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Endothelial-cell FAK targeting sensitizes tumours to DNA-damaging therapy

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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Figure 1: Endothelial-cell FAK deletion sensitizes cancer cells to DNA-damaging therapies in vivo.
Figure 2: Loss of endothelial-cell FAK sensitizes tumour cells to DNA-damaging therapy in vivo and in vitro.
Figure 3: FAK deficiency inhibits doxorubicin-stimulated endothelial-cell p65 activity, phosphorlyation and nuclear translocation.
Figure 4: Loss of endothelial-cell FAK inhibits doxorubicin-induced, NF-κB-dependent production of endothelial cytokines.

References

  1. 1

    De Vita, V. T, Hellman, S. & Rosenberg S. A Cancer: Principles and Practice of Oncology (Lippincott Williams and Wilkins, 2001)

    Google Scholar 

  2. 2

    Rottenberg, S. & Jonkers, J. Modeling therapy resistance in genetically engineered mouse cancer models. Drug Resist. Updat. 11, 51–60 (2008)

    CAS  Article  Google Scholar 

  3. 3

    Straussman, R. et al. Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500–504 (2012)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Wilson, T. R. et al. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors. Nature 487, 505–509 (2012)

    ADS  CAS  Article  Google Scholar 

  5. 5

    Sun, Y. et al. Treatment-induced damage to the tumor microenvironment promotes prostate cancer therapy resistance through WNT16B. Nature Med. 18, 1359–1368 (2012)

    CAS  Article  Google Scholar 

  6. 6

    Nakasone, E. S. et al. Imaging tumor-stroma interactions during chemotherapy reveals contributions of the microenvironment to resistance. Cancer Cell 21, 488–503 (2012)

    CAS  Article  Google Scholar 

  7. 7

    Gilbert, L. A. & Hemann, M. T. DNA damage-mediated induction of a chemoresistant niche. Cell 143, 355–366 (2010)

    CAS  Article  Google Scholar 

  8. 8

    Acharyya, S. et al. A CXCL1 paracrine network links cancer chemoresistance and metastasis. Cell 150, 165–178 (2012)

    CAS  Article  Google Scholar 

  9. 9

    Lu, J. et al. Endothelial cells promote the colorectal cancer stem cell phenotype through a soluble form of Jagged-1. Cancer Cell 23, 171–185 (2013)

    ADS  CAS  Article  Google Scholar 

  10. 10

    Mitra, S. K. & Schlaepfer, D. D. Integrin-regulated FAK–Src signaling in normal and cancer cells. Curr. Opin. Cell Biol. 18, 516–523 (2006)

    CAS  Article  Google Scholar 

  11. 11

    Lim, S. T. et al. Nuclear-localized focal adhesion kinase regulates inflammatory VCAM-1 expression. J. Cell Biol. 197, 907–919 (2012)

    CAS  Article  Google Scholar 

  12. 12

    McLean, G. W. et al. Specific deletion of focal adhesion kinase suppresses tumor formation and blocks malignant progression. Genes Dev. 18, 2998–3003 (2004)

    CAS  Article  Google Scholar 

  13. 13

    Shibue, T. & Weinberg, R. A. Integrin β1-focal adhesion kinase signaling directs the proliferation of metastatic cancer cells disseminated in the lungs. Proc. Natl Acad. Sci. USA 106, 10290–10295 (2009)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Tavora, B. et al. Endothelial FAK is required for tumour angiogenesis. EMBO Mol. Med. 2, 516–528 (2010)

    CAS  Article  Google Scholar 

  15. 15

    Nakamura, J. et al. Biphasic function of focal adhesion kinase in endothelial tube formation induced by fibril-forming collagens. Biochem. Biophys. Res. Commun. 374, 699–703 (2008)

    CAS  Article  Google Scholar 

  16. 16

    Stokes, J. B. et al. Inhibition of focal adhesion kinase by PF-562,271 inhibits the growth and metastasis of pancreatic cancer concomitant with altering the tumor microenvironment. Mol. Cancer Ther. 10, 2135–2145 (2011)

    CAS  Article  Google Scholar 

  17. 17

    Fisher, R. I. et al. Comparison of a standard regimen (CHOP) with three intensive chemotherapy regimens for advanced non-Hodgkin’s lymphoma. N. Engl. J. Med. 328, 1002–1006 (1993)

    CAS  Article  Google Scholar 

  18. 18

    Hagemeister, F. B. Treatment of relapsed aggressive lymphomas: regimens with and without high-dose therapy and stem cell rescue. Cancer Chemother. Pharmacol. 49 (suppl. 1). 13–20 (2002)

    Article  Google Scholar 

  19. 19

    Hambardzumyan, D. et al. PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev. 22, 436–448 (2008)

    CAS  Article  Google Scholar 

  20. 20

    Perkins, N. D. The diverse and complex roles of NF-κB subunits in cancer. Nature Rev. Cancer 12, 121–132 (2012)

    CAS  Article  Google Scholar 

  21. 21

    Zhang, H. M. et al. Induced focal adhesion kinase expression suppresses apoptosis by activating NF-κB signaling in intestinal epithelial cells. Am. J. Physiol. Cell Physiol. 290, C1310–C1320 (2006)

    CAS  Article  Google Scholar 

  22. 22

    Tseng, W. P., Su, C. M. & Tang, C. H. FAK activation is required for TNF-α-induced IL-6 production in myoblasts. J. Cell. Physiol. 223, 389–396 (2010)

    CAS  PubMed  Google Scholar 

  23. 23

    Petzold, T. et al. Focal adhesion kinase modulates activation of NF-κB by flow in endothelial cells. Am. J. Physiol. Cell Physiol. 297, C814–C822 (2009)

    CAS  Article  Google Scholar 

  24. 24

    Funakoshi-Tago, M. et al. Tumor necrosis factor-induced nuclear factor κB activation is impaired in focal adhesion kinase-deficient fibroblasts. J. Biol. Chem. 278, 29359–29365 (2003)

    CAS  Article  Google Scholar 

  25. 25

    Ben-Neriah, Y. & Karin, M. Inflammation meets cancer, with NF-κB as the matchmaker. Nature Immunol. 12, 715–723 (2011)

    CAS  Article  Google Scholar 

  26. 26

    DiDonato, J. A., Mercurio, F. & Karin, M. NF-κB and the link between inflammation and cancer. Immunol. Rev. 246, 379–400 (2012)

    Article  Google Scholar 

  27. 27

    Pikarsky, E. et al. NF-κB functions as a tumour promoter in inflammation-associated cancer. Nature 431, 461–466 (2004)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Reynolds, L. E. & Hodivala-Dilke, K. M. Primary mouse endothelial cell culture for assays of angiogenesis. Methods Mol. Med. 120, 503–509 (2006)

    PubMed  PubMed Central  Google Scholar 

  29. 29

    May, T. et al. Establishment of murine cell lines by constitutive and conditional immortalization. J. Biotechnol. 120, 99–110 (2005)

    CAS  Article  Google Scholar 

  30. 30

    Rocha, S., Campbell, K. J. & Perkins, N. D. p53- and Mdm2-independent repression of NF-κB transactivation by the ARF tumor suppressor. Mol. Cell 12, 15–25 (2003)

    CAS  Article  Google Scholar 

Download references

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.

Corresponding author

Correspondence to Kairbaan M. Hodivala-Dilke.

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The authors declare no competing financial interests.

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.

ac, 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.

ac, 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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