The prolyl hydroxylase enzymes are positively associated with hypoxia-inducible factor-1α and vascular endothelial growth factor in human breast cancer and alter in response to primary systemic treatment with epirubicin and tamoxifen

The hypoxic response through hypoxia inducible factor (HIF) is recognised as one of the most important microenvironmental influences on tumour behaviour that enables tumours to acquire an aggressive phenotype and become resistant to both chemotherapy and radiotherapy [1, 2]. Although HIF-1α is transcribed continuously, in normoxia its levels are kept low by a rapid degradation process through the ubiquitin-proteasome system [3]. This process is achieved through hydroxylation of two prolyl residues at Pro-402 and Pro-564 in the oxygen-dependent HIF-1α [4], and leads to recognition by the von Hippel-Lindau tumour suppressor protein and targeting for degradation [5]. Three isoenzymes - prolyl hydroxylase (PHD)1, PHD2 and PHD3 - are responsible for the modification of HIF-1α, the activity of which is dependent on the presence of oxygen as a co-substrate together with iron, ascorbate and 2-oxoglutarate as essential co-factors [6]. Therefore as the abundance of molecular oxygen for hydroxylation decreases, which occurs in the hypoxic microenvironment of a tumour with its disordered and leaky vasculature, a reduction in PHD enzymatic activity occurs, allowing HIF-1α accumulation. HIF-1α is then able to translocate to the nucleus, where it dimerises with its constitutively expressed partner HIF-1β (also known as aryl nuclear hydrocarbon translocator) and then binds to the hypoxic response element of genes that enhance tumour cell survival such as glycolysis (Glut1), angiogenesis (for example, vascular endothelial growth factor (VEGF)), iron metabolism (transferrin), pH control (carbonic anhydrase (CAIX)) and haemoglobin synthesis (erythropoietin) (reviewed in [1]).

HIF-1α and its downstream genes such as VEGF and CAIX are associated with advanced tumour stage, metastases and a shorter survival in breast cancer [7‐9]. The HIF pathway also upregulates several genes that lead to a proapoptotic phenotype, showing the complicated effects of HIF in tumours. This paradox is observed in breast cancer, where there appears to be a pivotal switch in the HIF-induced gene for BNIP3 with progression from in situ to invasive carcinoma [10]. This apparent dual action of HIF is also emphasised in tumour models that have demonstrated HIF is able to enhance both tumour growth and angiogenesis, and also in data to show that HIF has tumour-suppressive activity [11‐13]. Since the PHDs directly regulate and are regulated by HIF-1α, and have been shown to control a number of other significant intracellular factors including the adrenergic receptor and NF-κB [14], the above apparent opposing actions potentially being due to the action of the PHDs.

Although many of the downstream HIF genes have been studied extensively in breast tumours, however, there are limited data for the PHDs [15]. We therefore decided to assess the role of the PHDs in breast cancer patients treated by neoadjuvant chemotherapy in the setting of a phase II randomised trial. This assessment provides the optimal opportunity for investigating the effect of the PHDs, since HIF has been shown to mediate resistance to chemotherapy and radiotherapy and biopsies can be compared both pre treatment and post treatment. Our aims were to investigate the expression of the regulatory hydroxylases, PHD1, PHD2 and PHD3 in tumours from patients at baseline and post treatment, to correlate the expression of these factors with clinicopathological parameters, to explore the potential of these factors as biomarkers for predicting tumour response, and to explore the association between changes in their expression and survival.

HIF signalling is critically important for cell survival in low-oxygen environments, as occurs in tumours [24]. HIF is rapidly degraded after hydroxylation by the PHDs in the presence of molecular oxygen [25]. We hypothesised that tumours with high baseline levels of the PHDs and/or that are able to induce PHD after chemotherapy could modulate HIF-1α levels and thereby alter HIF signalling. The resultant effect could change the biological behaviour of the tumour and may be useful as a predictive marker. We also hypothesised that certain chemotherapeutic agents may be able to alter PHDs expression in tumours either directly through their effect on tumour cells or indirectly through changes in vascularity and oxygen delivery, and thereby also modulate HIF activity.

We observed frequent expression of the PHDs in invasive breast carcinoma that ranged from ~25 to 50% of cancer cells, suggesting that the PHDs are important in human breast cancer. Expression of PHD2 and PHD3 was stronger than PHD1, which is in keeping with these being the most potent isoforms that regulate HIF-1α and HIF-2α [26] - although PHD1 (and PHD3) may cooperate with PHD2, the major oxygen sensor [27]. Although PHD1 levels are regulated by oestrogen [28], we observed no association between ER and PHD1 either at baseline or in patients treated with tamoxifen - suggesting this is not a major control mechanism for PHD1 in breast cancer. This observation also supports the notion of PHD1 not having a significant role in breast tumorigenesis. Nevertheless, PHD1 and PHD3 were significantly positively associated with HIF-1α and the HIF-1α-regulated gene VEGF. It is recognised that PHD2 and PHD3 expression is modulated by directly by hypoxia, but less appreciated is that PHD1 levels may be altered through suppression of its mRNA under hypoxia [29]. This is in keeping with the upregulation of all PHDs in the breast tumours in this series. Although one might anticipate that elevated levels of PHDs would lead directly to lower HIF through proteosomal degradation, the final effect is not predictable because, if chronic tumour hypoxia still persists, PHD hydroxylation will be abrogated. Furthermore, even in the presence of oxygen, there is some evidence to suggest that the scaffold protein map organiser 1 may block the hydroxylated HIF-1α from entering the degradation pathway, thus retaining some of its transcriptional activity [30]. Other mechanisms may also be present that may enable HIF to escape proteosomal degradation, such as generation of reactive oxygen species [31]. The absence of a correlation of PHDs with CAIX is likely to be due to its significantly longer half-life than HIF-1α, in the order of ~35 hours [32]. This is because both markers need to be present at the same time for an association to be identified. Thus, unlike HIF-1α that has a half-life measured in minutes, the prolonged half-life of CAIX means it would remain in the tumour when other proteins with a shorter half-life are no longer present.

We also observed a significant increase all PHDs post chemotherapy in both trial arms. This increase is potentially hypoxically mediated, since epirubicin has been reported to result in a reduction of blood flow and to have anti-angiogenic effects [33]. Furthermore, despite all PHDs generally increasing post chemotherapy, there was a general trend for upregulation of PHD2 and downregulation of PHD1 and PHD3 to be associated with response. Although this differential regulation is unexpected, one should note that although widely expressed they have differing tissue distributions (for example, PHD3 is highly expressed in the heart whereas PHD1 is highly expressed in the testes) and that it is unclear why the three different isoforms exist and what their specific or overlapping activities might be in HIF-related and HIF-independent biology.

Although all three PHDs may have similar roles in some biological functions, they thus also have function specific to individual PHDs. For example, PHD2 appears to be the sole PHD responsible for myocardial development since Phd2^-/- knockouts are embryonic lethal and the myocardium is severely underdeveloped, amongst other major defects [34]. The same phenotype is not observed with Phd1^-/- or Phd3^-/- knockouts. Furthermore, PHD3 is the only PHD that appears to mediate apoptosis [35], the latter requiring the catalytic activity of PHD3 (as cells are not rescued by HIF-1α and/or HIF-2α), suggesting that PHD3 has non-HIF targets [35]. The effects of PHDs, however, also appear to be cell type specific. The PHDs thus promote cell survival rather than cell death in chondrocytes. A further level of complexity is through the ability of particular PHDs to modulate HIF-independent pathways. PHD1, through hydroxylation of IκB kinase-β, thus fails to result in disassociation of IκB from NF-κB, altering this transcriptional response [36].

The above data support the notion that the resultant decrease in PHD1 could result in a tumour responding via HIF-independent mechanisms. Loss of PHD1 may thus be anticipated to downregulate cyclin D₁ levels and suppress mammary tumour proliferation [37]. However, the biological effect of the expression of particular PHDs in individual tumours may also depend on the type of hypoxia (acute and/or chronic) to which it is exposed. Desensitisation and loss of HIF-1α (and HIF-2α) via hydroxylation by the PHDs has thus been reported to occur under long-term hypoxia, as intracellular oxygen availability is increased by inhibiting mitochondrial respiration [38].