demonstrated reduced survival correlated with CA IX expression in breast cancer . In breast cancer, high BNIP3 expression was associated with good survival outcome in invasive carcinoma but with an increased risk of recurrence and shorter disease-free survival in ductal carcinoma in situ , while in non-small lung cancer, high expression was an independent factor for overall survival . Further investigation is required to obtain a better appreciation of the value of HIF or HIF-related marker immunohistochemistry for prognosis.
Harnessing phenotype in combating tumour growth
The understanding of how hypoxia drives tumour progression is attracting substantial investigation, and an impressive number of reviews have ensued; however, a lot remains to be done to clarify not only the mechanisms involved but also the implication for diagnosis and treatment. Further investigation into the relevance of HIF-induced gene products as markers of prognosis should follow. The development of anti-angiogenic agents with significant potential as a cancer therapy has led the way in demonstrating that the hypoxic response of tumours can be targeted. Additional targets involved in HIF signalling and in its consequences should also prove beneficial in slowing cancer progression and metastasis.
The laboratory is funded by grants from the Ligue Nationale Contre le Cancer (Equipe labellisée), the Centre A. Lacassagne, the Centre National de la Recherche Scientifique (CNRS), the Ministère de l’Education, de la Recherche et de la Technologie, the Institut National de la Santé et de la Recherche Médicale (Inserm), and the Institut National du Cancer (INCA). We apologize to the many research groups whose work was cited indirectly by reference to review articles.
- 20.Dayan F, Roux D, Brahimi-Horn MC, Pouyssegur J, Mazure NM (2006) The oxygen sensor factor-inhibiting hypoxia-inducible factor-1 controls expression of distinct genes through the bifunctional transcriptional character of hypoxia-inducible factor-1alpha. Cancer Res 66:3688–3698PubMedCrossRefGoogle Scholar
- 24.Mazure NM, Bellot G, Garcia-Medina R, Gounon P, Roux D, Pouyssegur J (2007) Hypoxia-induced autophagy is mediated through the HIF-dependent induction of BNIP3 and BNIP3L. Bull Cancer 94:534Google Scholar
- 36.Ivanov S, Liao SY, Ivanova A, Danilkovitch-Miagkova A, Tarasova N, Weirich G, Merrill MJ, Proescholdt MA, Oldfield EH, Lee J, Zavada J, Waheed A, Sly W, Lerman MI, Stanbridge EJ (2001) Expression of hypoxia-inducible cell-surface transmembrane carbonic anhydrases in human cancer. Am J Pathol 158:905–919PubMedGoogle Scholar
- 40.Chiche J, Laferrière J, Trottier E, Roux D, Mazure N, Brahimi-Horn MC, Pouysségur J (2007) The hypoxia-induced tumor marker carbonic anhydrase IX plays a critical role in tumor microenvironmental pH homeostasis. Keystone Symposium Conference. Molecular Targets for Cancer. Abstract # 134, Whistler, British Columbia, 18–23 MarchGoogle Scholar
- 43.Gort EH, van Haaften G, Verlaan I, Groot AJ, Plasterk RH, Shvarts A, Suijkerbuijk KP, van Laar T, van der Wall E, Raman V, van Diest PJ, Tijsterman M, Vooijs M (2007) The TWIST1 oncogene is a direct target of hypoxia-inducible factor-2alpha. Oncogene (in press).. DOI 10.1038/sj.onc.1210795
- 49.Cherk MH, Foo SS, Poon AM, Knight SR, Murone C, Papenfuss AT, Sachinidis JI, Saunder TH, O, Keefe GJ, Scott AM (2006) Lack of correlation of hypoxic cell fraction and angiogenesis with glucose metabolic rate in non-small cell lung cancer assessed by 18F-Fluoromisonidazole and 18F-FDG PET. J Nucl Med 47:1921–1926PubMedGoogle Scholar
- 50.Rajendran JG, Mankoff DA, O, Sullivan F, Peterson LM, Schwartz DL, Conrad EU, Spence AM, Muzi M, Farwell DG, Krohn KA (2004) Hypoxia and glucose metabolism in malignant tumors: evaluation by [18F]fluoromisonidazole and [18F]fluorodeoxyglucose positron emission tomography imaging. Clin Cancer Res 10:2245–2252PubMedCrossRefGoogle Scholar
- 53.Van den Eynden GG, Van der Auwera I, Van Laere SJ, Colpaert CG, Turley H, Harris AL, van Dam P, Dirix LY, Vermeulen PB, Van Marck EA (2005) Angiogenesis and hypoxia in lymph node metastases is predicted by the angiogenesis and hypoxia in the primary tumour in patients with breast cancer. Br J Cancer 93:1128–1136PubMedCrossRefGoogle Scholar
- 54.Hussain SA, Ganesan R, Reynolds G, Gross L, Stevens A, Pastorek J, Murray PG, Perunovic B, Anwar MS, Billingham L, James ND, Spooner D, Poole CJ, Rea DW, Palmer DH (2007) Hypoxia-regulated carbonic anhydrase IX expression is associated with poor survival in patients with invasive breast cancer. Br J Cancer 96:104–109PubMedCrossRefGoogle Scholar