Nuclear FAK and Runx1 Cooperate to Regulate …...was by high-speed centrifugation (16,000 g at 4 C for 15minutes). Immunoblotting and immunoprecipitation Cell lysates or cell fractions

$Page 1: Nuclear FAK and Runx1 Cooperate to Regulate …...was by high-speed centrifugation (16,000 g at 4 C for 15minutes). Immunoblotting and immunoprecipitation Cell lysates or cell fractions$
Molecular and Cellular Pathobiology

Nuclear FAK and Runx1 Cooperate toRegulate IGFBP3, Cell-Cycle Progression, andTumor GrowthMarta Canel1,2, Adam Byron1, Andrew H. Sims1, Jessy Cartier3, Hitesh Patel1,Margaret C. Frame1, Valerie G. Brunton1, Bryan Serrels1, and Alan Serrels1,2

Abstract

Nuclear focal adhesion kinase (FAK) is a potentially importantregulator of gene expression in cancer, impacting both cellularfunction and the composition of the surrounding tumor micro-environment. Here, we report in a murine model of skin squa-mous cell carcinoma (SCC) that nuclear FAK regulates Runx1-dependent transcription of insulin-like growth factor bindingprotein 3 (IGFBP3), and that this regulates SCC cell-cycle pro-gression and tumor growth in vivo. Furthermore, we identified a

novel molecular complex between FAK and Runx1 in the nucleusof SCC cells and showed that FAK interacted with a numberof Runx1-regulatory proteins, including Sin3a and other epige-netic modifiers known to alter Runx1 transcriptional functionthrough posttranslational modification. These findings provideimportant new insights into the role of FAK as a scaffoldingprotein in molecular complexes that regulate gene transcription.Cancer Res; 77(19); 5301–12. �2017 AACR.

IntroductionFocal adhesion kinase (FAK) is a non–receptor protein tyrosine

kinase that controls diverse cellular functions, including celladhesion, migration, invasion, polarity, proliferation, and sur-vival (1, 2). FAK is therefore involved in a number of processesthat can impact on the malignant phenotype. Deletion of fak inmouse models of cancer has shown a requirement for FAK intumor formation and progression (3–9). Overexpression of FAKhas also been reported in a number of human epithelial tumors(10–12), and it has therefore emerged as a potential target forcancer therapy, with a number of FAK kinase inhibitors currentlybeing developed (13).

FAK was identified as a protein highly phosphorylated inresponse to integrin activation and primarily located at cell–extracellular matrix adhesion sites termed focal adhesions (1).Recent reports have also identified that FAK contains nuclearlocalization signals within the F2 lobe of the four-point-one,ezrin, radixin, moesin (FERM) domain (14) and a nuclear exportsignal within the kinase domain (15). Therefore, FAK can trans-

locate to the nucleus, where its function remains poorly charac-terized.Within the nucleus, the FAKFERMdomain canbind to thetranscription factors p53 and GATA4, resulting in their turnoverand inactivation, thereby controlling cell survival and inflamma-tory signals (14, 16). Recently, we reported that nuclear FAK wastethered to chromatin and regulated the expression of chemo-kines and cytokines, including Ccl5 and TGFb2, that contributeto establishment of an immunosuppressive tumor environmentthrough driving elevated intratumoral regulatory T-cell num-bers (17). Therefore, nuclear FAK protein complexes can act toregulate transcriptional programs important in controllingcellular responses and the composition of the tumor immuneenvironment.

Runt-related transcription factor 1 (Runx1; also known asAML1) is one of a family of three transcription factors (Runx1–3) that can either activate or repress transcription depending onthe target gene, cell type, and associated cofactors (18, 19). It hasbeen shown to have a critical role in hematopoiesis and hemato-poietic function (20) and is essential for mammalian develop-ment (21). In the context of cancer, Runx1 is best known for itsrole in acute myeloid leukemia where it is frequently foundmutated (18). In recent years, it has also become clear that Runx1plays an important role in solid epithelial malignancies. Forexample, Runx1 deficiency impairs mouse skin tumorigenesis(22), while in contrast, it acts as a tumor suppressor in the ApcMin

mouse model of colorectal carcinogenesis (23). Therefore, it hasan important but context-dependent function in cancer. Here, weidentify a novel molecular complex between FAK and Runx1 inthe nucleus of skin squamous cell carcinoma (SCC) cells. Weshow that nuclear FAK and Runx1 cooperate to regulate expres-sion of IGFBP3, and that IGFBP3 regulates cell-cycle progressionand tumor growth in vivo. Using proteomics and network biologyapproaches, we identify that nuclear FAK interacts with a numberof Runx1-regulatory proteins that can alter Runx1 transcriptionalfunction through posttranslational modification. Furthermore,we identify a novel interaction between nuclear FAK and Sin3a, acore component of the Sin3a/HDAC corepressor complex, and

1Cancer Research UK Edinburgh Centre, Institute of Genetics and MolecularMedicine, University of Edinburgh, Edinburgh, United Kingdom. 2MRC Centre forInflammation Research, The Queen's Medical Research Institute, University ofEdinburgh, Edinburgh, United Kingdom. 3British Heart Foundation Centre forCardiovascular Science, University of Edinburgh, The Queen's Medical ResearchInstitute, Edinburgh, United Kingdom.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Canel and A. Byron contributed equally to this article as first authors.

B. Serrels and A. Serrels contributed equally to this article as senior authors.

Corresponding Authors: Alan Serrels, University of Edinburgh, EdinburghEH42XR, United Kingdom. Phone: 44-1-31-242-9100; Fax: 44-1-131-242-6578;E-mail: [email protected]; and Bryan Serrels, [email protected]

doi: 10.1158/0008-5472.CAN-17-0418

�2017 American Association for Cancer Research.

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show that Runx1/Sin3a interaction is enhanced in SCC FAK-wtcells. This study provides new insights into the potential mechan-isms through which nuclear FAK regulates transcription factorfunction to control cell behavior.

Materials and MethodsMaterials

All antibodies used are listed in Supplementary Materials andMethods. All siRNA was purchased from Dharmacon and shRNAfrom Open Biosystems.

Cell linesCell lines used in this study were generated, authenticated,

and characterized as described previously (17, 24). Cellswere pathogen tested in September 2016 using the ImpactIII test(Idex Bioresearch) and were negative for all pathogens. Celllines are routinely tested for mycoplasma every 2 to 3 monthsin-house and have never been found to be mycoplasma pos-itive. Cell lines are cultured for no more than 3 monthsfollowing freeze thawing. Runx1 or scrambled siRNA wastransiently transfected into SCC cells using HiPerFect (Qiagen).Cells were left for 3 days and then immunoblotting or quan-titative qRT-PCR analysis was carried out. Cell lines stablyexpressing fluorescent ubiquitination-based cell-cycle indicator(FUCCI), Runx1, or empty vector shRNA were generated bylentiviral infection and selected in 2 mg/mL puromycin.

Generation of nuclear-targeting FAK mutantsPoint mutations were introduced into wild-type FAK in three

different combinations: (i) R177A and R178A, (ii) K190A andK191A, and (iii) K216A and K218A, using site-directed mutagen-esis as described previously (17). Primer sequences are listed inSupplementary Materials and Methods.

Quantitative RT-PCRRNA extracts were obtained using an RNeasy Kit (Qiagen),

following the manufacturer's instructions. cDNA was made usinga First-Strand cDNA Synthesis Kit (Invitrogen). qRT-PCR wasperformed as described previously (17). Analysis was performedusing Rotor-Gene software, and expression relative to B2M wascalculated using Excel (Microsoft). Standard PCR was performedusing the above conditions but substituting SensiFAST for a 2 �PCR Master Mix. Primers used are listed in Supplementary Mate-rials and Methods.

Nuclear fractionation and total cell lysatesCells were collected by scraping and low-speed centrifugation

(1,000� g at 4�C for 5minutes), followed by twowashes with ice-cold PBS. Cell pellets were resuspended in buffer A (10 mmol/LKCl, 10mmol/LHEPES, pH7.9, 0.5mmol/LDTT, and1.5mmol/LMgCl2) and incubated on ice for 15 minutes. Cells were lysedusing a 25 g needle and nuclei were isolated by centrifugation at12,000 � g at 4�C for 30 seconds. The nuclear pellet was washedtwice in buffer A and incubated in buffer C [25% (v/v) glycerol, 0.2mmol/L EDTA, pH 8.0, 0.42 mol/L NaCl, 0.5 mmol/L PMSF, 20mmol/LHEPES, pH7.9, 0.5mmol/LDTT, and1.5mmol/LMgCl2]at 4�C for 1 hour. Clarification of the nuclear extracts was by high-speed centrifugation (16,000 � g at 4�C for 5minutes). Alterna-tively, cells were washed with PBS and lysed in RIPA lysis buffer(50mmol/L Tris-HCl, pH7.4, 150mmol/L sodiumchloride, 0.1%SDS, and 1% sodium deoxycholate) with inhibitors (Complete

Protease Inhibitor Cocktail and PhosSTOP; Roche). Clarificationwas by high-speed centrifugation (16,000 � g at 4�C for15minutes).

Immunoblotting and immunoprecipitationCell lysates or cell fractions [10–20mg protein, as measured by

Micro BCA Protein Assay Kit (Pierce)] were supplemented withSDS sample buffer (Tris, pH6.8, 20% glycerol, 5% SDS, b-mer-captoethanol, and bromophenol blue), separated by SDS-PAGE,transferred to nitrocellulose and immunoblotted with specificantibodies at 1:1,000 dilution. For immunoprecipitation experi-ments, 0.25 to 1mg of cell lysate or cell fraction was immuno-precipitated with either 5 mL of mouse Runx1 antibody, 10 mL ofagarose-conjugated mouse FAK antibody, 10 mL of agarose-con-jugated Myc-tag, or 10 mL of agarose-conjugated control IgG, andimmune complexes collected. Beads were washed three timeswith lysis buffer, once with 0.6mol/L lithium chloride, and thenadded to SDS sample buffer.

2D gel electrophoresisTwo-dimensional SDS-PAGE was performed using the ZOOM

IPGRunner System (Thermo Fisher Scientific) according to themanufacturer's protocol. Gels were transferred onto nitrocellu-losemembranes, blocked (5%BSA inTBST) andprobedwith anti-RUNX1 antibody.

Protein capture arraysSCC cells (2 � 106) were plated onto a 90-mm tissue culture

dish and allowed to adhere overnight. Growth medium wasremoved and replaced with 5 mL of normal growth medium.Cells were cultured for a further 24 hours before conditionedmedium was removed and centrifuged at 1,000 rpm for 5 min-utes. Supernatant was collected and used for analysis. To controlfor cell number, the 90-mmdish containing SCC cells was lysed inRIPA buffer and protein quantified using BCA protein assay asdescribed above. This was used to normalize loading of condi-tioned media onto the protein capture arrays. Secreted proteinanalysiswas performedusingmouse angiogenesis protein capturearrays (R&D Systems) according to the manufacturer's protocol.The only modification was the use of a streptavidin DyLight-800antibody for detection (1:10,000 dilution, Rockland Biochem-icals). Image acquisition and analysis was performed using a LicorImager. Mean spot intensities were calculated from duplicatearrayed spots per cell line. Data were median centered andsubjected to unsupervised agglomerative hierarchical clusteringon the basis of Pearson correlation computed with a complete-linkage matrix using Cluster 3.0 (C Clustering Library, version1.50; ref. 25). Clustering results were visualized using Java Tree-View (version 1.1.6; ref. 26).

Blood vessel immunostainingSubcutaneous tumors were surgically excised, placed in an

aluminum foil boat, submerged in OCT compound, and snapfrozen using dry ice andmethanol bath. OCT-embedded sampleswere stored long term at�80�C. For analysis, sections were cut at�20�C onto siliconized microscope slides. For staining, sectionswere removed from the �20�C freezer, and 50 mL of fixative wasapplied directly onto the tissue. Sections were incubated for 8minutes at 4�C, and then, the fixative removed and the slidesallowed to equilibrate to room temperature for approximately 10to 20 minutes. Sections were then rehydrated in PBS for 10minutes. The area of tissue was surrounded with a hydrophobic

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Cancer Res; 77(19) October 1, 2017 Cancer Research5302




barrier pen and blocked using 1% horse serum in PBS for 30minutes at room temperature. Tissue was then incubated withanti-CD31 antibody [BD Biosciences, 1:100 dilution in incuba-tion buffer (1% BSA, 1% normal donkey serum, 0.3% TritonX-100, 0.01% sodium azide in PBS)] overnight at 4�C. Slides werewashed three times for 15 minutes with PBS, and then incubatedwith anti-rat Alexa Fluor 594–conjugated secondary antibody(Molecular Probes, Thermo Fisher Scientific) diluted at 1:200 inincubation buffer for 1 hour at room temperature. Slides werewashed three times for 15minutes in PBS and then counterstainedwith DAPI for 5minutes. Slides were washed once for 15minutesin PBS and mounted in VectaShield anti-fade mounting media(Vector Labs). Images were acquired using an Olympus FV1000confocal microscope.

Transcription factor network analysisTranscription factors were extracted from the DECODE data-

base (Qiagen), selecting the most relevant transcription factorspredicted by text mining to bind between 20 kb upstreamand 10 kb downstream of the human transcription starting site.The transcription factor Runx1 was used to seed a network of1,000 related proteins using the GeneMANIA plugin (version3.4.1; human interactions) in Cytoscape (version 3.3.0; ref. 27),onto which proteins specifically isolated in nuclear FAK proteincomplexes (17) were mapped. The resulting interactome wasextracted, andproteinswithphysical or predicted direct or indirectinteractions with Runx1 were analyzed and topological networkparameters computed using theNetworkAnalyzer plugin (version2.7). Networks were clustered using the yFiles Organic algorithmimplemented in Cytoscape. Pathway enrichment analysis wasperformed using the Database for Annotation, Visualization andIntegrated Discovery (version 6.8; ref. 28). Pathway terms withBenjamini–Hochberg-corrected P < 0.01 were considered signif-icantly overrepresented.

Subcutaneous tumor growthCells (2.5� 105) were injected subcutaneously into each flank

of immunocompromised CD-1 nude mice, and tumor growthmeasured twice weekly using calipers. Measurements were takenfrom 3mice (each bearing two tumors) for each cell line, and thevolume of the tumor (v) was calculated in Excel using the formulav¼ 4/3� p� r3. Data were graphed and statistics calculated usingPrism (GraphPad).

In vivo cell-cycle analysesOptical window chambers were implanted onto CD-1 nude

mice as described previously (29). All animal work was carriedout in compliance with UK Home Office guidelines. FUCCI-expressing SCC cells (1 � 106) were injected into the dermisand at the time of window implantation. Twenty-four hourslater, mice were anaesthetized using an isoflurane–oxygen mixand three-dimensional image stacks acquired using an Olym-pus FV1000 confocal microscope. Image analysis was per-formed using the spot detection tool in Imaris (Bitplane). Thenumber of green, red, and double-positive nuclei were countedand calculated as a percentage of the total number of cellswithin the image stack.

Longitudinal in vivo imaging of tumor angiogenesisOptical window chambers were implanted onto CD-1 nude

mice as described above. Prior to sealing the window with a

coverslip, a tagRFP-expressing tumor fragment (�1 mm in diam-eter) was placed into the window. To obtain tumor fragments, adonor animal was injected with 2.5� 105 tagRFP-expressing SCCcells 10 days prior to optical window implantation. Tumors wereremoved, cut into small pieces using a scalpel, and fluorescencechecked using an Olympus OV110 whole-animal imaging sys-tem. For longitudinal imaging of tumor angiogenesis, mice bear-ing optical windows were anaesthetized using an isoflurane–oxygen mix. Images were acquired 2, 4, 7, and 9 days postim-plantation using an Olympus OV110 whole-animal imagingsystem set to acquire both GFP and RFP signals using the zoomlens set to �1.6 magnification. For image analysis and identifi-cation of blood vessels, autofluorescence images acquired usingtheGFP channel were inverted in ImageJ. The tubeness pluginwasused to detect blood vessel structures and apply an image maskover these structures. The accuracy of this was checked manually.The vascular densitywas determined by calculating the percentageof the field of view covered by the vascular imagemask. Data weregraphed and statistics calculated using Prism.

ResultsFAK is required for cell-cycle progression and angiogenesisin SCC tumors

We have previously shown using a murine model of skin SCCthat depletion of FAK expression can result in immune-mediatedtumor regression in syngeneic mice, and a growth delay inimmunodeficient mice (17, 24). We therefore sought to furtherdissect the complex role of FAK in regulating SCC tumor growth.We measured tumor growth in CD-1 nude mice following injec-tion of 0.25 � 106 FAK-deficient cells (SCC FAK�/�) and FAK-deficient cells that reexpressed wild-type FAK (SCC FAK-wt) atclose to endogenous levels. As previously reported, SCC FAK�/�

tumors exhibited a growth delay when compared with SCCFAK-wt tumors (Fig. 1A; ref. 24) that was associated with anincreased tumor doubling time (Fig. 1B). FAK is a protein thatplays a pleiotropic role in regulating cancer development andprogression, and we hypothesized that a number of its reportedfunctions, including regulation of proliferation/cell cycle andtumor angiogenesis (1, 2, 30), may contribute to the observedtumor growth delay in SCC FAK�/� tumors when established onan immune deficient host background. Thus, we set out to useintravital imaging to analyze, in real time, the cell-cycle distribu-tion and kinetics of tumor neoangiogenesis in SCC FAK-wt andSCC FAK�/� tumors when grown on CD-1 nude mice.

Tomeasure cell-cycle distribution in real time, SCC FAK-wt andSCC FAK�/� cells expressing the FUCCI reporter (31) wereimplanted via intradermal injection under dorsal-skinfold imag-ing windows. The FUCCI probe is based on the differentialproteolysis of geminin and cdt1 at specific phases of the cellcycle, allowing differential profiling of cells at G1 (red), G1–S(yellow), and S–G2–M (green) phases. Images of FUCCI-expres-sing SCC FAK-wt and SCC FAK�/� tumors revealed an increasednumber of red fluorescent cells in SCC FAK�/� tumors (Fig. 1C).Analysis of the proportion of red, yellow, and green nuclei fromthree-dimensional image stacks acquired 24hours after tumor cellimplantation identified a specific delay in the G1 phase of the cellcycle in SCC FAK�/� tumor cells (Fig. 1D).

FAK, expressed in cancer cells, has been reported to influencetumor angiogenesis through regulating the expression of VEGF(30). To ascertain whether defective tumor neoangiogenesis may

Cooperation of Nuclear FAK and Runx1 in IGFBP3 Regulation

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also be occurring in SCC FAK�/� tumors, we performed longitu-dinal imaging of tagRFP-labeled SCC FAK-wt and SCC FAK�/�

tumors under dorsal-skinfold windows. Using acquisition para-meters similar to those required to image GFP, we acquiredimages of tissue autofluorescence, in which blood vessels appearas dark nonfluorescent structures that can be readily visualized.Longitudinal imaging of neoangiogenesis revealed a significantdecrease in vascular density in SCC FAK�/� tumors when com-paredwith SCC FAK-wt tumors (Fig. 1E and F). Thus, SCC FAK�/�

tumors exhibit defective cell-cycle progression and neoangiogen-

esis that may contribute to defective tumor growth in CD-1 nudemice.

FAK negatively regulates transcription of IGFBP3We and others have previously identified that FAK can regulate

the expression of secreted proteins that have the potential to act inboth an autocrine and paracrine manner to influence cancer cellbehavior and the surrounding tumor microenvironment (17, 32,33). To investigate whether FAK-dependent regulation of secretedfactors in SCC cancer cells could influence cell-cycle progression

Figure 1.

FAK regulates SCC tumor growth, cell cycle, and angiogenesis in vivo.A,Growth of SCCFAK-wt and SCCFAK�/� tumor xenografts in CD-1 nudemice.n¼ 5–6 tumorsper group. B, SCC FAK-wt and SCC FAK�/� tumor doubling time. Unpaired t test, �� , P < 0.0001. C, Intravital imaging of FUCCI expressing SCC FAK-wtand SCC FAK�/� cells 24 hours postimplantation under dorsal skinfold windows. D, Quantitation of FUCCI cell-cycle distribution from 3-dimensional image stacksshown in C. Sidak corrected two-way ANOVA, ��, P <0.001. n¼ 4 tumors per group. E, Longitudinal imaging of tumor angiogenesis following implantation of tumorfragments under dorsal skinfold windows. Red, tagRFP labeled SCC tumor; green, tissue autofluorescence. F, Quantitation of blood vessel density at day 9.Unpaired t test, � , P ¼ 0.0306. Data in all graphs represented as mean � SEM. n ¼ 3 tumors per group.

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and tumor angiogenesis, we screened for secreted factors impli-cated inbothprocesses usingprotein capture arrays. Analysis of 53secreted factors revealed both positive andnegative regulation as aconsequence of FAK expression (Fig. 2A; Supplementary Fig. S1).Applying a 4-fold cutoff for differential regulation, we identifiedIGFBP3 as the most highly upregulated protein in SCC FAK�/�

conditioned medium, while PDGF-AA, PDGF-AB/BB, FGF-1, andMMP-3 were the most highly upregulated proteins identified in

SCC FAK-wt conditioned medium. Using these findings, wefocused on IGFBP3, a protein belonging to the family of insu-lin-like growth factor binding proteins (IGFBP), as this factor haspreviously been reported to negatively regulate both cell-cycleprogression (34, 35) and angiogenesis (36–38). Western blotanalysis of conditioned media confirmed an upregulation ofIGFBP3 in SCC FAK�/� conditioned samples (Fig. 2B), andq(RT)-PCR identified similar regulation of Igfbp3 gene transcript

Figure 2.

FAKnegatively regulates the expression of IGFBP3 but not other IGFBP familymembers.A,Relative secreted levels of 53 angiogenesis-related proteinsmeasured byantibody capture array frommedia conditioned by SCC FAK-wt and FAK�/� cells. Proteins are ordered by fold change. Dotted gray lines indicate 4-fold enrichment;proteins changed by at least 4-fold are indicated. Box-and-whisker plot summarizes the median (line), 25th and 75th percentiles (box), and 5th and 95thpercentiles (whiskers). B, Representative anti-IGFBP3 Western blot from concentrated conditioned media. C, qRT-PCR analysis of Igfbp3 transcript levels in SCCFAK-wt and FAK�/� cells. n ¼ 3. D, Representative PCR analysis of Igfbp3 family transcript levels in SCC FAK-wt and FAK�/� cells. E, qRT-PCR analysis ofIgfbp3 transcript levels in SCC FAK-wt and FAK�/� cells. n ¼ 3. F, qRT-PCR analysis of Igfbp3 transcript levels in SCC FAK-wt and FAK�/� cells. n ¼ 3.G, Representative anti-IGFBP3 Western blot showing secreted IGFBP3 protein levels from SCC FAK-wt, SCC FAK�/�, and SCC FAK-kd cells. Data in all graphsrepresented as mean � SEM. Unpaired t test, �� , P < 0.01.






levels (Fig. 2C). Thus, we conclude that FAK negatively regulatesthe transcription of Igfbp3, leading to reduced levels of extracel-lular secreted IGFBP3 protein.

IGFBP3 is amember of a family of six proteins, IGFBP1–6 (39).However, only antibody capture pairs specific for IGFBP1, 2, and3were present on the protein capture arrays used. Therefore, wesought to determine whether FAK regulated the expression ofother IGFBP family members. Employing sequence-specific pri-mers designed for eachof the six IGFBPs,weusedPCR to screen forexpression in cDNA libraries prepared from SCC FAK-wt and SCCFAK�/� cells (Fig. 2D). Aside from Igfbp3, only expression of Igfbp4and Igfbp6 could be detected following 20 cycles of PCR. Allprimer sets were confirmed to produce single products of thecorrect size using cDNA prepared from mouse liver (Supplemen-tary Fig. S2). qRT-PCR analysis of Igfbp4 expression revealed nodifferential regulation (Fig. 2E), whereas Igfbp6 expression wasobserved to exhibit a small but significant decrease in SCCFAK�/�

cells when compared with SCC FAK-wt (Fig. 2F). Therefore,IGFBP3 was the only IGFBP family member to be negativelyregulated in response to FAK expression.

FAK kinase activity is not required for regulation of IGFBP3There has been considerable interest in targeting FAK function

as a cancer therapy, and a number of small-molecule FAK kinaseinhibitors are now in early-phase clinical development (13). Todetermine the role of FAK kinase activity in regulation of IGFBP3,we tested IGFBP3 protein expression using an SCC FAK�/� cellline in which a FAK kinase–deficient mutant (FAK-kd) had beenreexpressed to levels comparable with SCC FAK-wt cells. Anti-IGFBP3 Western blotting from conditioned media revealed thatIGFBP3 expression in SCC FAK-kd cells was similar to thatobserved in SCC FAK-wt cells (Fig. 2G), implying that FAK-dependent regulation of IGFBP3 expression was independent ofFAK kinase activity.

IGFBP3 regulates cell-cycle progression but not angiogenesis toinfluence tumor growth

To determine whether increased IGFBP3 expression in SCCFAK�/� cells contributed to the defective growth of SCC FAK�/�

tumors, we generated SCC FAK�/� cells with a stable knockdownof IGFBP3 using shRNA (Fig. 3A). Analysis of subcutaneoustumor growth showed that knockdown of IGFBP3 in SCCFAK�/� cells partially restored tumor growth in vivo (Fig. 3B),resulting in an average tumor volume of approximately doublethe size when compared with SCC FAK�/� tumors on day 12.Using intravital imaging togetherwith the FUCCI cell-cycle report-er, we identified that the G1 arrest observed in SCC FAK�/� cellsin vivo was overcome following IGFBP3 depletion (Figs. 3C andD).However, thedefect inblood vessel formationwas still evidentin the SCC FAK�/� shRNA-IGFBP3 tumors as measured by thepresence of CD31þ vessels (Figs. 3E and F). Therefore, IGFBP3likely contributes to regulation of SCC tumor growth throughcontrolling cell-cycle progression, but not angiogenesis. Further-more, depletion of IGFBP3 in SCC FAK�/� cells was not sufficientto fully restore tumor growth, implying that regulation of angio-genesis is likely also important.

FAK nuclear localization is required for regulation of IGFBP3transcription

We have recently reported in SCC cells that nuclear FAK canbind to transcription factors and transcriptional regulators

with the potential to influence gene expression (17). To inves-tigate the requirement for FAK nuclear localization in regula-tion of Igfbp3, we made a series of FAK nuclear targetingmutants according to previously reported findings that iden-tified putative nuclear localization sequences within the F2lobe of the FERM domain (40), and expressed these in SCCFAK�/� cells. A total of three nuclear targeting mutants wereconstructed as follows: (i) by replacing arginine residues atpositions 177 and 178 with alanines (FAK-177/178); (ii) byreplacing lysine residues at positions 190 and 191 with ala-nines (FAK-190/191); and (iii) by replacing lysine residues atpositions 216 and 218 with alanines (FAK-216/218). We havepreviously reported a fourth mutant deficient in nuclear target-ing, in which all six of these amino acid residues have beenreplaced with alanines (17). Biochemical fractionation fol-lowed by Western blotting was used to assess nuclear/cyto-plasmic distribution of the resulting mutants when reexpressedin SCC FAK�/� cells, revealing that all four mutant FAKproteins were deficient in their ability to localize to the nucleus(Fig. 4A). Given that all of the double mutants appearedas defective in nuclear translocation as the mutant harboringall six mutations, we chose to move forward with these forfurther analysis. qRT-PCR analysis of Igfbp3 expression in celllines expressing these mutant FAK proteins revealed signifi-cantly increased levels of Igfbp3 transcript in all three cell lines(Fig. 4b), implying a crucial role for FAK nuclear targeting inthe transcriptional regulation of Igfbp3. The inability of thesemutants to completely restore Igfbp3 transcript levels to that ofSCC FAK�/� cells is likely the consequence of low levels ofresidual nuclear FAK (Fig. 4A).

Runx1 is required for FAK-dependent transcriptionalregulation of IGFBP3

To better define the link between nuclear FAK and regulationof Igfbp3 transcription, we next sought to identify transcriptionfactors with predicted binding sites in the promoter of theIgfbp3 gene. Using the DECODE database (Qiagen), we gen-erated a list of transcription factors predicted by text miningto bind between 20 kb upstream and 10 kb downstream ofthe human transcription start site (Fig. 4C). Of these transcrip-tion factors, three, Sp1, Runx1, and components of the tran-scription factor IID complex (TFIID), were detected by massspectrometry in a nuclear FAK interactome in SCC cells(Fig. 4C, dark gray bars; ref. 17). We noted that the transcrip-tion factor Runx1, which has been reported to regulate Igfbp3gene expression (41), had the most Igfbp3 promoter bindingsites predicted by text mining (Fig. 4C). Moreover, Runx1 wasnot among the top transcription factors predicted to bindto the gene promoters of the other secreted proteins in thecluster highly upregulated in SCC FAK�/� conditioned medi-um (Supplementary Fig. S1, top cluster, and SupplementaryFig. S3), suggesting that these other angiogenesis-related pro-teins may be transcriptionally regulated by a distinct mecha-nism to Igfbp3.

Using shRNA-mediated stable knockdown of Runx1 expres-sion, we next determined whether Runx1 was required forIGFBP3 expression in SCC FAK-wt and SCC FAK�/� cells.Biochemical fractionation followed by Western blotting con-firmed a substantial knockdown of Runx1 expression in bothSCC FAK-wt shRNA-Runx1 and SCC FAK�/� shRNA-Runx1 cells(Fig. 4D) when compared with controls. Analysis of Igfbp3

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expression in these cell lines using qRT-PCR showed a loss ofIgfbp3 gene expression in SCC FAK�/� shRNA-Runx1 cells,reverting expression levels down to those observed in SCC

FAK-wt vector-only control cells (Fig. 4E). Thus, Runx1 isrequired for increased Igfbp3 transcription following FAK lossin SCC cells. Using an independent method of expression

Figure 3.

IGFBP3 regulates cell-cycle progression but not tumor angiogenesis. A, Representative anti-IGFBP3 Western blot from concentrated media conditioned by eitherSCC FAK-wt, SCC FAK�/�, or SCC FAK�/� IGFBP3 shRNA cells. Anti-FAK Western blot shows FAK expression status and anti-tubulin Western blot was used as aloadingcontrol.B,Left, growthofSCCFAK-wt, SCCFAK�/�, andSCCFAK�/� IGFBP3 shRNAtumor xenografts inCD-1 nudemice; right, averagevolumeofSCCFAK�/�

and SCC FAK�/� IGFBP3 shRNA tumors at day 12. Unpaired t test, ��, P < 0.001. n¼ 6 tumors per group. C, Intravital imaging of FUCCI expressing SCC FAK-wt, SCCFAK�/�, and SCC FAK�/� IGFBP3 shRNA cells 24 hours postimplantation under dorsal skinfold windows. D, Quantitation of FUCCI cell-cycle distribution from3-dimensional imagestacksshown inC. Values shownfor SCCFAK-wtandSCCFAK�/� cells are repeated fromFig. 1D.n¼4 tumorspergroup.E,Fluorescent stainingoffrozen tissue sections using anti-CD31 antibody (red). Nuclei labeled usingDAPI (blue).F,Quantitation of the% area occupied by CD31þ cells. Tukey corrected one-wayANOVA, �� , P < 0.0001. Data in all graphs represented as mean � SEM. n ¼ 3 tumors per group with an average of three fields measured per tumor.






knockdown, similar results were observed following depletionof Runx1 using siRNAs (Supplementary Fig. S4).

FAK is in a complex with Runx1 in the nucleusAnalysis of Runx1 expression in SCC FAK-wt and FAK�/� cells

revealed that Runx1 is exclusively expressed in the nucleus and

that its expression level is not regulated by FAK (Fig. 4D). Hence,regulation of Runx1 expression/degradation is unlikely themech-anism through which FAK controls Runx1 transcriptional activityin SCC cells. Runx1 can act as either a transcriptional activator orrepressor, and the composition of the proteins interacting withRunx1 at a given gene can modulate this function (18). Using a

Figure 4.

Nuclear FAK and RUNX1 regulate IGFBP3 expression. A, Representative anti-FAK Western blot of cytoplasmic and nuclear fractions prepared from a series of SCCcells expressing FAK nuclear localization signal mutants. B, qRT-PCR analysis of Igfbp3 expression in SCC cells expressing FAK NLS mutants. Tukey corrected one-way ANOVA, �� , P < 0.0001. Data in all graphs represented as mean � SEM. n ¼ 3. C, Predicted transcription factor–binding sites in the promoter of Igfbp3.Transcription factors that interact with nuclear FAK in SCC cells are displayed in dark gray. D, Representative Western blot showing Runx1 depletionusing shRNA. E, qRT-PCR analysis of Igfbp3 expression in control and Runx1-depleted SCC FAK-wt and SCC FAK�/� cells. Sidak corrected two-way ANOVA,�� , P < 0.0001. ns, not significant. n ¼ 3.

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mass spectrometry dataset of the nuclear FAK interactome inSCC cells (17), we identified a potential FAK/Runx1 interaction.Coimmunoprecipitation of Runx1 and FAK in SCC FAK-wtbut not in SCC FAK�/� cells confirmed a novel associationbetween FAK and Runx1 (Figs. 5A and B). Thus, nuclear FAKexists in complex with Runx1 under steady-state conditions inSCC cancer cells.

A key mechanism of Runx1 regulation is posttranslationalmodification. Runx1 interacts with a number of proteins, includ-

ing kinases, histone acetyltransferases, arginine methyltrans-ferases, and histone deacetylases, that can posttranslationallymodify Runx1, switching it between transcriptional activationand repression (18, 19). To further explore how FAK influencesRunx1 function, we examined the Runx1 interaction landscape inthe context of FAK. To do this, we constructed a Runx1 proteininteraction network in silico, onto which we mapped the exper-imentally derived nuclear FAK interactome. This integrated inter-action network identified a subnetwork of Runx1 regulators and

Figure 5.

Nuclear FAK interacts with Runx1. A, Representative Western blot of anti-FAK immunoprecipitation probed with anti-Runx1 antibody. B, Representative Western blotof anti-Runx1 immunoprecipitation probed with anti-FAK antibody. Ctrl, IgG control. C, Interaction network analysis of physical or predicted direct binders ofRunx1 that interact with FAK in the nucleus of SCC cells (see Supplementary Table S2). Runx1 is shown as a square node. Protein node size is proportional to foldenrichment in nuclear FAK immunoprecipitations. Node color indicates significance of enrichment in nuclear FAK immunoprecipitations.D,Pathway enrichment analysisof KEGG terms in the nuclear FAK interactome of Runx1 binders (Q < 0.01; see Supplementary Fig. S5). E, RepresentativeWestern blot of anti-phospho-tyrosine (pTyr)immunoprecipitation probed with anti-Runx1 antibody. F, 2D gel electrophoresis probed with anti-Runx1 antibody. G, Representative Western blot of SCC FAK-wtand FAK�/� nuclear lysates probed with anti-Sin3a antibody. H,Western blot of anti-Sin3a immunoprecipitation from SCC FAK-wt and FAK�/� nuclear lysates probedwith anti-FAK antibody. IgG control (Ctrl) was done using SCC FAK-wt nuclear lysates. I, Western blot of anti-Runx1 immunoprecipitation from SCC FAK-wt,SCC FAK�/�, and SCC FAK-wt Runx1 shRNA nuclear lysates probed with anti-Sin3a antibody. IgG control (Ctrl) was done using SCC FAK-wt nuclear lysates.






associatedproteins that interactwith FAK in thenuclei of SCCcells(Fig. 5C; Supplementary Fig. S5). Interestingly, a number of theseproteins, including DNA (cytosine-5)-methyltransferase 1(Dnmt1), Sin3a, histone deacetylases 1, 2, and 3 (HDAC1, 2,and 3), and nuclear receptor corepressor 1 and 2 (NCoR1 and 2),are linked to repression of Runx1 transcriptional activity (18, 19).Therefore, nuclear FAK interactswithproteins that can regulate theposttranslational modification and transcriptional function ofRunx1. KEGG analysis also identified an enrichment for proteinswith roles in the cell cycle (Fig. 5D; Supplementary Table S1),consistent with a complex that may play a wider role in theregulation of cell-cycle progression.

Todeterminewhether FAK regulated Runx1 tyrosine phosphor-ylation, we used immunoprecipitation of tyrosine phosphorylat-ed proteins from SCCFAK-wt and FAK�/� cell lysates, followed byWestern blotting and detection with an anti-Runx1 antibody. Noregulation of Runx1 tyrosine phosphorylation was observed (Fig.5E). To assess Runx1 posttranslational modification on a widerscale, we performed 2D gel electrophoresis using nuclear extractsprepared from SCC FAK-wt and FAK�/� cells and probed thesegels with an anti-Runx1 antibody. This identified a number ofpotential differences in both the migration and intensity of spotsdetected (Fig. 5F, red arrows highlight changes), consistent withan altered state of posttranslational modification.

We next investigated whether FAK-regulated proteins associat-edwith the posttranslationalmodification of Runx1. On the basisof data presented in Fig. 5C,we focusedonSin3a, a transcriptionalcorepressor known to interact with a number of HDACs (42) andsuppress Runx1 transcriptional activity (43). Western blottingusing an anti-Sin3a antibody identified reduced Sin3a levels inthe nucleus of SCCFAK�/� cellswhen comparedwith SCCFAK-wtcells (Fig. 5g). Coimmunoprecipitation from SCC FAK-wt andFAK�/� nuclear extracts confirmed a novel association betweenSin3a and FAK in SCC FAK-wt cells (Fig. 5H). Thus, FAK is incomplex with and regulates the nuclear levels of a Sin3a. Immu-noprecipitation of Runx1 from SCC FAK-wt and FAK�/� nuclearextracts followed by Western blotting and detection with an anti-Sin3a antibody confirmed an association between Runx1 andSin3a in SCCFAK-wt cells (Fig. 5i).No interactionwas observed inSCC FAK�/� cells. Therefore, we propose that one potentialmechanism through which FAK may influence the posttransla-tional modification and transcriptional activity of Runx1 isthrough regulating the expression and subsequent recruitmentof Sin3a to Runx1 transcriptional complexes.

DiscussionNumerous studies havedemonstrated the importance of FAK in

regulating tumor cell behavior,whichhas largely been linked to itsrole in integrating signals from adhesion sites and growth factorreceptors at the cell periphery to control cell adhesion, migration,and survival (1, 2). In addition, it is now becoming clear that FAKcan also play a role within the nucleus to control gene expression(14, 17, 44). However, the mechanisms underpinning this func-tion remain to be fully characterized. A number of nuclear FAK-binding proteins have now been identified, including the tran-scription factors p53 and GATA4 (14, 16). Recently, we haveshown that nuclear FAK is associatedwith chromatin and interactswith transcription factors, including the TBP-associated factorTAF9, and transcriptional regulators reported or predicted toregulate expression of the chemokine Ccl5. In doing so, it controls

the transcription of chemokines that regulate the composition ofthe immunosuppressive tumor environment required to evadethe CD8þ T-cell antitumor immune response (17). Here, weidentify a novel interaction between nuclear FAK and the tran-scription factor Runx1. We show that nuclear FAK controls theRunx1-dependent expression of IGFBP3, which in turn regulatescell-cycle progression and SCC tumor growth in vivo. Furthermore,we identify that FAK interacts with and regulates the nuclear levelsof Sin3a and that FAK is required for recruitment of Sin3a intocomplex with Runx1. Therefore, nuclear FAK can regulate tran-scription factor activity, potentially via recruitment of proteins totranscriptional complexes that can alter transcription factor post-translational modification, thereby controlling expression of spe-cific genes that can play an important role in regulating bothtumor cell behavior, and how tumor cells influence the compo-sition of the tumor immune environment.

Runx1 is a transcription factor with an important role in bothnormal development anddisease (20, 21).Deregulationof Runx1function contributes to the development of hematologic malig-nances, and in solid epithelial cancers, it has been identified asboth a tumor promoter and a tumor suppressor (19). Runx1 caneither activate or repress transcription, depending on the com-position of the protein complexes with which it is associated at agiven gene (18), and this likely contributes to the complexity of itsrole in regulating tumorigenesis. It is known to interact with anarray of proteins, including kinases, histone acetyltransferases,arginine methyltransferases, histone deacetylases, and ubiquitinligases, all of which can posttranslationally modify Runx1 toregulate its transcriptional function (18, 19). Using mass spec-trometry, we show that FAK interacts with a number of theseproteins, includingDnmt1, Sin3a,HDACs1, 2, and3, andNCoR1and 2, all of which are known to repress Runx1 function (18, 19).Coimmunoprecipitation experiments confirmed a novel interac-tion between Sin3a and FAK, and further identified a requirementfor FAK to promote interaction of Runx1 with Sin3a. Supportingthis as a potential mechanism of FAK-dependent Runx1 regula-tion, 2D gel analysis of nuclear extracts from SCC FAK-wt andFAK�/� cells using an anti-Runx1 antibody identified severalprotein species that show differential migration, consistent withaltered posttranslational modification. Notably, posttranslation-al modification is an important mechanism in the general regu-lation of transcription factor function (45). Thus, we concludethat this may represent a previously unknown mode of FAK-dependent regulation of gene expression.

FAK is known to regulate a number of cellular processesimportant for the malignant phenotype (1, 2). Here, we identifya new role for nuclear FAK in regulation of SCC cell-cycle pro-gression in vivo via controlling Runx1-dependent expression ofIGFBP3. IGFBP3 has been linked to cell-cycle arrest in tumor cellspreviously, where a G1 arrest was accompanied by reduction in anumber of cyclins, including cyclin D1, CDKs, and increased p21expression (34, 35). IGFBP3 has also been linked to regulation ofangiogenesis (36–38). However, knockdown of IGFBP3 in theSCC cells had no effect on tumor angiogenesis. Analysis ofsecreted proteins present in SCC conditioned media identifiedanumber of changes, includingmultiple factors that can influencetumor angiogenesis, implying that other factors may be moreimportant in the regulation of angiogenesis in our SCC model.Interestingly, analysis of the predicted transcription factor–bind-ing sites in the promoters of other angiogenesis-related genesregulated by FAK identified that their mechanism of regulation is

Canel et al.





likely distinct to that of IGFBP3, highlighting the potential mech-anistic diversity underpinning FAK-dependent transcriptionalregulation.

Thedata presented here shednew light on the possiblemechan-isms underpinning nuclear FAK-dependent regulation of geneexpression, highlighting the importance of FAK as a scaffold forprotein interactions in the nucleus. Future work should focus onFAK-associated transcription factors to define how their interac-tomemay change in the absence of FAK, or its kinase activity, andwhat the biological and potential clinical relevance of thesechanges may be.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Canel, M.C. Frame, V.G. Brunton, B. Serrels,A. SerrelsDevelopment of methodology: M. Canel, A. Byron, B. Serrels, A. SerrelsAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Canel, J. Cartier, H. Patel, M.C. Frame, B. Serrels,A. Serrels

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Canel, A. Byron, A.H. Sims, H. Patel, B. Serrels,A. SerrelsWriting, review, and/or revision of the manuscript: M. Canel, A. Byron,M.C. Frame, B. Serrels, A. SerrelsAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Cartier, B. SerrelsStudy supervision: B. Serrels, A. Serrels

AcknowledgmentsWe would like to thank Arkadiusz Welman for assistance in manuscript

preparation and submission.

Grant SupportThis work was supported by Cancer Research UK program grants C157/

A11473 and C157/A15703 (awarded to M.C. Frame), and the EuropeanResearch Council Advanced Investigator grant no. 294440, Cancer Innovation(awarded to M.C. Frame). J Cartier was supported by an MRC Confidence inConcept award (MRC/CIC/11).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 15, 2017; revised June 23, 2017; accepted August 4, 2017;published OnlineFirst August 14, 2017.

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2017;77:5301-5312. Published OnlineFirst August 14, 2017.Cancer Res Marta Canel, Adam Byron, Andrew H. Sims, et al. Progression, and Tumor GrowthNuclear FAK and Runx1 Cooperate to Regulate IGFBP3, Cell-Cycle

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Nuclear FAK and Runx1 Cooperate to Regulate …...was by high-speed centrifugation (16,000 g at 4 C for 15minutes). Immunoblotting and immunoprecipitation Cell lysates or cell fractions