Of the identified HIF prolyl hydroxylases, PHD2 is thought to be the key oxygen sensor regulating HIF (Berra et al, 2003
). Silencing PHD2
through RNA interference increased HIF-1α
levels under normoxic conditions. This effect was not observed with either PHD1
. Furthermore, several groups independently generated genetic knockout mice of the different Phd
s (Takeda et al, 2006
; Aragones et al, 2008
; Minamishima et al, 2008
). Phd1 and Phd3 homozygous knockout mice appeared phenotypically normal with the expected Mendelian ratios (Takeda et al, 2006
). Meanwhile, homozygous knockout of Phd2 was embryonic lethal between days 12.5 and 14.5, because of vascular defects of the placenta and the heart. Moreover, conditional knockout of Phd2 increased vascular and capillary density, vessel branching, and recruitment of vascular smooth muscle cells, whereas conditional knockout of Phd1 and Phd3 did not have these vascular effects (Takeda et al, 2007
). Taken together, these data suggest that PHD2 may have an important role in regulating HIF and angiogenesis.
We recently identified PHD2 as a mediator of potent tumour angiogenesis pathways (Chan et al, 2009
) whereas Mazzone et al (2009
) reported a different function of PHD2 in vessel normalisation. We began our studies by analysing mRNA and protein expression levels of PHD2 in human tumours. In colorectal carcinomas, PHD2 levels of mRNA and protein were both decreased in the tumour compared with non-involved, adjacent normal colon tissue, suggesting that loss of PHD2 may influence the tumour development. To further investigate, we used shRNA to stably silence PHD2 in several different human cell lines, including three colorectal cell lines and a pancreatic cell line. In each of these lines, knocking down PHD2 levels did not affect in vitro
cell growth. It should be noted that we did not challenge these cells to hypoxic stress or additional PHD inhibition. Henze et al (2010
) found that inhibiting PHD activity by hypoxia or DMOG reduced glioma tumour cell survival in vitro
. However, when we implanted our cell lines as xenografts into the flanks of immunocompromised mice, tumour growth was significantly and dramatically increased compared with wild-type control cells. As the best-characterised target of PHD2 is HIF, we then investigated whether the enhanced tumour growth of PHD2 loss is dependent on HIF
Using HCT116 cells, a colon carcinoma, deleted for HIF-1α
(Dang et al, 2006
), we then silenced PHD2 and implanted these cells as tumours. Tumours that lacked HIF and PHD2 grew faster than control tumours that only lacked HIF. This provocative finding suggests that PHD2 has additional functions that are independent of HIF.
To determine the mechanism of why tumours with PHD2 silenced grew faster, we sectioned the tumours. TUNEL analysis showed no difference between control and knockdown tumours, showing that the wild-type tumours were not smaller because of an increase in apoptosis. However, Ki67 expression, a marker of proliferation, indicated that the PHD2-silenced tumours grew better than wild-type tumours in vivo
. These results suggested that PHD2 disruption in tumour cells altered their interaction with the tumour microenvironment, allowing for more efficient growth. We further stained the tumour sections for CD31, a marker of blood vessels, which showed a three- to four-fold increase in tumour blood vessels in the tumours with PHD2 silenced. Similarly, several additional groups have also found that PHD2 can influence tumour growth through its effect on angiogenesis. Lee et al (2008
) showed that a reduction in PHD2 leads to enhanced tumour growth and enhanced tumourigenesis. In reciprocal experiments, Matsumoto et al (2006
) found that 2-oxogluturate, a substrate of PHD2, reduced both tumour growth and angiogenesis. Examining the effect of Phd2 deletion on endothelial cells directly, Takeda and Fong (2007
) found that loss of Phd2 impaired proliferation. Thus, it seemed likely that the enhanced tumour blood supply of the PHD2 knockdown tumour was providing the necessary components to increase tumour growth.
We then investigated whether the PHD2 knockdown cells were secreting factors capable of influencing angiogenesis. Using a standard in vitro angiogenesis assay of endothelial cell tube formation, conditioned media from PHD2 knockdown HCT116 cells were able to cause primary endothelial cells plating on matrigel to aggregate, forming complex, tube-like structures. Conditioned media from control HCT116 cells, which do not have PHD2 knocked down, however, lacked the components to induce the endothelial cells to branch. Subjecting the conditioned media to an angiogenesis antibody array, we revealed that PHD2 consistently regulates angiogenin (ANG) and IL-8, two known soluble pro-angiogenic factors. Conditioned media from HCT116 cells that had PHD2 knocked down had elevated protein levels of ANG and IL-8. Notably, the levels of VEGF were unchanged by PHD2 levels. Silencing either ANG or IL-8 impaired both angiogenesis and tumourigenesis. These results suggested that PHD2 normally functions to inhibit angiogenesis and that silencing PHD2 promotes angiogenesis.
Tumour vasculature is actually regulated by two complementary processes: angiogenesis, which is local sprouting, and vasculogenesis, which is the de novo production of new blood vessels. Angiogenesis is the formation of new blood vessels from pre-existing vessels. This local angiogenesis can also send signals to the bone marrow, which can in turn release precursor or progenitor cells to induce neovascularisation. This process of blood vessel formation by de novo production of endothelial cells, or vasculogenesis, requires tumour cells to interact with stromal cells and circulating bone marrow-derived cells (BMDCs). We then investigated whether PHD2 knockdown affected recruitment of BMDCs to the growing tumour vasculature. To determine this we stained for two BMDC markers, CD11b and CD45. CD11b is a marker of myeloid–monocytic precursors, whereas CD45 is a myeloid marker. PHD2 silencing increased mobilisation of BMDCs to the growing tumour, whereas silencing of ANG and IL8 impaired this mobilisation. Thus, PHD2 functions to regulate both angiogenesis and vasculogenesis through ANG and IL-8.
The PHD2 regulation of tumour vasculature through ANG and IL-8 is independent of HIF. Cummins et al (2006
) showed that PHD1 could regulate IKKβ
, an inhibitor of NF-κ
B, which suggested that PHD2 might also regulate NF-κ
B. In PHD2-silenced cells, NF-κ
B activity was elevated and mutation of the NF-κ
B sites in the promoters of ANG and IL-8 impaired NF-κ
B activation. Furthermore, IL-8 is a well-characterised NF-κ
B target and NF-κ
B bound to the promoter of ANG, as determined by chromatin immunoprecipitation, showing that the influence of PHD2 on the tumour vasculature is mediated through NF-κ
B activity on ANG and IL-8, two known pro-angiogenic factors. Transient silencing of p65, an essential subunit of the NF-κ
B complex, impaired in vitro
angiogenesis. Using breast cancer data sets, there was a strong, inverse correlation between PHD2 mRNA levels and NF-κ
B activity profiles as well as negative relationship between PHD2 and CD31 mRNA levels, as determined by microarray analysis. Furthermore, NF-κ
B activity profiles positively correlated with an increase in CD31 mRNA. Taken together, these data illustrate that PHD2 can regulate the tumour vasculature through an HIF-independent mechanism but relies in part on inhibition of NF-κ
B and the downstream targets of ANG and IL-8.