Abstract
Chemoresistance is a serious limitation of cancer treatment1. Until recently, almost all the work done to study this limitation has been restricted to tumour cells2. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.
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Acknowledgements
We thank A. Papachristodoulou, J. Holdsworth and B. Williams for their help with immunostaining and animal husbandry. Also M. Hemann for his critical appraisal of the manuscript. The work was funded by CR-UK (C9218/A12007), AICR (12-1068), Medical Research Council (G0901609), National Cancer Institute (P01 CA95426:JGG); Leukemia Lymphoma Research (11022); and CR-UK PhD studentship (C1443/A9215).
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The following authors are listed in the author list in alphabetical order: S.B., F.D., I.F., T.L., D.M.L. and P.-P.W. for their equal and combined contribution to the paper. B.T. and K.M.H.-D. designed the experiments. B.T. performed the experiments. L.E.R. did the GM-CSF rescue experiments in vivo, vessel perfusion, doxorubicin delivery, p-STAT3 staining and hypoxia assays. S.B. performed some of the tumour growth and treatment experiments, CD45 analysis, human lymphoma staining and irradiated cytokine responses; F.D. carried out the conditioned media experiments and MTS assays and several histological analyses; I.F. conducted the primary endothelial cell assays; T.L. measured endothelial-cell FAK and blood-vessel FAK levels in human lymphoma; D.M.L. did the transfections and nuclear fractionation experiments; P.-P.W. carried out the transfected cell cytokine arrays. G.E. carried out the histology; A.C. and J.G.G. provided human lymphoma tissue sections and advice; A.L., J.H. and N.P. performed the NF-κB activation assays and A.A. carried out the survival analysis. B.T. and K.M.H.-D. wrote the paper with substantial input from the co-authors.
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Extended data figures and tables
Extended Data Figure 1 Loss of endothelial-cell FAK in established tumours.
Pdgfb-iCreER;Fakfl/fl mice14 and wild-type control mice (Pdgfb-iCreER;non-floxed) were injected subcutaneously with B16F0-melanoma cells. At day 7 after tumour-cell injection, once tumour growth was established, mice were given tamoxifen to induce, or not, endothelial-cell FAK deletion (generating ECFAKKO and ECFAKWT mice) and tumours continued to grow until they reached the legal size limit at day 24 after tumour-cell injection. Immunofluorescence staining of tumour sections from ECAFKWT and ECAFKKO mice for FAK (green) and endomucin (red) shows efficient deletion of endothelial FAK in tumour blood vessels when FAK deletion is induced after tumour growth has begun. DAPI staining is shown in blue. Endothelial-specific FAK deletion in vivo was confirmed for all experiments. Representative images are given for a minimum of 5 mice per genotype. Scale bar, 75 μm.
Extended Data Figure 2 Loss of endothelial-cell FAK in established tumours does not affect tumour blood vessel density, perfusion or endothelial apoptosis.
a–c, Pdgfb-iCreER;Fakfl/fl mice and wild type control mice (Pdgfb-iCreER;non-floxed) were injected subcutaneously with B16F0-melanoma cells. At day 7 after tumour-cell injection, once tumour growth was established, mice were given tamoxifen to induce, or not, endothelial-cell FAK deletion (generating ECFAKKO and ECFAKWT mice, respectively) and tumours continued to grow until they reached the legal size limit at day 24 after tumour-cell injection. Blood vessels were analysed histologically in midline tumour sections. a, Tumour blood vessel density was not affected by the deletion of FAK after tumour growth had begun. Immunofluorescence of endomucin-stained blood vessels and quantitation of number of blood vessels per mm2 of tumour section are given. b, In an ante-mortem procedure, tumour burdened ECFAKKO and ECFAKWT mice were injected intravenously with PE-conjugated PECAM antibody. Midline sections were immunostained to detect endomucin-positive vessels. Examination of the percentage of endomucin-positive blood vessels that are PE-PECAM-positive gives an indication of blood vessel perfusion. Tumour blood vessel perfusion was not affected significantly by the deletion of FAK after tumour growth had begun. c, Double immunostaining for tumour endothelial cells (PECAM), and either cleaved caspase 3 (CC3) or TUNEL, and DAPI in tumour sections from ECFAKWT and ECFAKKO mice. Apoptotic tumour cells are clearly visible (arrowheads). In contrast, apoptosis is not detectable in the endothelium of either genotype (arrows). Quantitation of the percentage of CC3-positive or TUNEL-positive tumour endothelial cells showed no significant difference between genotypes. Bar charts show means ± s.e.m. n = 5–7 mice per group. NS, not significant, Student’s t-test. Scale bars, 100 μm.
Extended Data Figure 3 Increased tumour-cell DNA damage without changes in blood vessel permeability, doxorubicin delivery, hypoxia or CD45 infiltration in ECFAKKO mice.
a, Quantitation of γH2AX immunostaining indicates that the level of DNA damage in the tumour-cell compartment is increased in treated ECFAKKO mice when compared with ECFAKWT mice. Bar chart shows mean percentage of γH2AX-positive tumour cell nuclei ± s.e.m. n = 3 mice per group. b, Mice were injected via the tail vein with Hoechst dye and PE-PECAM, in an ante-mortem process, and tumour sections were analysed for blood vessel permeability. Representative images of tumour sections showing Hoechst uptake in tumour cells and PE-PECAM-positive blood vessels are given. Bar chart shows mean number of Hoechst-positive nuclei per field of view for tumours grown in ECFAKWT and ECFAKKO mice + s.e.m. n = 8 mice per group. c, Mice were injected via the tail vein with doxorubicin (20 mg kg−1) and analysed for levels of autofluorescent doxorubicin delivery. Representative images of autofluorescent doxorubicin are given. Bar chart shows mean percentage of doxorubicin-positive area proportional to tumour section area + s.e.m. n = 7 mice per group. d, Mice were treated or not with doxorubicin at 9, 11 and 13 days, injected via the tail vein with pimonidazole at day 14 and killed 1 h thereafter. Tumour sections were immunostained to detect hypoxia using an anti-pimonidazole antibody. Bar chart shows percentage hypoxic tumour section area + s.e.m. n = 7 mice per genotype. e, Mice were treated or not with doxorubicin as in c and tumour sections were immunostained for CD45-positive immune cells. Bar chart shows mean CD45 infiltration as a percentage of CD45-positive cell area over tumour section area + s.e.m. n = 4–6 mice per group. NS, not significant, Student’s t-test. Scale bar, 100 μm.
Extended Data Figure 4 Endothelial-cell FAK deletion enhances the mean survival of doxorubicin-treated mice in experimental metastasis models of melanoma and lymphoma.
a–c, Pdgfb-iCreER;Fakfl/fl mice and wild-type control mice (Pdgfb-iCreER;non-floxed) were injected via the tail vein (intravenously) with either B16F10 melanoma cells (a, b) or EumycBCL2 lymphoma cells (c) to establish experimental metastasis models. After tumour growth was established endothelial-cell FAK deletion was induced, or not, by treatment of Pdgfb-iCreER;Fakfl/fl and Pdgfb-iCreER;non-floxed mice with tamoxifen (generating ECFAKKO and ECFAKWT mice, respectively). Mice were then either treated with placebo (a) or doxorubicin (b, c) and survival of the mice was recorded. Data show that endothelial-cell FAK deletion, after tumour growth was established, had no effect on survival per se (a). In contrast, in both the B16F10 and EumycBCL2 experimental metastasis assays, the deletion of endothelial-cell FAK was sufficient to significantly extend median survival after treatment with doxorubicin (b, c). Dashed lines represent median survival. Timelines for tamoxifen and doxorubicin treatment are given in the horizontal bars below each graph. n = 10–20 mice per genotype per test. *P = 0.0209, **P = 0.0055, Gehan–Breslow–Wilcoxon Test. NS, not significant.
Extended Data Figure 5 Alterations in FAK distribution but not expression levels in doxorubicin-stimulated wild-type endothelial cells in vitro and in vivo.
a, Double immunofluorescence staining of cultured wild-type (WT) and FAK-null endothelial cells, with or without doxorubicin treatment, for FAK (green) and DAPI (blue). Staining confirms that FAK is not detectable in FAK-null endothelial cells. In contrast FAK is redistributed in doxorubicin-treated wild-type endothelial cells when compared with untreated wild-type controls. Experiments are representative of three repeats. b, Western blot analysis confirms that FAK levels are not significantly changed in doxorubicin-treated wild-type endothelial cells. HSC70 acts as a loading control. Bar chart represents mean densitometric readings of FAK levels relative to controls. n = 3. c, FAK expression levels are not different after doxorubicin treatment in vivo. Image J analysis of endothelial-cell FAK intensity was performed on tumour sections from PBS or doxorubicin-treated ECFAKWT mice stained for endomucin and FAK. Graph shows average FAK fluorescence pixel intensity levels in endomucin-positive endothelium for individual mice, with means per group ± s.e.m. n = 13–15 mice per treatment group. NS, not significant, Student’s t-test. Scale bar, 50 μm.
Extended Data Figure 6 FAK deficiency inhibits doxorubicin-induced p65 nuclear localization in primary endothelial cells.
a, Doxorubicin-induced p65 nuclear translocation is inhibited in FAK-null primary endothelial cells in vitro. Wild-type (WT) and FAK-null primary lung endothelial cells were treated for 24 h with tamoxifen and 0.25 μM doxorubicin. Immunostaining for p65 (red) was performed. DAPI staining is shown in blue. Arrows indicate cytoplasmic p65; arrowheads indicate nuclear p65. Scale bar, 50 μm. b, Bar charts show the percentage of endothelial cells with nuclear p65 (fold increase) after doxorubicin treatment (0.125 μM or 0.25 μM) for 24 h (T24) or 48 h (T48). n = 91–205 cells per test group.***P < 0.0001, Student’s t-test. NS, not significant.
Extended Data Figure 7 Phosphorylation of IκBα is reduced in doxorubicin-treated FAK-null endothelial cells.
Wild-type (WT) and FAK-null endothelial cells were treated with 0.125 μM doxorubicin for the time indicated. Representative western blot of cytosolic phospho-S32-IκBα and total IκBα. Bar chart shows mean densitometric readings from two biological replicates.
Extended Data Figure 8 Loss of endothelial-cell FAK inhibits the production of irradiation-induced endothelial cytokines.
Wild-type (WT) and FAK-null endothelial cells were treated with 5 Gy irradiation. Seventy-two hours later, whole-cell lysates were extracted and used in proteome profiler cytokine arrays. Bar chart shows the fold difference in cytokine expression between irradiated and non-irradiated wild-type and FAK-null endothelial cells + s.e.m., n = 4 experimental repeats. †P = 0.06, *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test.
Extended Data Figure 9 Cytokine production is similar in untreated wild-type and FAK-null endothelial cells and DNA damage does not increase tumour-cell p-STAT3 expression in ECFAKKO mice.
a, Wild-type (WT) and FAK-null endothelial-cell whole-cell lysates were extracted and used in proteome profiler cytokine arrays. Bar chart shows baseline cytokine expression in untreated wild-type and FAK-null endothelial cells + s.e.m., n = 4 experimental repeats. NS, not significant. b, Pdgfb-iCreER;Fakfl/fl mice and control mice (Pdgfb-iCreER;non-floxed) were injected subcutaneously with CMT19T tumour cells (day 0). At 7–8 days post-inoculation, that is, once tumour growth was established, mice were given tamoxifen to induce endothelial-cell FAK deletion in Pdgfb-iCreER;Fakfl/fl but not Pdgfb-iCreER;non-floxed mice, generating ECFAKKO and ECFAKWT mice, respectively. Thereafter CMT19T bearing mice were given 5 Gy gamma irradiation (day 10). Immunostaining for p-STAT3 in tumour sections revealed that although irradiation increased the percentage of p-STAT3-positive perivascular tumour cells this was not evident in ECFAKKO mice. Representative images of double immunostaining for p-STAT3 and PECAM are given. Bar chart shows mean percentage of p-STAT3-postive perivascular tumour cells + s.e.m. n = 6 mice per group. ***P < 0.005, Student’s t-test. NS, not significant.
Extended Data Figure 10 In vivo rescue of chemosensitization phenotype.
Mouse melanoma B16F0 cells (1 × 106) were injected subcutaneously in the flank of Pdgfb-iCreER;Fakfl/fl mice and control mice (Pdgfb-iCreER;non-floxed). Seven days after tumour-cell injection, that is, once tumour growth was established, these mice were given tamoxifen to induce endothelial-cell FAK deletion in Pdgfb-iCreER;Fakfl/fl but not Pdgfb-iCreER;non-floxed mice, generating ECFAKKO and ECFAKWT mice, respectively. Intratumoral injection of a low dose of recombinant GM-CSF (15 ng) (top graph), or IL-6 (3 ng) (bottom graph), restored doxorubicin-treated tumour growth in ECFAKKO mice to wild-type levels. Top graph shows mean tumour volumes over time + standard deviation. Bottom graph shows mean tumour volumes over time + s.e.m. n = 10–18 mice per group. NS, not significant, Student’s t-test.
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Tavora, B., Reynolds, L., Batista, S. et al. Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy. Nature 514, 112–116 (2014). https://doi.org/10.1038/nature13541
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DOI: https://doi.org/10.1038/nature13541
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