Here, we provide evidence that loss of endothelial FAK in adult mice can inhibit tumour angiogenesis and tumour growth implicating a direct role for FAK in tumour angiogenesis. Indeed, our study suggests that this role for FAK extends to at least VEGF- and bFGF-dependent angiogenic responses.
We have evidence that the basis of the angiogenic defect in the Pdgfb
-iCreER;FAKfl/fl mice likely involves the effect of endothelial FAK deletion inhibiting EC directional migration and proliferation both in vivo
and in vitro
. These changes in cell behaviour correlate with abnormal actin bundling in FAK−/− ECs. Similar to findings by Ilic et al (1995
) using FAK−/− embryo fibroblasts, we observed dense actin fibres around the cell periphery of FAK−/− ECs. This abnormal architecture of the actin cytoskeleton is usually found in the early stages of abnormal non-polar cell spreading. In contrast to this abnormal architecture, we provide data demonstrating that focal contact formation and paxillin incorporation is normal in FAK−/− ECs. These data imply that endothelial FAK is responsible not for focal contact formation, but rather for the overall organization of the actin cytoskeleton. We speculate that the lack of proper actin organization is likely to be related to the poor proliferation and migration of FAK−/− ECs.
Sprouting angiogenesis involves several phases: (1) an initial increase in EC proliferation, migration and tube formation, followed by (2) maturation of vessels which includes both the recruitment of supporting cells and the deposition of an intact basement membrane. We show that tumour blood vessels in ECFAKKO mice in vivo have reduced proliferation in vivo and that FAK−/− ECs also show reduced proliferation and enhanced apoptosis. In contrast however, the deposition of a laminin-rich basement membrane and the numbers of tumour vessels with associated α-SMA-positive supporting cells is normal in ECFAKKO. Our results have thus dissected the role of FAK in the phases of angiogenesis and suggest that the in vivo role for FAK in pathological angiogenesis is to support the initial phase of neovascularization but not maturation of the vessels.
Our observations that postnatal retinal blood vessel outgrowth is impaired in vivo
provides a second example of how the deletion of endothelial FAK can inhibit angiogenesis. Endothelial tip cell filopodia detect proangiogenic growth factor gradients and these are thought to guide tip cell migration during angiogenesis in the retina (Gerhardt et al, 2003
). FAK is also known to be important in filopodia formation (Braren et al, 2006
; Hsia et al, 2003
). We observed blunted tip cell formation in ECFAKKO
retinae, and thus provide in vivo
evidence that endothelial FAK deletion may cause dysfunctional EC migration during angiogenesis in the whole organism and thus inhibit angiogenesis.
How does FAK regulate endothelial proliferation= Previous studies, using fibroblasts, have shown a dominant role for FAK upstream of the PI3K/Akt signalling pathway (Xia et al, 2004
). In addition, this pathway has been implicated in EC survival (Ackah et al, 2005
; Chen et al, 2005
; Gerber et al, 1998
; Phung et al, 2006
; Somanath et al, 2006
; Sun et al, 2005
; Yang et al, 2003
). We observed poor VEGF-stimulated phosphorylation of p85 and Akt in FAK-deleted ECs with no apparent effect on Src, ERK or PDK phosphorylation. This decrease in p85 and Akt phosphorylation provides a possible explanation for the increased cellular apoptosis and decreased proliferation in FAK-deleted ECs.
Our results in adult mice corroborate previous data, which show that deletion of FAK can inhibit developmental angiogenesis (Braren et al, 2006
; Shen et al, 2005
). In contrast however, the inducible deletion of endothelial-specific FAK using OHT treated FAK fl/fl;End-SCL-Cre-ER(T)/+ mice was reported to not affect adult angiogenic responses (Weis et al, 2008
). How can we explain the apparent discrepancies between these data and our own?
Firstly, OHT treatment of FAK fl/fl;End-SCL-Cre-ER(T)/+ mice results in molecular compensation by the FAK-related molecule Pyk2. In our study, using Pdgfb
-iCreER;FAKfl/fl mice, we saw no overexpression of Pyk2, either in vitro
or in vivo
, or compensation by Pyk2 and others have also shown that this molecule does not always compensate for FAK (Park et al, 2009
). Secondly, since in our study Pdgfb
drives CreERT, but in the Weis study End-SCL
drives CreERT, it is possible that the two different promoters have different spatial and temporal activities in ECs and possibly generate different off-target effects. Our results suggest that off-target effects in the Pdgfb
-iCreER;FAKfl/fl mice are unlikely since FAK levels in platelets isolated from Pdgfb
-iCreER;FAKfl/fl mice appear normal and that bone marrow transplants from Pdgfb
-iCreER;FAKfl/fl do not affect tumour growth or angiogenesis. Rather, it is tempting to speculate that the deletion of FAK in FAK fl/fl;End-SCL-Cre-ER(T)/+ mice versus Pdgfb
-iCreER;FAKfl/fl mice could highlight the different requirements for FAK in different subpopulations of ECs during adult pathological angiogenesis. Lastly, the difference in duration of induction required for activation of Cre in End-SCL-Cre-ER(T);FAKfl/fl versus Pdgfb
-iCreER;FAKfl/fl mice might actually contribute to the lack of an angiogenic phenotype in the former (Weis et al, 2008
). The Cre recombinase (CreERT2
) used in the Pdgfb
-iCreER mice has been reported to be ten-times more sensitive to OHT than the Cre recombinase used in the (CreERT
) End-SCL-Cre-ER(T) mice (Indra et al, 1999
; Shimshek et al, 2002
). This is illustrated by the fact that efficient FAK deletion requires over 20 days of tamoxifen treatment in End-SCL-Cre-ER(T);FAKfl/fl mice but only 2 days in Pdgfb
-iCreER;FAKfl/fl mice. Thus taken together, detailed reading indicates that the two systems used are sufficiently different to give apparently opposing effects of endothelial FAK deletion.
Our study has clinical relevance. The role of FAK in tumourigenesis has been based mainly on studies of FAK in tumour cells (Gabarra-Niecko et al, 2003
; Luo et al, 2009
; McLean et al, 2004
; Provenzano et al, 2008
). The majority of these studies have shown that FAK is required for tumour cell proliferation, migration, survival, and invasion. However, tumour ECs, together with other tumour stromal cells, interact with transformed cells to generate a microenvironment that supports tumour development. Importantly, the stromal compartment recently has been recognized as playing an important role in overall tumour behaviour (Anton & Glod, 2009
). Given the recent development of FAK inhibitors as potential anti-cancer agents (Brunton & Frame, 2008
), our results additionally identify endothelial FAK as a possible anti-angiogenic target thus highlighting further the importance of the anti-cancer efficacy of such drugs.