BRCA1 tumours correlate with a HIF-1α phenotype and have a poor prognosis through modulation of hydroxylase enzyme profile expression

BACKGROUND: There are limited data regarding the hypoxia pathway in familial breast cancers. We therefore performed a study of hypoxic factors in BRCA1, BRCA2 and BRCAX breast cancers. METHODS: Immunoperoxidase staining for HIF-1alpha, PHD1, PHD2, PHD3, VEGF and FIH was carried out in 125 (38 BRCA1, 33 BRCA2 and 54 BRCAX) breast carcinomas. These were correlated with clinicopathological parameters and the intrinsic breast cancer phenotypes. RESULTS: BRCA1 tumours correlated with positivity for HIF-1alpha (P=0.008) and negativity for PHD3 (P=0.037). HIF-1alpha positivity (P=0.001), PHD3 negativity (P=0.037) and nuclear FIH negativity (P=0.011) was associated with basal phenotype. HIF-1alpha expression correlated with high tumour grade (P=0.009), negative oestrogen receptor (ER) status (P=0.001) and the absence of lymph node metastasis (P=0.028). Nuclear FIH expression and PHD3 correlated with positive ER expression (P=0.024 and P=0.035, respectively). BRCA1 cancers with positive HIF-1alpha or cytoplasmic FIH had a significantly shorter relapse-free survival (P=0.007 and P=0.049, respectively). CONCLUSIONS: The aggressive nature of BRCA1 and basal-type tumours may be partly explained by an enhanced hypoxic drive and hypoxia driven ER degradation because of suppressed PHD and aberrantly located FIH expression. This may have important implications, as these tumours may respond to compounds directed against HIF-1alpha or its downstream targets

Cytoplasmic location of factor-inhibiting hypoxia-inducible factor is associated with an enhanced hypoxic response and a shorter survival in invasive breast cancer

INTRODUCTION: Hypoxia-inducible factor (HIF)-1alpha levels in invasive breast carcinoma have been shown to be an adverse prognostic indicator. Cellular HIF-1alpha activity is regulated by factor-inhibiting hypoxia-inducible factor 1 (FIH-1). In hypoxia, FIH-1 hydroxylation of Asn803 within the C-terminal transactivation domain does not occur and HIF-1alpha forms a fully active transcriptional complex. The present study investigates the role of FIH-1 in invasive breast carcinoma and its correlation with hypoxia. METHODS: Microarrayed tissue cores from 295 invasive carcinomas were stained for FIH-1, for HIF-1alpha and for carbonic anhydrase 9. FIH-1 expression was correlated with standard clinicopathological parameters and with the expression of the surrogate hypoxic markers HIF-1alpha and carbonic anhydrase 9. RESULTS: FIH-1 was positive in 239/295 (81%) tumours, 42/295 (14%) exclusively in the nucleus and 54/295 (18%) exclusively in the cytoplasm. Exclusive nuclear FIH-1 expression was significantly inversely associated with tumour grade (P = 0.02) and risk of recurrence (P = 0.04), whereas exclusive cytoplasmic FIH-1 was significantly positively associated with tumour grade (P = 0.004) and carbonic anhydrase 9 expression (P = 0.02). Patients with tumours that excluded FIH-1 from the nucleus had a significantly shorter survival compared with those with exclusive nuclear expression (P = 0.02). Cytoplasmic FIH-1 expression was also an independent poor prognostic factor for disease-free survival. CONCLUSION: FIH-1 is widely expressed in invasive breast carcinoma. As with other HIF regulators, its association between cellular compartmentalization and the hypoxic response and survival suggests that tumour regulation of FIH-1 is an additional important mechanism for HIF pathway activation

A Dialogue between the Hypoxia-Inducible Factor and the Tumor Microenvironment

The hypoxia-inducible factor is the key protein responsible for the cellular adaptation to low oxygen tension. This transcription factor becomes activated as a result of a drop in the partial pressure of oxygen, to hypoxic levels below 5% oxygen, and targets a panel of genes involved in maintenance of oxygen homeostasis. Hypoxia is a common characteristic of the microenvironment of solid tumors and, through activation of the hypoxia-inducible factor, is at the center of the growth dynamics of tumor cells. Not only does the microenvironment impact on the hypoxia-inducible factor but this factor impacts on microenvironmental features, such as pH, nutrient availability, metabolism and the extracellular matrix. In this review we discuss the influence the tumor environment has on the hypoxia-inducible factor and outline the role of this factor as a modulator of the microenvironment and as a powerful actor in tumor remodeling. From a fundamental research point of view the hypoxia-inducible factor is at the center of a signaling pathway that must be deciphered to fully understand the dynamics of the tumor microenvironment. From a translational and pharmacological research point of view the hypoxia-inducible factor and its induced downstream gene products may provide information on patient prognosis and offer promising targets that open perspectives for novel “anti-microenvironment” directed therapies

Factor inhibiting HIF (FIH-1) promotes renal cancer cell survival by protecting cells from HIF-1α-mediated apoptosis.

BACKGROUND: Clear cell renal cell carcinoma (CCRCC) is the commonest form of kidney cancer. Up to 91% have biallelic inactivation of VHL, resulting in stabilisation of HIF-α subunits. Factor inhibiting HIF-1 is an enzyme that hydroxylates HIF-α subunits and prevents recruitment of the co-activator CBP/P300. An important question is whether FIH-1 controls HIF activity in CCRCC. METHODS: Human VHL defective CCRCC lines RCC10, RCC4 and 786-O were used to determine the role of FIH-1 in modulating HIF activity, using small interfering RNA knockdown, retroviral gene expression, quantitative RT-PCR, western blot analysis, Annexin V and propidium iodide labelling. RESULTS: Although it was previously suggested that FIH-1 is suppressed in CCRCC, we found that FIH-1 mRNA and protein are actually present at similar levels in CCRCC and normal kidney. The FIH-1 inhibition or knockdown in the VHL defective CCRCC lines RCC10 and RCC4 (which express both HIF-1α and HIF-2α) resulted in increased expression of HIF target genes. In the 786-O CCRCC cell line, which expresses only HIF-2α, FIH-1 attenuation showed no significant effect on expression of these genes; introduction of HIF-1α resulted in sensitivity of HIF targets to FIH-1 knockdown. In RCC4 and RCC10, knockdown of FIH-1 increased apoptosis. Suppressing HIF-1α expression in RCC10 prevented FIH-1 knockdown from increasing apoptosis. CONCLUSION: Our results support a unifying model in which HIF-1α has a tumour suppressor action in CCRCC, held in check by FIH-1. Inhibiting FIH-1 in CCRCC could be used to bias the HIF response towards HIF-1α and decrease tumour cell viability

Regulation of HIF: prolyl hydroxylases.

Hypoxia inducible factor (HIF) is an alpha/beta heterodimeric transcriptional complex that plays a key role in directing cellular responses to hypoxia. Recent studies have defined novel oxygen-sensitive signal pathways that regulate the activity of HIF by post-translational hydroxylation at specific residues within the alpha subunits. HIF prolyl hydroxylation regulates proteolytic degradation of HIF whereas HIF asparaginyl hydroxylation modulates interaction with transcriptional co-activators. These hydroxylations are catalysed by a set of non-haem Fe(II)- and 2-oxoglutarate (2-OG)-dependent dioxygenases. During catalysis, the splitting of molecular oxygen is coupled to the hydroxylation of HIF and the oxidative decarboxylation of 2-OG to give succinate and CO2. Hydroxylation at two prolyl residues within the central 'degradation domain' of HIF-alpha increases the affinity for the von Hippel-Lindau (pVHL) E3 ligase complex by at least three orders of magnitude, thus directing HIF-alpha polypeptides for proteolytic destruction by the ubiquitin/proteasome pathway. Since the HIF hydroxylases have an absolute requirement for molecular oxygen this process is suppressed in hypoxia allowing the HIF-alpha to escape destruction and activate transcription. Co-substrate and co-factor requirements for Fe(II), ascorbate, and the Krebs cycle intermediate 2-OG, and inducible changes in the cellular abundance of three closely related HIF prolyl hydroxylases (PHD1-3) provide additional interfaces with cellular oxygen status that may be important in regulating the oxygen-sensitive signal

Genetic analysis of the role of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (FIH) in regulating hypoxia-inducible factor (HIF) transcriptional target genes [corrected].

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that directs a broad range of cellular responses to hypoxia. Recent studies have defined a set of 2-oxoglutarate and Fe(II)-dependent dioxygenases that modify HIF-alpha subunits by prolyl and asparaginyl hydroxylation. These processes potentially provide a dual system of control, down-regulating both HIF-alpha stability and transcriptional activity. Although genetic analyses in both primitive organisms and mammalian cells have demonstrated a critical role for the prolyl hydroxylase pathway in the regulation of HIF, analogous studies have not been performed on the HIF asparaginyl hydroxylase pathway, and its role in directing the expression of endogenous HIF transcriptional targets has not yet been clearly defined. Here we demonstrate, using small interfering RNA-mediated FIH suppression and controlled overexpression by a doxycycline-inducible system, that alterations in FIH expression in both directions have reciprocal effects on the expression of a range of HIF target genes. These effects were observed in normoxic and severely hypoxic cells but not anoxic cells. Evidence for FIH activity in severely hypoxic cells contrasted with results for the prolyl hydroxylase PHD2, suggesting that these enzymes display different oxygen dependence in vivo, with PHD2 requiring higher levels of oxygen for biological activity. Our results demonstrate an important physiological role for FIH in regulating HIF-dependent target genes over a wide range of oxygen tensions and indicate that inhibition of FIH has the potential to augment HIF target gene expression even in severe hypoxia

Interaction of hydroxylated collagen IV with the von hippel-lindau tumor suppressor.

The von Hippel-Lindau tumor suppressor (pVHL) targets hydroxylated alpha-subunits of hypoxia-inducible factor (HIF) for ubiquitin-mediated proteasomal destruction through direct interaction with the hydroxyproline binding pocket in its beta-domain. Although disruption of this process may contribute to VHL-associated tumor predisposition by up-regulation of HIF target genes, genetic and biochemical analyses support the existence of additional functions, including a role in the assembly of extracellular matrix. In an attempt to delineate these pathways, we searched for novel pVHL-binding proteins. Here we report a direct, hydroxylation-dependent interaction with alpha-chains of collagen IV. Interaction with pVHL was also observed with fibrillar collagen chains, but not the folded collagen triple helix. The interaction was suppressed by a wide range of tumor-associated mutations, including those that do not disturb the regulation of HIF, supporting a role in HIF-independent tumor suppressor functions