RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent ... · Mehlika Hazar-Rethinam1, Lilia Merida de Long1, Orla M. Gannon1, Samuel Boros2, Ana Cristina Vargas 2 , Marcin Dzienis

Cancer Biology and Signal Transduction

RacGAP1 Is a Novel Downstream Effector ofE2F7-Dependent Resistance to Doxorubicinand Is Prognostic for Overall Survival inSquamous Cell CarcinomaMehlika Hazar-Rethinam1, Lilia Merida de Long1, Orla M. Gannon1, Samuel Boros2,Ana Cristina Vargas2, Marcin Dzienis3, Pamela Mukhopadhyay1, Natalia Saenz-Ponce1,Daniel D.E. Dantzic1, Fiona Simpson1, and Nicholas A. Saunders1

Abstract

We have previously shown that E2F7 contributes to drugresistance in head and neck squamous cell carcinoma (HNSCC)cells. Considering that dysregulation of responses to chemother-apy-induced cytotoxicity is one of themajor reasons for treatmentfailure in HNSCC, identifying the downstream effectors thatregulate E2F7-dependent sensitivity to chemotherapeutic agentsmay have direct clinical impact. We used transcriptomic profilingto identify candidate pathways that contribute toE2F7-dependentresistance to doxorubicin. We thenmanipulated the expression ofthe candidate pathway using overexpression and knockdown in invitro and in vivo models of SCC to demonstrate causality. Inaddition, we examined the expression of E2F7 and RacGAP1 ina custom tissuemicroarray (TMA) generated fromHNSCCpatientsamples. Transcriptomic profiling identified RacGAP1 as a poten-tial mediator of E2F7-dependent drug resistance. We validated

E2F7-dependent upregulation of RacGAP1 in doxorubicin-insensitive SCC25 cells. Extending this, we found that selectiveupregulation of RacGAP1 induced doxorubicin resistance inpreviously sensitive KJDSV40. Similarly, stable knockdown ofRacGAP1 in insensitive SCC25 cells induced sensitivity todoxorubicin in vitro and in vivo. RacGAP1 expression wasvalidated in a TMA, and we showed that HNSCCs that over-express RacGAP1 are associated with a poorer patient overallsurvival. Furthermore, E2F7-induced doxorubicin resistancewas mediated via RacGAP1-dependent activation of AKT. Final-ly, we show that SCC cells deficient in RacGAP1 grow slowerand are sensitized to the cytotoxic actions of doxorubicin invivo. These findings identify RacGAP1 overexpression as a novelprognostic marker of survival and a potential target to sensitizeSCC to doxorubicin. Mol Cancer Ther; 14(8); 1939–50. �2015 AACR.

IntroductionCutaneous squamous cell carcinomas (CSCC) and head and

neck SCC (HNSCC) are among the most common malignanciesafflictingman (1, 2). Current treatment options for advanced SCCinclude adjuvant chemotherapy with platinum-based drugs, suchas taxanes, 5-Fluorouracil, or therapeutic antibodies against EGFR(3, 4). However, the response is generally transient and charac-

terizedby thedevelopment of drug resistance. Thus, there is aneedto identify new therapeutic strategies that can bypass the emer-gence of a drug-resistant phenotype.

The E2F transcription factor complex comprises a family ofactivating (E2F1, 2, 3a) or repressive/inhibitory (E2F3b, 4, 5, 6, 7,or 8) E2Fs that regulate key cellular functions, such as transcrip-tion, differentiation, and apoptosis. In the context of keratino-cytes (KC), the E2F transcription factor family has been shown tocontrol (i) proliferation, (ii) differentiation, (iii) stress responses,and (iv) apoptosis (5–10). Consistent with their roles in KCs,dysregulationofE2F is a commonoccurrence in SCC(11, 12), andoverexpression of E2Fs, such as E2F1 and E2F7, occurs in themajority of CSCCs and HNSCCs (12–15). E2F1 and E2F7 areknown to have opposing actions in the regulation of proliferation(5), differentiation (14), and apoptosis (9, 14). For example,recent reports have shown that treatment of wild-type cells withDNA-damaging agents, such as doxorubicin or etoposide, inducesE2F7 protein levels and subsequent inhibition of the E2F1-medi-ated DNA damage response (9, 10). In the context of KCs, E2F7was shown to causally modify responses to conventional che-motherapeutics (15) and UV responses in vitro (14). Thus, sen-sitivity to common cytotoxic agents and stimuli appear to beregulated by the relative ratio of E2F1 to E2F7 in the tissue. Giventhat both E2F1 and E2F7 are known to be overexpressed in SCC(14, 15), it is reasonable to speculate that this may also

1Epithelial Pathobiology Group, University of Queensland DiamantinaInstitute, PrincessAlexandraHospital,Translational Research Institute,Woolloongabba, Queensland, Australia. 2Department of Pathology,Princess Alexandra Hospital, Woolloongabba, Queensland, Australia.3DepartmentofMedicalOncology,PrincessAlexandraHospital,Wool-loongabba, Queensland, Australia.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Current address for P. Mukhopadhyay: The QIMR Berghofer Medical ResearchInstitute, Brisbane, Queensland, Australia.

Corresponding Author: Nicholas A. Saunders, The University of QueenslandDiamantina Institute, Translational Research Institute, 37 Kent St, Woolloon-gabba,Queensland4102, Australia. Phone: 61-7-34437098; Fax: 61-7-34436966;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-15-0076

�2015 American Association for Cancer Research.

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contribute to drug resistance in SCC. In this regard, we recentlyshowed that the sphingosine kinase 1 (Sphk1) gene is a directtarget of E2F7 in SCC (15). E2F7-dependent overexpression ofSphk1 in SCC induces increased production of the antiapop-totic phospholipid, sphingosine-1-phosphate (S1P), which inturn invokes anthracycline resistance via activation of the PI3K/AKT pathway (15). Thus, E2F dysregulation in SCC induced theactivation of a Sphk1/S1P-dependent drug-resistant phenotype(15). Identification of this novel pathway was noteworthybecause anthracyclines such as doxorubicin are not in clinicaluse for the treatment of SCC, and thus the ability to sensitizeSCCs to an existing class of chemotherapeutics would be ofclinical value. However, the activation of the Sphk1 pathwaywas clearly only part of the explanation for the anthracyclineresistance observed in SCC. Thus, other pathways that controldrug resistance in SCC were likely to exist.

In the present study, we used transcriptomic profiling to iden-tify a novel druggable E2F7/RacGAP1/AKT pathway that selec-tively induces anthracycline resistance in SCC.

Materials and MethodsChemicals and viability assays

The following drugs were purchased: AZA1 (Millipore), doxo-rubicin (Sigma Aldrich), NSC23766 (Abcam), S1P (CaymanChemicals), Y-27632 (Sigma Aldrich), and ZVAD-fmk (AlexisBiochemicals). BGT26 was provided by Novartis, and stocks ofBGT226 were prepared as described (16). ZVAD-fmk was added30 minutes before other treatments. Cell viability was performedby trypan blue exclusion or using Cell Titer 96 Aqueous OneSolution Cell Proliferation Assay (Promega).

Tissue culture, adenovirus infection, and transfectionMurine epidermal keratinocytes (MEK) and human epider-

mal keratinocytes (HEK) were isolated and cultured asdescribed (17, 18). E2F7 KO KCs were generated by ready-to-use adenovirus harboring Cre recombinase infection ofmurine epidermal keratinocytes as per the manufacturer'srecommendations (MOI of 50; Vector Biolabs). Detroit562and SCC25 cells were obtained from the American Type CultureCollection, and cultures were not passaged for longer than 6months. SCC15 was a kind gift from Dr. Elizabeth Musgrove(Garvan Institute, New South Wales, Australia) and was verifiedby short tandem repeat (STR) genotyping (12). KJDSV40 cellswere maintained as described previously (12) and were verifiedby STR genotyping. STR-verified cells were not passaged forlonger than 6 months after verification. All cell lines used wereregularly tested and validated to be Mycoplasma free. Controland overexpression plasmids used for manipulating E2F7 andthe siRNA used for targeting E2F7 have been described previ-ously (9, 14). SureSilencing shRNA plasmids directed againstRacGAP1 or Sphk1 were purchased from SuperArray BioscienceCorp (SA Biosciences). A RacGAP1 expression (TrueORF GoldClones) and control plasmids were purchased from OriGeneTechnologies (Australian Biosearch).

RNA isolation and quantitative RT-PCRTotal RNAwas isolated, cDNAwas prepared, and qRT-PCRwas

performed as described (15). Primer sequences were E2F7 For-ward: GTCAGCCCTCACTAAACCTAAG, E2F7 Reverse: TGCGTT-GGATGCTCTTGG; RacGAP1 Forward: GACGTTGAATAGGATG-

AGTCATGG, RacGAP1 Reverse: GCTCAAACAGATTCCGCACA;Sphk1 Forward: AAGACCTCCTGACCAACTGC, Sphk1 Reverse:GGCTGAGCACAGAGAAGAGG.

Gene expression analysisEach sample was analyzed in duplicate. Complementary RNA

was generated from samples using the Illumina TotalPrep RNAAmplification Kit and hybridized with Illumina HumanHT-12 v4Expression BeadChips (Illumina) as the per manufacturer's pro-tocol. Expression data from the microarrays were analyzed aspreviously described (19). Only genes with a fold change of 1 (ineither direction) or greater and a B value of greater than 3(exceeding the 95% probability of differential expression) wereconsidered to be differentially expressed and further analyzed.Differentially expressed probe sets were analyzed as pair-wisecontrasts. Microarray data have been uploaded to Gene Expres-sion Omnibus under the reference: GSE58074.

Colony-forming assayKnownnumber of SCC cells was plated and allowed to grow for

15 days. Plates were fixed and stained with Coomassie Blue andcounted as previously described (20). Colony-forming efficiencywas expressed as the total number of colonies/total number ofcells plated � 100.

DNA synthesisDNA synthesis was measured using a colorimetric ELISA 5-

bromo-2-deoxyuridine (BrdUrd) incorporation assay (RocheDiagnostics) in accordance with the manufacturer's instructions.

Chromatin immunoprecipitationChromatin immunoprecipitation (ChIP) was performed using

the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads;Cell Signaling Technology) in accordancewith themanufacturer'sinstructions. ChIP enrichment was determined by conductingqRT-PCR as described above. The primers used were as follows:50-GAAGTGAGTAGTGGGGGTGC-30 (RacGAP1 Forward); 50-TCCATCTTTCACACGAACACTCT-30 (RacGAP1 Reverse).

ImmunoblotImmunoblotting was carried out according to previously pub-

lished procedures (21) using the following primary antibodies:Anti-RacGAP1 (EPR9018; 1:2,000; Abcam), Anti-Sphk1 (1:1,000;Sigma Aldrich), PARP (1:1,000; Cell Signaling Technology),phospho-Akt (Ser473; D9E; XP; 1:2,000; Cell Signaling Technol-ogy), Akt (1:2,000; Cell Signaling Technology), phospho-p44/42MAPK (Erk1/2; Thr202/Tyr204; E10; 1:2,000; Cell SignalingTechnology), ERK 1 (C-16; 1:2,000; Santa Cruz), and b-actin(1:10,000; Sigma Aldrich).

ImmunohistochemistryImmunohistochemistry was carried out according to previous-

ly published procedures (21, 22) using the following primaryantibodies: Anti-PCNA (1:3,000; Sigma Aldrich), Anti-RacGAP1(EPR9018; 1:100; Abcam), cleaved caspase-3 (Asp 175; 1:50; CellSignaling Technology), and phospho-Akt (Ser473; D9E; XP; 1:50;Cell Signaling Technology). Secondary antibody was Starr TrekUniversal HRP Detection System (Biocare Medical) followed bycolorimetric immunohistochemical staining with CardassianDAB Chromogen as per the manufacturer's instructions (BiocareMedical).

Hazar-Rethinam et al.

Mol Cancer Ther; 14(8) August 2015 Molecular Cancer Therapeutics1940




Tissue microarraysGeneration and composition of the patient tissue microarrays

(TMA) have been previously described (15). Immunohistochem-istry was conducted using a Dako EnVisionþ System-HRP (DAB)kit in accordance with the manufacturer's instructions (DAKO).Sectionswere incubatedwithAnti-E2F7 (1:250; Abcam) andAnti-RacGAP1 (EPR9018; 1:100; Abcam) antibodies. Staining inten-sitywas evaluated by twopathologists in a blinded fashionusing amodified quickscore method as described (23).

Determination of RhoA and Rac1 activityRhoA and Rac1 activities were measured with RhoA/Rac1/

Cdc42 activation assay combo biochem kit (Cytoskeleton) inaccordance with the manufacturer's instructions.

Animal studiesAll animal experiments were approved by the Institutional

Animal Ethics Committee. In vivo tumor studies used 6-week-oldfemale nonobese diabetic/severe combined immunodeficientmice. Mice were injected subcutaneously on the flank with 2 �106 cells. Groups of 6 mice received treatments (intraperitonealinjections twice/week) when tumors were around 4 mm indiameter. Animal weight and tumor growth were monitoredfor a period of up to 3 weeks, and animals were sacrificed whentumors reached 10 mm in diameter.

Statistical analysisStatistical significance was calculated by a Student t test with a

95% confidence level using GraphPad Prism v5 (GraphPadsoftware).

ResultsRacGAP1 is a novel downstream effector of E2F7

We generated E2f7 knockdown (KD) murine KCs via adeno-virus-mediated Cre deletion of floxed sequences in primaryKCs isolated from E2f7-floxed mice (15). KD KCs were treatedfor 48 hours with increasing concentrations of doxorubicin(0–1 mmol/L), etoposide (0–100 mmol/L), and cisplatin (0–20mmol/L). Figures 1A–C show that E2F7 deficiency sensitizes KCsto doxorubicin, modestly to cisplatin, but not at all to etoposide.These data suggest that E2F7-mediated doxorubicin resistance isnot attributable to topoisomerase inhibition because etoposidesensitivity was not modified by E2F7. Moreover, pan-caspaseinhibition significantly protected E2F7-deficient cells from doxo-rubicin-induced cytotoxicity (Fig. 1D), indicating that apoptoticpathways are being activated.

We undertook a screen of doxorubicin sensitivity inHEKs and 4SCC cell lines. The KJDSV40 cell line exhibited the highest sen-sitivity to doxorubicin (IC50 of 0.082 mmol/L; Fig. 1E), whereasSCC25 cells displayed the least sensitivity (IC50 of 0.55 mmol/L;Fig. 1E) and HEKs displayed intermediate sensitivity (IC50 of0.29 mmol/L; Fig. 1E). We have previously shown that the insen-sitive SCC25 cell line expresses high levels of E2F7, whereas thesensitiveKJDSV40cell line expresses low levelsof E2F7(15). Basedon these data, we selected the sensitive KJDSV40 cell line and theinsensitive SCC25 cell line for transcriptomic profiling.

Specifically, we generated a list of genes that were poorlyexpressed in KJDSV40 cells and highly expressed in SCC25(Fig. 2A). We also generated a second list of genes that weredifferentially regulated in SCC25 cells in which E2F7 had been

silenced with siRNA (Fig. 2A). We then used these two lists toidentify those transcripts (referred to as List A; SupplementaryTable S1) that displayed E2F7-dependent expression between theSCC25 cell lines (Fig. 2A). We also generated an additional list oftranscripts for SCC25 cells or SCC25 cells in which E2F7 issilenced by siRNAs that have been treated with 1 mmol/L ofdoxorubicin. The transcripts that were found to be E2F7-depen-dant in the context of doxorubicin-treated SCC25 cells were thenreferred to as List B (Fig. 2A; Supplementary Table S1). Bycombining Lists A and B, we identified RacGAP1 as the mostdifferentially overexpressed genes with a B value of 17 (Supple-mentary Table S1).

RacGAP1 (also known as MgcRacGAP and CYK4) is an evolu-tionarily conservedGTPase activating protein (GAP) that displaysactivity toward the Rho family of GTPases. The Rho family ofGTPases is a subfamily of the Ras superfamily and consists ofsmall signaling G proteins: Rho (A, B, and C isoforms), Rac (1,2,3isoforms andRhoG), andCdc42 (Cdc42, Tc10, TCL, Chp/Wrch-2,and Wrch-1; ref. 24), which function as molecular switchesbetween a GTP-loaded "ON" and a GDP-loaded "OFF" state(24). Thus, RacGAP1 has the potential to regulate a diverse arrayof cellular functions through its central role as a regulator of theactivation state of the Rho family of GTPases. In particular,RacGAP1 is known to play important roles in the completion ofcytokinesis (25), cell transformation, motility, migration, andmetastasis (26–29). RacGAP1 is also involved in IL6-inducedmacrophage differentiation (30) and nuclear transport ofSTAT3/5 transcription factors (31). However, a role for RacGAP1in SCC or doxorubicin sensitivity has not been shown previously.

Quantitative RT-PCR andWestern blottingwere used to confirmthat RacGAP1 was more highly expressed in SCC25 (doxorubicininsensitive) cells than in KJDSV40 (doxorubicin sensitive) cells(Fig. 2B and C). Similarly, we showed that knockdown of E2F7 bysiRNA in SCC25 cells caused a reduction in RacGAP1 mRNA (Fig.2D) andprotein level (Fig. 2E).Conversely, overexpressionofE2F7in KJDSV40 cells resulted in elevated levels of RacGAP1 transcript(Fig. 2F) as well as RacGAP1 protein (Fig. 2G). These data suggestthat RacGAP1 is a downstream target of E2F7 in SCC cells.Supporting this, ChIP analysis of E2F7 binding showed thatE2F7 could bind the RacGAP1 promoter, suggesting that RacGAP1is a direct transcriptional target of E2F7 (Fig. 2H). This is the firstreport to show that RacGAP1 is a downstream effector of E2F7.

RacGAP1expression is elevated in SCCsWe examined RacGAP1 expression levels by immunohis-

tochemistry using a TMA consisting of 35 paired normal, primarytumor, and matched metastasis from HNSCC patients treated atthePrincess AlexandraHospital (PAH). The TMAswere stained forE2F7 and RacGAP1 protein expression and scored blinded by twopathologists. All matched adjacent "normal" epithelia demon-strated either negative or weak staining for RacGAP1, which waspredominantly nuclear in location (Fig. 3A). Conversely, mod-erate to high levels of RacGAP1 expression were consistentlyrecorded for the primary tumor (Fig. 3B) and its matched lymphnode metastasis (Fig. 3C). The tumor epithelial cells showednuclear and cytosolic expression for RacGAP1. RacGAP1 wassignificantly overexpressed in 73% of primary and metastatichuman SCCs compared with matched adjacent normal tissue(P < 0.0001; Fig. 3D). In addition, our analyses showed that E2F7expression is significantly upregulated in HNSCC compared withmatched adjacent normal tissue (P < 0.0001; Fig. 3D). The

RacGAP1 Is Overexpressed in Drug-Resistant SCC

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Kaplan–Meier analysis revealed an inverse relationship betweenRacGAP1 expression levels and progression-free survival (PFS) ofHNSCC patients studied over a period of 42 months whosesamples were arrayed on the TMA (Fig. 3E). These data show,for the first time, that RacGAP1 is overexpressed in HNSCC and isassociated with a poorer PFS.

RacGAP1 expression/activity determines sensitivity todoxorubicin

We examined the effect of shRNA-mediated knockdown orRacGAP1 overexpression on sensitivity to doxorubicin. RacGAP1gene silencing was achieved using 4 different constructs of whichshRNA.3 displayed the greatest knockdown in RacGAP1 proteinlevel (Fig. 4A). For subsequent experiments, the shRNA complexshRNA.3 was employed. Consistent with previous reports (32),RacGAP1 shRNA-transfected SCC25 cells displayed significantreductions in proliferation (Fig. 4B), colony-forming efficiencyin vitro (Fig. 4C), and induced amodest increase in cleaved PARP1(Fig. 4D) compared with control vector–transfected cells. Finally,silencing RacGAP1 significantly enhanced the sensitivity ofSCC25 cells to doxorubicin (Fig. 4E). Conversely, overexpression

of RacGAP1 in sensitive KJDSV40 cells resulted in increases inRacGAP1 protein level (Fig. 4F), and reduced sensitivity to doxo-rubicin compared with vector control (Fig. 4G). Combined, thesedata indicate that RacGAP1 can promote proliferation and inhibitdoxorubicin-induced cell death in SCCs.

RacGAP1 differentially regulates the GTP-loaded state of RhoAand Rac1 in SCC cells

We examined whether the overexpression of RacGAP1 in theSCC cell lines was reflected in alterations of the GTP loading(activation status) of the model targets RhoA and Rac1. Specif-ically, RhoA GTP loading was constitutively higher in KJDSV40cells, which express very low levels of E2F7 and RacGAP1, com-pared with SCC25 cells which express high levels of E2F7 andRacGAP1 (Fig. 5A). In contrast, GTP loading of Rac1was higher inSCC25 cells when comparedwithKJDSV40 cells (Fig. 5B), and theGTP loading of Rac1 was significantly reduced in SCC25 cellsfollowing RacGAP1 knockdown (Fig. 5B). Finally, knockdown ofRacGAP1 in SCC25 cells resulted in an increase in theGTP loadingof RhoA (Fig. 5A). These data suggest that E2F/RacGAP1-depen-dent resistance to doxorubicinmay bemediated by activated Rac1

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Figure 1.Cytotoxic responses to doxorubicinin E2F7-deficient murine KCs. E2F7-floxed KCs were incubated with(squares) or without (circles) Ad-Cre-GFP for 48 hours and then incubatedwith varying doses of doxorubicin (A),etoposide (B), or cisplatin (C).Viability (absorbance 490 nm) wasassessed 48 hours after treatmentand is expressed as the mean � SEMobtained from triplicatedeterminations of three independentexperiments. D, Ad-Cre-GFP–uninfected E2F7-floxed or Ad-Cre-GFP–infected E2F7-deficientproliferative KCs were treated with 1mmol/L doxorubicin in the presence orabsence of ZVAD-fmk and viabilitydetermined 48 hours later. Viabilityis plotted as a percentage ofdoxorubicin-treated uninfected E2F7-floxed MEKs and represents the mean� SEM obtained from triplicatedeterminations of three independentexperiments. E, HEK, Detroit562,KJDSV40, SCC15, and SCC25 cellswere treated with doxorubicin for48 hours and viability plotted aspercentage of untreated cells. Inset,estimated IC50 values for doxorubicinin HEK, KJDSV40, and SCC25 cellsdetermined by nonlinear regressionanalysis in Prism. ns, not significant;� , P < 0.05; �� , P < 0.01; ��, P < 0.001;�� , P < 0.0001.






or inactivated RhoA. To determine which, if any, of these possi-bilities may apply, we coincubated SCC25 cells with doxorubicinþ/– an established selective Rho/ROCK1 inhibitor (10 mmol/L Y-27632; ref. 33), an established Rac1/Cdc42 inhibitor (10 mmol/LAZA1; ref. 34), or a Rac1-selective inhibitor (25 mmol/LNSC23766; ref. 35) for 48 hours and established viability (Fig.5C). These experiments showed that doxorubicin resistance wasunaltered following incubation with a Rho/ROCK1 inhibitor andwas moderately reduced following incubation with the Rac1/cdc42 inhibitor (Fig. 5C). In contrast, treatment of SCC25 cellswith the Rac1-selective inhibitor significantly increased sensitivityof SCC25 cells to doxorubicin (Fig. 5C). These results indicate anumber of important points. Firstly, RhoA appears to be a

preferred substrate for RacGAP1 in SCC cells. This is reflected bythe high level of RhoA-GTP loading compared with Rac1-GTPloading as well as the increase in GTP loading observed followingRacGAP1 knockdown in the SCC25 cells. Secondly, althoughRac1-GTP loading behavior is not indicative of it being a preferredsubstrate of RacGAP1, it is clear that alterations in RacGAP1activity modify Rac1-GTP loading. Thirdly, use of a Rac1-selectiveinhibitor phenocopies the inhibition of doxorubicin resistanceobserved with RacGAP1 knockdown (Fig. 4E). Finally, the pref-erence for RhoA by RacGAP1, in SCC cells, is consistent with aprevious report showing that the conventional preference for Rac1can be switched to RhoA following phosphorylation of the Serine387 site of RacGAP1 by Aurora B kinase (36, 37).

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Figure 2.RacGAP1 is a downstream effector ofE2F7. A, summary of the strategy usedto identify E2F7-dependenttranscripts that associate withdoxorubicin resistance in SCC cells. Band C, RacGAP1 mRNA and proteinlevels were determined in KJDSV40and SCC25 cells by qRT-PCR andimmunoblotting, respectively. D andE, the expression of RacGAP1transcripts and protein wasdetermined by qRT-PCR andimmunoblotting, respectively, ofextracts derived from SCC25 andSCC25 cells in which E2F7 wassilenced with siRNA for 48 hours.F and G, RNA was extracted fromKJDSV40 and KJDSV40 cells in whichE2F7 was overexpressed from anexpression plasmid. The expression ofRacGAP1 transcripts and protein wasdetermined 48 hours aftertransfection by qRT-PCR andimmunoblotting, respectively. H, toquantitate E2F7 binding to theRacGAP1 promoter, ChIPs wereperformed using an E2F7 antibody ornonimmune IgG as control in HEK,KJDSV40, and SCC25 cells. Each ChIPand qRT-PCR was repeated 2 or 3times, respectively. Data, mean� SEMof duplicate determinants normalizedfor expression of the housekeepinggene TBP for B, D, and F; n¼ 3. b-Actinis provided as a loading control for C,E, and G. Western blot figure isrepresentative of three independentexperiments. �� , P < 0.01;�� , P < 0.0001.






RacGAP1 modulates doxorubicin sensitivity via downstreamactivation of the PI3K/AKT pathway

There is an existing literature showing that the PI3K/AKTpathway is an important component of RacGAP1 signaling(38). However, whether PI3K/AKT signaling lies upstream ordownstream of the Rho family of GTPases remains less clear andappears to be context-specific (38). Dysregulation of the PI3K/AKT pathway is a common event in HNSCC, which can beattributed to multiple factors, such as mutations, amplifications,and signal-induced activation of the pathway (39). For example,we recently showed that E2F7 overexpression or knockdowncaused an increase and decrease in p-AKT levels in SCC cellsrespectively (15). Since RacGAP1 is a downstreameffector of E2F7in SCC cells, we examined whether RacGAP1 could modify thePI3K/AKT signaling pathway in SCC cells. In the first instance, wenoted that knockdown of RacGAP1 in SCC25 cells had no impacton the activation status of the ERK pathway (Fig. 5D). In contrast,RacGAP1 knockdown in SCC25 cells significantly reduced thelevel of p-AKT (Fig. 5E), whereas RacGAP1 overexpression inKJDSV40 cells increased p-AKT levels (Fig. 5F).We had previouslyshown that the PI3K/mTOR inhibitor, BGT226, was able to ablate

AKT signaling and induce apoptosis in SCC cell lines (16). Wecompared the sensitivity of SCC25 cells with BGT226 in SCC25cells or SCC25 cells in which RacGAP1 was knocked down. Figure5G indicates that knockdown of RacGAP1 is able to reduce SCC25cell viability to 70% that of control cells. Similarly, inhibition ofPI3K activity using a dose of BGT226 known to induce maximalinhibition (16) reduced SCC25 cell viability to approximately50% (Fig. 5G). Finally, exposure of RacGAP1-deficient SCC25cells to BGT226 resulted in a further decrease in viability to below20% (Fig. 5G). These data indicate that RacGAP1 participates inAKT-dependent and AKT-independent events.

We recently reported that E2F7 is able to directly activate theSphk1/S1P axis in SCC cells, which induces doxorubicin resis-tance (15). It is also interesting to note that both the E2F7/RacGAP1 pathway identified in this study and the E2F7/Sphk1/S1P pathway (15) induced doxorubicin resistance and convergedon the AKT pathway. Therefore, we examined whether the Sphk1and RacGAP1 pathwaysmay interact with one another. Figure 5Hshows that knockdown of Sphk1 can induce loss of RacGAP1mRNA, whereas knockdown of RacGAP1 induces loss of Sphk1mRNA expression. Although the mechanism controlling this

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Figure 3.Expression of RacGAP1 in HNSCCand adjacent normal tissue andits association with PFS.Representative images of adjacentnormal tissue (A) and HNSCCspecimens stained for RacGAP1 (Band C). D, quantitation of E2F7 andRacGAP1 staining intensities inmatched samples of primary tumor,normal squamous epithelium, andlymph node metastases (n ¼ 35).Tissue sections were scored using amodified quickscore method todetermine the percentage of cellsstained (0%–100%) and the intensityof staining (1þ to 3þ). E, Kaplan–Meier analysis of PFS stratified byRacGAP1 expression in the HNSCCpatient cohort. ns, not significant,��, P < 0.0001.






feedback is unknown, it is clear that targeted inhibition of eitherthe RacGAP1 pathway or the Sphk1 pathway is likely to impactone another. To illustrate this point, knockdown of RacGAP1 orSphk1 in SCC25 cells results in reduced p-AKT levels (Fig. 5I) andincreased sensitivity to doxorubicin (Fig. 5J), which can bereversed by the addition of exogenous S1P (Fig. 5I and J).

RacGAP1 suppression enhances sensitivity of SCC25 todoxorubicin in vivo

SCC25 cells were generated to stably express either vectorcontrol or RacGAP1 shRNAand inoculated intoNOD/SCIDmice.

When tumors were around 4 mm in diameter, mice were ran-domized into four groups and treated with vehicle, dimethylsulfoxide (DMSO), or 0.5 mg/kg doxorubicin by i.p. injectionstwice per week. RacGAP1 knockdown was confirmed by Westernblotting immediately before the inoculation of SCC25 cells(Fig. 6A). Treatment with doxorubicin was well tolerated by theNOD/SCIDmice, and the bodyweights remained stable through-out the study (Fig. 6A). RacGAP1-deficient cells showed reducedtumor growth rate (Fig. 6B). Treatment of mice bearing vectorcontrol SCC25 tumors with/without 0.5 mg/kg doxorubicinhad minimal effect on tumor growth rates (Fig. 6B). However,

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Figure 4.The sensitivity to doxorubicin ismediated via an E2F7/RacGAP1 axis inSCC. A, SCC25 cells were transfectedwith 4 different constructs coding forshRNAs directed against RacGAP1.After 48 hours, RacGAP1 proteinexpression was determined byimmunoblotting. b-Actin is providedas a loading control. SCC25 cellswere transfected with theRacGAP1shRNA.3 or a scrambledshRNA construct. After 48 hours,BrdUrd incorporation (B) and CFE (C)were determined. Data are expressedas a percentage of that observed forcontrol shRNA. D, cleavage of PARPwas determined by immunoblottingextracts of RacGAP1shRNA andcontrol shRNA–transfected SCC25cells. E, RacGAP1shRNA and controlshRNA–transfected SCC25 cells weretreated with doxorubicin (1 mmol/L)for 48 hours and viability estimated bytrypan blue exclusion. Viability isplotted as percentage control(untreated). F, KJDSV40 cells weretransfected with RacGAP1 expressionplasmid or a noncoding empty vector.After 48 hours, RacGAP1 proteinexpression was determined byimmunoblotting. b-Actin is a loadingcontrol. G, RacGAP1 expressionplasmid or empty vector–transfectedKJDSV40 cells were treated withdoxorubicin (1 mmol/L) for 48 hoursand viability estimated by trypanblue exclusion. Viability wasexpressed as percentage control(untreated). Western blot figures arerepresentative of three independentexperiments. All quantitative datapresented as mean � SEM. Forviability, values represent triplicatedeterminations of three independentexperiments; for CFE, expressionvalues represent duplicatedeterminations from at least threeindependent experiments; for BrdUrd,values represent triplicatedeterminations from 4 independentexperiments. �� , P < 0.01;�� , P < 0.001; �� , P < 0.0001.






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Figure 5.Alterations in RacGAP1 activity modify Rac1-GTP loading, and RacGAP1 lies upstream of AKT. Rhotekin-binding domain (RBD; A) binding and p21 activatedkinase I-binding domain (PAK-PBD; B) assays were performed on the extracts from KJDSV40, SCC25, and SCC25 cells in which RacGAP1 had beensilenced by shRNA, as indicated. The amount of activated or total RhoA and Rac1 was detected by immunoblotting the RBD and PAK-PBD samplesand the whole cell lysate with RhoA or Rac1 antibodies. To confirm equal input, the membrane was reprobed with an actin antibody. C, SCC cells were treatedwith increasing doses of doxorubicin in combination with 10 mmol/L AZA1 or 10 mmol/L NSC23766 or 25 mmol/L NSC23766 for 48 hours. Viabilitywas then assessed and plotted as percentage doxorubicin only treated. SCC25 cells were transfected with the RacGAP1shRNA or a scrambled shRNAconstruct. D, after 48 hours p-ERK and total ERK protein levels and (E) p-AKT and total AKT protein levels were determined by immunblotting. F, p-AKTand total AKT protein levels are shown for KJDSV40 cells in which RacGAP1 was overexpressed. G, RacGAP1shRNA and control shRNA–transfectedSCC25 cells were treated with BGT226 (300 nmol/L) for 48 hours and viability estimated by trypan blue exclusion and plotted as percentage control shRNA.H, SCC25 cells were transfected with Sphk1shRNA (left) and RacGAP1shRNA (right) as well as control shRNA. After 48 hours, RacGAP1 (left) andSphk1 mRNA (right) levels were determined by qRT-PCR. I, SCC25 cells were transfected with RacGAP1 or Sphk1 shRNAs. Forty-eight hours aftertransfection, cells were treated with 1 mmol/L S1P for 24 hours. p-AKT and total AKT protein levels were then determined by immunoblotting. J, SCC25 andSCC25 in which RacGAP1 or Sphk1 had been silenced were treated with 1 mmol/L doxorubicin and 1 mmol/L S1P for 24 hours. Viability was thenassessed and plotted as percentage doxorubicin-only treated. Western blot figures are representative of three independent experiments. All quantitative datapresented as mean � SEM obtained from triplicate determinations of three independent experiments for C, G, and J. Data are the mean � SEM ofduplicate determinants normalized for expression of the housekeeping gene TBP for H; n ¼ 3. �� , P < 0.01; �� , P < 0.001; ��, P < 0.0001.






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Figure 6.RacGAP1 suppression enhanced sensitivityof SCC25 to the cytotoxic actions ofdoxorubicin in vivo. A, on the day ofsubcutaneous injections, RacGAP1deficiency was confirmed byimmunoblotting using protein extractsfrom SCC25 cells in which RacGAP1 hadstably silenced with shRNA. b-Actin isprovided as a loading control. All animalswere inoculated subcutaneously with2� 106 SCC25 cells expressing vector alone(scrambled shRNA) or Sphk1 shRNA andtumors allowed to establish until theyreached the indicated sizes. Establishedtumors were treated with vehicle or 0.5mg/kg doxorubicin twice per week. A,animal weight was determined twice perweek. B, tumor volumes were monitoredtwice weekly. The inset includes vehicle or0.5 mg/kg doxorubicin-treated SCCtumors harboring RacGAP1shRNA. C, after13 days of treatment, animals weresacrificed and tumors excised.Representative results fromdistinct groupsare shown. þ, tumors formed from controlshRNA–transfected SCC25 cells; �, tumorsformed from RacGAP1shRNA–transfectedSCC25 cells. D, immunostaining forRacGAP1, PCNA, cleaved caspase-3, andp-AKT or normal Rabbit IgG as a negativecontrol. Representative images of at leastthree independent tumors are shown foreach group (Bar, 100 mm). Data presentedas mean � SEM of individualmeasurements from 6 mice per group.E, schematic model showing howdisruption of the novel E2F/RacGAP1/Rac1/AKT pathway can lead to doxorubicinresistance in SCC.






RacGAP1-deficient SCC25 tumors treated with doxorubicinstarted to regress by day 4 after treatment, which continued fora further 7 days at which time all mice were sacrificed due to thetumor burden in control mice. The subcutaneous tumors wereexcised, photographed, and examined histologically (Fig. 6C).

Immunohistochemical examination of the excised tumorsshowed that knockdownof RacGAP1wasmaintained throughoutthe study (Fig. 6D). Tumors from vehicle and doxorubicin-treatedcontrol mice stained strongly for PCNA, indicating a higherproportion of proliferating cells in control tumors compared withRacGAP1-deficient tumors (treated or untreated; Fig. 6D). Incontrast, doxorubicin induced higher apoptosis indices in tumorsderived from SCC25/RacGAP1shRNA than in the SCC25/vectorcontrol as estimated by immunostaining for cleaved caspase-3(Fig. 6D). Ser473 p-AKT levels in RacGAP1-deficient cells werealso decreased (Fig. 6D). Collectively, these results suggest thatRacGAP1 contributes to the growth of HNSCC in vivo and thattargeted inhibition of RacGAP1-overexpressing tumors may sen-sitize them to the cytotoxic actions of doxorubicin.

DiscussionThis is the first study to identify an E2F7/RacGAP1/AKT axis

through which SCC cells acquire resistance to doxorubicin(Fig. 6E). Specifically, we show that (i) RacGAP1 is a noveldownstream effector of E2F7, (ii) RacGAP1 is overexpressed inpatient SCC and is associated with poor PFS, (iii) RacGAP1overexpression is associated with inactivation of RhoA and acti-vation of Rac1, (iv) pharmacologic inhibition of Rac1, but notRhoA, reinstates doxorubicin sensitivity similar to that observedfollowing RacGAP1 knockdown in SCC cells, and (v) E2F7-dependent doxorubicin resistance is mediated via induction ofRacGAP1/Rac1 and Sphk1, which in turn activates AKT-depen-dent and AKT-independent pathways in vitro and in vivo.

The E2F transcription factor family is involved in a diverse arrayof cellular functions that are controlled by the relative ratio ofatypical E2F (e.g., E2F7) to activating E2F (e.g., E2F1). Forexample, the apoptotic actions of E2F1, in SCC cells, can beantagonized by E2F7 overexpression (14). Similarly, E2F7 inhi-bits doxorubicin-induced cytotoxicity by inducing the expressionof Sphk1 resulting in increased levels of S1P which enhance theSer473 p-AKT–dependent prosurvival response (15). These dataare of particular relevance because we know that the majority ofhuman SCCs express high levels of both E2F1 and E2F7. In thepresent study, we found that high levels of RacGAP1 in advancedSCC patients were associated with a poor PFS. Moreover, wedemonstrated that forced overexpression of E2F7 was able toinduce RacGAP1 overexpression and doxorubicin resistance,whereas knockdown of E2F7 reduced RacGAP1 expression andinduced sensitivity to doxorubicin in vitro and in vivo. These dataindicate that RacGAP1 is a direct downstream transcriptionaltarget of E2F7. TheRacGAP1promoter contains E2F-binding sites,and E2F activation has been reported to be required for theinitiation of transcription at the RacGAP1 promoter in humanlymphocyte cell line (40). Consistent with this, we showed thatelevated E2F7 levels in SCC are associated with increased bindingof E2F7 to the RacGAP1 promoter and increased expression ofRacGAP1 in SCCs. E2F7 is traditionally considered to be atranscriptional repressor; however, it has also been shown thatE2F7 can function as a direct transcriptional activator of theVEGFA promoter via the formation of an E2F7–HIF1a transcrip-

tional complex (41). Similarly, E2F7 has been shown to bind theSphk1 promoter in SCC cells and is associated with increasedSphk1 transcription (15). The precise mechanism by which E2F7regulates the transcription of RacGAP1 and Sphk1 is currentlyunder examination. Regardless of the mechanism, our functionaldata show that E2F7 regulates doxorubicin-induced cytotoxicityvia transcriptional induction of RacGAP1.

To our knowledge, this is the first report showing overexpres-sion of RacGAP1 in HNSCC tumor samples. This is also the firstreport to show that overexpression of tumor-associated RacGAP1is directly controlled by E2F7, which itself is known to be over-expressed in SCC and to induce drug resistance (15). Overexpres-sion of RacGAP1 has been reported in high-grade meningiomas,non–small cell lung cancer, gastric cancer, hepatocellular carci-noma, breast cancer (24, 38, 42–44) as well as in the moreaggressive tumor phenotypes of epithelial ovarian cancer, high-grade breast cancer, and invasive cervical cancer (45–47). How-ever, it is unknown whether the overexpression of RacGAP1 inthese tumors is linked to overexpression of E2F7. Although theoverexpression of RacGAP1 is not considered to be simply a"passenger" in other cancer types (26–29), its contribution toHNSCC is unknown. Our data show that loss of RacGAP1expression is able to reduce SCC growth in a xenotransplantmodel via inhibition of proliferation and increased basal apo-ptosis. In addition, we show that high levels of expression areassociated with poor PFS of HNSCC patients. Thus, our clinicaland preclinical data would suggest that E2F7-dependent over-expression of RacGAP1 is likely to be a driver of tumor growth anddrug resistance in HNSCC.

The functional consequences of RacGAP1 overexpression arenot reflective of a generalized loss of GTP loading of the Rho/Racfamily of GTPases. Our data indicated that RacGAP1 favored theconversion of RhoGTP to RhoGDP in HNSCC cells. However, itwas clear that the GTP-loading status of Rac1 was also responsiveto changes in RACGAP1 expression. For example, RacGAP1appeared to negatively regulate the GTP-loading status of Rac1such that knockdown of RacGAP1 resulted in a reduction in theGTP-loading state of Rac1. Although this seemed counterintui-tive, it has been shown that phosphorylation of RacGAP1, byAurora B kinase, on Serine 387 shifts its GAP activity from Rac toRho, resulting in increased GTP loading (activation) of Rac1 andreduced GTP loading of Rho (inactivation; ref. 36). Significantly,we show that incubation of doxorubicin-resistant SCC cells with aselective Rac1 inhibitor was able to reinstate doxorubicin sensi-tivity, whereas incubationwith a RhoA/ROCK1 selective inhibitordid not. These data suggest that the resistance to doxorubicincorrelates with the GTP-loaded "active" state of Rac1. This con-clusion is supported by a previous study showing that the anti-apoptotic protein, Bcl-2, interacts with Rac1 to protect tumor cellsfrom the cytotoxic actions of etoposide and daunorubicin (48).Moreover, our conclusion is also consistent with a previous reportsuggesting that Rac1 was a potential therapeutic target in chemor-adioresistant HNSCC (49). Thus, the E2F7-dependent drug-resis-tant phenotype we observed in SCC cells may result from anAurora-B kinase–mediated switch of RacGAP1 activity from Rac1to RhoA leading to Rac1-dependent activation of AKT-mediateddoxorubicin resistance.

There is an existing literature on the role of PI3K/AKT incontrolling RacGAP1 activity and the GTP loading of theRho family of GTPases. In particular, it has been shown thatAKT directly binds to and activates RacGAP1 activity via






phosphorylation of T249 (50). In the present study, we show thatE2F7 induces RacGAP1 expression, which is associatedwith (i) anincrease in activated Rac1, (ii) a decrease in RhoA activity, (iii) anincrease in Ser473 p-AKT, and (iv) resistance to doxorubicin.These findings are consistent with the observation that PI3K/AKTsignaling is among the most significantly altered canonical path-ways following RacGAP1 silencing in HCC (38). Our observa-tions also suggest that the overexpression of RacGAP1 in SCCmaycontribute to the activation of the AKT pathway that is seen inmore than 40% of all HNSCCs. However, how RacGAP1 con-tributes to AKT activation remains unclear.We certainly know thatoverexpressing or knocking down E2F7 or RacGAP1 modifiesSer473 phosphorylation of AKT. However, we also know thatE2F7 directly induces Sphk1 expression and S1P levels in SCC,leading to increased Ser473 phosphorylation of AKT (15). Finally,we now show that Sphk1 and RacGAP1 indirectly modify oneanother's expression. Thus, it is difficult to determine whetherRacGAP1-dependent effects on AKT phosphorylation status aremodified by RacGAP1 or indirectly via changes in Sphk1/S1P.Regardless of the mechanism, our results demonstrate the exis-tence of a novel, complex, and interdependent network betweenE2F7, RacGAP1, and Sphk1/S1P, and the importance of such anetwork in chemosensitivity.

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

Authors' ContributionsConception and design: M. Hazar-Rethinam, N.A. SaundersDevelopment of methodology: M. Hazar-Rethinam, O.M. Gannon, A.C. Var-gas, N. Saenz-Ponce, F. Simpson, N.A. Saunders

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Hazar-Rethinam, S. Boros, M. Dzienis, D.D.E.DantzicAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Hazar-Rethinam, P. Mukhopadhyay, N.A.SaundersWriting, review, and/or revision of themanuscript:M.Hazar-Rethinam, O.M.Gannon, M. Dzienis, F. Simpson, N.A. SaundersAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases):M. Hazar-Rethinam, L.M. de Long, A.C. Vargas,N. Saenz-Ponce, N.A. SaundersStudy supervision: F. Simpson, N.A. Saunders

AcknowledgmentsThe authors acknowledge the generous gift of E2f7Flox/Flox or E2f8Flox/Flox

mice from professor Gustavo Leone, The Ohio State University (Columbus,OH). They also acknowledge the generous donations of tissue samples from thepatients without which this project could not have been possible.

Grant SupportN.A. Saunders is supported by grants from the Australian NHMRC

(#APP1049182) and the Cancer Council Queensland (#APP1025479). He isalso supported by a Senior Research Fellowship awarded by the Cancer CouncilQueensland. O.M. Gannon is supported by a grant from theWesley St AndrewsResearch Institute (#WRI2011-17). M. Hazar-Rethinam is supported by anAustralian Postgraduate Award.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January 27, 2015; revised May 13, 2015; accepted May 15, 2015;published OnlineFirst May 27, 2015.

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2015;14:1939-1950. Published OnlineFirst May 27, 2015.Mol Cancer Ther Mehlika Hazar-Rethinam, Lilia Merida de Long, Orla M. Gannon, et al. Squamous Cell CarcinomaResistance to Doxorubicin and Is Prognostic for Overall Survival in RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent

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RacGAP1 Is a Novel Downstream Effector of E2F7-Dependent ... · Mehlika Hazar-Rethinam1, Lilia Merida de Long1, Orla M. Gannon1, Samuel Boros2, Ana Cristina Vargas 2 , Marcin Dzienis