Induction of angiogenesis through local sprouting and neovascularization is essential to the rapid growth of solid tumors. The identity and regulation of the factors responsible for these processes are areas of active investigation as these signaling pathways could provide valuable avenues for therapeutic invention. HIF and VEGF have garnered considerable attention for their roles in mediating tumor angiogenesis. Recently, interest has grown in the regulators of HIF and in alternative mechanisms of pathological angiogenesis (Fischer et al., 2007
; Shojaei et al., 2007
). In this report, we demonstrate that PHD2 functions as a tumor suppressor by limiting ANG and IL-8, which play roles in both angiogenesis and BMDC recruitment contributing to vasculogenesis. Despite the role of PHD2 in regulating HIF stability, the tumor suppressor function of PHD2 observed here does not solely depend on the presence of HIF. Eliminating HIF did not reduce tumor growth in the PHD2-silenced background in multiple cancer types. PHD2 knockdown elevated normoxic NF-κB activity and consequently, increased IL-8 and ANG, both of which contribute to enhance tumor blood supply and thus tumor growth. Moreover, analysis of clinical data reveals a reduction of PHD2 in human cancers, which is also correlated with an increase in tumor vasculature. Together, these findings highlight the importance of HIF- and VEGF-independent pathways in regulating tumor angiogenesis.
Bone marrow-derived cells are increasingly appreciated to be important contributors to the expansion of tumor vasculature (Gao et al., 2008
; Lyden et al., 2001
; Shojaei et al., 2007
; Yang et al., 2004
). However, the mechanisms by which tumors recruit these cells are not yet fully understood. A few factors have been implicated, such as the Id proteins, MMP-9, and Bv8, but the entire repertoire of signals responsible for the recruitment is largely unknown. Our current data provide evidence that the release of IL-8 and ANG is enhanced in tumors with reduced PHD2, and that these factors are important for both recruitment of BMDCs and for local angiogenesis. The precise mechanisms by which PHD2 restricts release of IL-8 and ANG and how vasculogenesis is mediated by BMDC recruitment in response to IL-8 and ANG remains to be determined. We demonstrate that NF-κB is required for enhanced angiogenesis upon PHD2 silencing. NF-κB, in turn, regulates ANG
. Intriguingly, inhibition of either ANG or IL-8 appears to be sufficient to abolish the effect of losing PHD2, suggesting potential clinical applications. In particular, inhibiting either ANG or IL-8 may be potential avenues for treatment of tumors that have become resistant to anti-VEGF therapies. Our findings demonstrate that a biological consequence of NF-κB regulation by PHD2 is to impair tumor growth through disruption of the blood supply, suggesting that this pathway may be important in human cancers.
How does PHD2 come to be downregulated in some tumors? One possibility is that PHD2 itself might be mutated in a way that reduces its activity, expression, or stability. A recent report describes a patient with a PHD2
mutation who presents with both polycythemia and multiple tumors (Ladroue et al., 2008
). In addition, PHD2
was found to be mutated at a high rate in endometrial cell lines (Kato et al., 2006
). Another possibility is that methylation of the PHD2
promoter could result in epigenetic silencing of PHD2
. Nevertheless, dysregulation of PHD2 has important consequences, contributing to tumorigenesis in multiple ways. Our analysis indicates that PHD2 levels are significantly reduced in certain clinical cases, such as colon and breast tumors. Although this reduction in PHD2 may contribute to tumor growth through enhanced stability of HIF, our work and the work of others indicate that PHD2 also has additional HIF-independent regulatory functions. For example, in agreement with the large variability of PHD2 protein levels in the NCI60 panel, simultaneous protein expression of HIF-1 and PHD2 has been found in a subset of head and neck tumors (Jokilehto et al., 2006
). Our data suggests that PHD2 can regulate HIF and NF-κB in a complementary, additive fashion with both pathways likely contributing to tumor growth. Both the HIF-dependent hypoxia response and the HIF-independent function of PHD2 can influence NF-κB and IL-8. It therefore remains to be seen in which contexts one PHD2 function can prevail over the other and whether HIF-dependent or HIF-independent pathways come to dominate tumorigenesis. One possibility is that these pathways cooperate to provide redundant pathways for activating NF-κB and promoting angiogenesis. Alternatively, HIF-dependent mechanisms may be predominant in hypoxic regions, while disruption of PHD2 may allow for HIF-independent mechanisms that promote angiogenesis in nonhypoxic regions. Future studies will elucidate whether PHD2 regulation of HIF is important for tumor initiation while regulation of NF-κB is important for tumor progression or dissemination.
As the search for HIF inhibitors is an active area of cancer biology, this study suggests that PHD2 also plays a critical role in tumor growth. However, HIF inhibition may not be sufficient to impede tumor growth especially in tumors in which PHD2 activity is lost. PHD2 has at least two tumor suppressor activities: 1) negatively regulating HIF through hydroxylation and 2) negatively regulating NF-κB in a HIF-independent manner, both of which in turn modulates tumor vasculature through angiogenesis and vasculogenesis. Further studies on the mechanism of PHD2 loss will be essential to gain new insights into which tumors may benefit clinically from anti-angiogenic therapies. Identifying other PHD2 targets and binding partners, as well as delineating how they influence tumorigenesis, will be a burgeoning field of study and will be critical in furthering our understanding of tumor progression.