TP53 Disruptive Mutations Lead to Head and Neck Cancer ... · measured by SA-b-gal staining, p21 expression, and release of ROS. The mitochondrial agent metformin ... biomarker for

Predictive Biomarkers and Personalized Medicine

TP53 Disruptive Mutations Lead to Head and Neck CancerTreatment Failure through Inhibition of Radiation-InducedSenescence

Heath D. Skinner1, Vlad C. Sandulache2,3, Thomas J. Ow2, Raymond E. Meyn1, John S. Yordy4,Beth M. Beadle1, Alison L. Fitzgerald2, Uma Giri1, K. Kian Ang1, and Jeffrey N. Myers2

AbstractPurpose: Mortality of patients with head and neck squamous cell carcinoma (HNSCC) is primarily

driven by tumor cell radioresistance leading to locoregional recurrence (LRR). In this study, we use a

classificationofTP53mutation (disruptive vs. nondisruptive) and examine impact on clinical outcomes and

radiation sensitivity.

Experimental Design: Seventy-four patients with HNSCC treated with surgery and postoperative

radiation and 38 HNSCC cell lines were assembled; for each, TP53 was sequenced and the in vitro

radioresistancemeasured using clonogenic assays. p53 protein expressionwas inhibited using short hairpin

RNA (shRNA) and overexpressed using a retrovirus. Radiation-induced apoptosis, mitotic cell death,

senescence, and reactive oxygen species (ROS) assays were carried out. The effect of the drug metformin

on overcoming mutant p53-associated radiation resistance was examined in vitro as well as in vivo, using an

orthotopic xenograft model.

Results: Mutant TP53 alone was not predictive of LRR; however, disruptive TP53 mutation strongly

predicted LRR (P ¼ 0.03). Cell lines with disruptive mutations were significantly more radioresistant

(P<0.05). Expression of disruptiveTP53mutations significantly decreased radiation-induced senescence, as

measured by SA-b-gal staining, p21 expression, and release of ROS. The mitochondrial agent metformin

potentiated the effects of radiation in the presence of a disruptive TP53 mutation partially via senescence.

Examination of our patient cohort showed that LRR was decreased in patients taking metformin.

Conclusions: Disruptive TP53 mutations in HNSCC tumors predicts for LRR, because of increased

radioresistance via the inhibition of senescence. Metformin can serve as a radiosensitizer for HNSCC

with disruptive TP53, presaging the possibility of personalizing HNSCC treatment. Clin Cancer Res; 18(1);

290–300. �2011 AACR.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is thesixth leading cause of cancer worldwide, with an estimated49,260 diagnoses each year in the United States alone (1).Although multimodality therapy is important in the man-

agement of HNSCC, the eradication of locoregional diseasein the primary or postoperative setting is primarily achievedby external beam radiation. As the vast majority of patientdeaths from HNSCC are due to locoregional recurrence(LRR), the survival of the patient largely depends on theradiosensitivity the tumor. Unfortunately, there are fewbiomarkers to determine radioresistance in HNSCC.

One candidate biomarker isTP53, which encodes the p53protein and is the most commonly altered gene in HNSCC,as shown most recently by our group and others throughwhole exomic sequencing of a large panel of HNSCCtumors (2). TP53 has been previously investigated as abiomarker for LRR following radiotherapy in HNSCC withmixed results (3–6). A possible explanation for this findingis mutation-specific functionality of the p53 protein. Onelarge study classifying TP53mutations inHNSCC describedTP53 mutation as "disruptive" or "nondisruptive" on thebasis of alteration of DNA binding (7). Any mutation ineither the L2 or L3 loop of the DNA-binding domain,resulting in a polarity change within the protein, or any

Authors' Affiliations:Departments of 1Radiation Oncology and 2Head andNeck Surgery, The University of Texas MD Anderson Cancer Center;3Bobby R. Alford Department of Otolaryngology - Head and Neck Surgery,Baylor College of Medicine, Houston; and 4Department of RadiationOncology, The University of Texas Southwestern Medical Center, Dallas,Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Jeffrey N. Myers, Department of Head & NeckSurgery, University of TexasMDAnderson Cancer Center, 1515 HolcombeBlvd., Unit 1445, Houston, TX 77030. Phone: 713-745-2667; Fax: 713-794-4662; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2260

�2011 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(1) January 1, 2012290

on February 15, 2021. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst November 16, 2011; DOI: 10.1158/1078-0432.CCR-11-2260

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stop codon, was classified as disruptive. In that study, thepresence of a disruptive mutation was associated with poorsurvival in a heterogeneously treated patient population.Other studies have examined alternative classifications ofTP53, which were also prognostic of survival in HNSCC(8; 9). Despite these results, there has been little study of therelationship between TP53 classification and LRR followingradiotherapy.The role of TP53 in in vitro radiosensitivity is also an area

of active investigation, with many of the previous studiesproducing contradictory results (10–16). Radiation isknown to result in p53 activation and apoptosis; however,the contribution of radiation-induced apoptosis to tumorresponse is not clear, and there are strong arguments that,for most tumor cells, apoptosis plays a minimal role in theradiation response (17; 18). In contrast, it has been hypoth-esized that mitotic death is the primary mechanism ofradiation-induced cell death (19). In this process, abnormalcell division due to DNA damage leads to the formation oflarge cells with multiple micronuclei (20). It is unclearwhether this form of cell death is associated with TP53(21–24). Furthermore, emerging data indicate that senes-cence may play a role in the radiation response, which, insome models, is dependent upon wild-type TP53 (25).Regardless, the role of TP53 in radiosensitivity is far fromclear.To investigate the role of TP53 in radiosensitivity, the

present study was done to determine whether a classifica-tion of TP53 (wild-type, nondisruptive, or disruptive muta-tion) predicts for radiosensitivity in vitro as well as inthe clinical setting. Furthermore, we wished to determinethe mechanisms involved in the observed radiation

response in vitro and use this knowledge to preferentiallyradiosensitize HNSCC cells.

Materials and Methods

Clinical samplesAll clinical studies reported in this study have been

approved by the Institutional Review Board of the Univer-sity of Texas MD Anderson Cancer Center. Seventy-foursnap-frozen, pretreatment tumor samples were collectedfrom patients with HNSCC at high risk for LRR treated withsurgical resection and postoperative radiotherapy from1992 to 2003. Exons 2-12 of the TP53 gene were sequencedusing genomic DNA. Standard Sanger sequencing usingBigDye Terminator chemistry (Applied Biosystems LifeTechnologies) was performed by Beckman Coulter Geno-mics (Beckman Coulter). Mutations were then classifiedaccording to the method of Poeta and colleagues (7).Patients were evaluated every 2 to 3 months for 1 yearfollowing treatment, every 3 to 4months the following year,and every 6 months thereafter.

Cell culture and constructsHNSCC cells were cultured in a 37�C incubator in 5%

CO2 atmosphere for this study as described previously (26).Cells stably expressing short-hairpin RNA (shRNA) specificfor p53 (shp53) were generated as described previously(27). Briefly, after infection with green fluorescent protein(GFP)-tagged empty lentiviral vector (LVTHM) or encodingan shRNA against p53 (LVUH-shp53; Addgene), cells werecultured for several passages, and then sorted using flowcytometry. GFP-positive cells were cultured normally. TP53constructs (C176F, R282W, R175H, and E336X) were gen-erated by extracting RNA from cell lines known to expressthese mutants. Reverse transcriptase PCR (RT-PCR) wasthen carried out using TP53-specific primers. The resultingproduct was purified and inserted into a pBABE retroviralvector containing a puromycin-resistance insert (pBABE-puro; Addgene) using standard cloning techniques. Theresulting vectors were verified by Sanger sequencing at theMD Anderson Cancer Center DNA core facility. After trans-fection and packaging in 293T cells, the viral supernatantwas centrifuged at 1,000 rpm to remove cellular debris andadded to UMSCC1 cells in combination with polybrene.After 1 passage, the cells underwent selection with puro-mycin. All cell lines used in this study have been authen-ticated against the parental recipient cell line via shorttandem repeat (STR) analysis.

Clonogenic assayHNSCC cells were seeded in 12-well plates at predeter-

mined densities for different radiation doses to allow for anapproximately equal number of resultant colonies. The nextday, cells were irradiated using a high-dose-rate 137Cs irra-diator (4.5 Gy/min) and cultured for 10 to 14 days to allowfor colony formation. Cells were then fixed in a 3% crystalviolet/10% formalin solution. Colonies of more than50 cells were then counted and survival fraction was

Translational Relevance

The lack of clinically used biomarkers in head andneck cancer is a significant problem in moving towardmore individualized therapy. TP53, the gene thatencodes the protein p53, is by far the most commonlymutated gene found in head and neck cancer. However,this finding has not been clinically useful in guidingtherapy. We have used a method of grading TP53muta-tion based on predicted functionality of the resultantprotein and shown that a particular class ofmutation hasa much higher rate of locoregional failure followingradiotherapy as well as a much higher rate of in vitroradioresistance by inhibiting radiation-induced cellularsenescence. Furthermore, we show that these resistantcells can be selectively radiosensitized through the use ofthe antidiabetic drug metformin. Finally, we show sig-nificant decreases in locoregional failure following radi-ation as well as improved survival in patients takingmetformin. Thus,weprovide a commonmarker for pooroutcome in head and neck cancer as well as a novelmeans by which to target these aggressive tumors.

TP53 Mutation and Senescence

www.aacrjournals.org Clin Cancer Res; 18(1) January 1, 2012 291




determined. All treatments were in triplicate or greater.For metformin (Sigma-Aldrich) or z-Vad-fmk (BD Phar-mingen) treatment, cells were pretreated for 2 hours beforeradiation with either drug or vehicle [PBS and dimethylsulfoxide (DMSO), respectively] and drug treatment wascontinued overnight. For N-acetyl cysteine (NAC) treat-ment, the cells were treated starting 2 hours followingradiation and the drug treatment was continued overnight.The cells were then washed and cultured in fresh media forthe remainder of the experiment.

ImmunofluorescenceHNSCC cells were plated on coverslips and treated as

indicated. Cells were washed twice with PBS and fixed usinga 1:1mixture ofmethanol and acetone for 10minutes. Cellswere thenwashed in TBS–Tween (TBST) and permeabilizedusing 0.1% Triton-X for 5minutes. Cells were again washedwith TBST and incubated with fluorescein isothiocyanate(FITC)–Phalloidin for 1 hour at room temperature. Cellswere washed with TBST and mounted on standard glassslides using 40,6-diamidino-2-phenylindole (DAPI)–Vecta-shield (Vector Laboratories). For assessment of mitoticdeath, the number of cells with 1 or more micronucleusper high-power field were counted and reported as a per-centage of total cells per field. Images were taken from 5quadrants, in duplicate, and pooled.

Senescence-associated-b-gal stainingSenescence-associated (SA)-b-gal staining was carried out

according to the manufacturer’s instructions (Cell Signal-ing). Briefly, HNSCC cells plated in 6-well plates wereirradiatedwith 4 to 6Gyon the next day. Cellswere culturednormally until the indicated times post treatment. Cellswere then fixed for 10minutes and stained overnight for SA-b-gal activity at 37�C. Blue-staining cells were scored assenescent and reported as a percentage of all the cellsobserved per high power field.

Annexin V stainingAnnexin V staining was carried out according to the

manufacturer’s instructions (BD Pharmingen). Briefly,HNSCC cells were cultured normally and treated as indi-cated. Media was collected, and adherent cells were brieflytrypsinized and added to the previously collected media.The cells were then centrifuged, the supernatant wasremoved, and the cells were washed in PBS. The cells werethen re-suspended in Annexin V binding buffer, andAnnexin V and 7-AAD were added. Samples were mixedgently and incubated at room temperature. Samples werethen analyzed for Annexin V and 7-AAD staining using aBeckman Coulter XL 4 color cytometer (Beckman Coulter),and the data were analyzed using Flo-Jo software (FloJo).Early apoptosis was determined by the proportion of cellsthat were Annexin V positive and 7-AAD negative.

Cell-cycle analysisHNSCC cells were seeded in a 6-well plate and incubated

for the indicated time. Media was collected; cells were

washed twice in PBS and trypsinized. The resulting cellsuspension was washed in PBS and fixed with 70% ethanolat room temperature for 30 minutes. Cells were thenwashed in PBS and stained with propidium iodide. Cell-cycle detection was then conducted using a Beckman Coul-ter XL 4 color cytometer, (Beckman Coulter) and the datawere analyzed using Flo-Jo software (FloJo).

Reactive oxygen species measurementIntracellular reactive oxygen species (ROS) levels were

measured according to previously published protocolsusing 5-(and-6)-carboxy-20,70-dichlorofluorescein (CM-H2DCFDA) dye (28). Briefly, cells were loaded with CM-H2DCFDA for 60 minutes in culture media. Media waschanged before irradiation to remove excess dye. Fluores-cence was measured at various times following irradiationusing a standard spectrophotometer, normalized to thecontrol condition and total amount of DNA (29). For flowcytometric experiments, cells were incubated with CM-H2DCFDA dye as described and trypsinized, and fluores-cence was analyzed using flow cytometry as describedabove.

ImmunoblottingCells were treated as indicated and washed 3 times with

cold PBS. Standard lysis buffer was then added to each plateand incubated on ice for 15 minutes. Cells were thencollected using a plastic scraper and centrifuged at 14,000rpm at 4�C for 10 minutes. The supernatant was removed,and total protein concentration was then calculated usingBio-Rad Protein Assay (Bio-Rad). Immunoblot analysis wascarried out as described previously (26). Membranes wereblocked for 1hour at room temperature using1%powderedmilk in 0.1% Tween 20 in TBS, then incubated overnightwith anti-p53 DO-1 (Santa Cruz Biotechnology), anti-bactin (Cell Signalling), or anti-p21 (BD Pharmingen) at4�C. The following day, membranes were washed with0.1% Tween 20 in TBS and incubated for 1 hour at roomtemperature with species-specific secondary antibody. Forp53 and actin, fluorescence-conjugated secondary antibo-dies (Invitrogen) were used and signal was analyzed usingan Odyssey Infrared Imaging System (LI-COR Biosciences)and the associated software (v3.0). For p21, horseradishperoxidase conjugated anti-rabbit immunoglobulin G(Santa Cruz Biotechnology) was used and signal wasgenerated using the SuperSignal West chemiluminescentsystem (Pierce Biotechnology).

p21 transcriptionInduction of p21 transcription was measured through

luciferase reporter activity using a vector containing the2.4-kb p21 promoter and firefly luciferase (pWWP-Luc;ref. 30; Addgene). UMSCC1 (p53-null) cells expressingvarious TP53 constructs were co-transfected with pWWP-Luc and a constitutively active Renilla luciferase constructusing Lipofectamine 2000 (Invitrogen). Cells were irra-diated the following day at the indicated doses andincubated for 24 hours before collection. Luciferase

Skinner et al.

Clin Cancer Res; 18(1) January 1, 2012 Clinical Cancer Research292




activity was measured with the Promega Dual-LuciferaseReporter Assay System.

Orthotopic mouse modelAll animal experimentation was approved by the Animal

Care andUse Committee (ACUC) of theUniversity of TexasMDAnderson Cancer Center. The orthotopic mousemodelhas been previously described (31). Briefly, mice wereinjected with 100,000 HN 31 cells in the anterior half ofthe oral tongue on day 0. After tumor growth was noted,tumors were treated with radiation using a Co60 irradiatorand custom lead blocks (5 Gy) on day 8 post-injection and/or metformin [250 mg/kg daily, intraperitoneal (IP)]. Eachtreatment group comprised 10mice.Mice received a total of8 daily metformin treatments during the experimentalperiod. Tumor measurements were obtained on days 6,13, 16, and 20 post injection. Tumor volumewas calculatedas previously described (31).

StatisticsLRR and overall survival (OS) for the patient population

was calculated using the Kaplan–Meier method, and com-parisons between groups were determined using log-rankstatistics.Multivariate analysis for LRRwas carried out usingforward step-wise Cox regression analysis. Variables includ-ed tumor and nodal stage, surgical margin status or extra-capsular extension, site, gender, smoking history, and TP53status. ANOVA with post hoc analysis or Student t tests werecarried out to analyze in vitro data and tumor volume. All Pvalues are 2-sided. P values less than 0.05 were consideredsignificant.

Results

Disruptive TP53 mutations are associated withincreased LRR following PORTDisruptive TP53 is associated with decreased survival in

HNSCC, which we hypothesized was due to higher ratesof LRR following PORT. To explore this hypothesis, wesequenced TP53 in HNSCC tumors from 74 patientstreated with PORT. Patient characteristics were similaramong different TP53 classification groups (Supplemen-tary Table S1). Median follow-up of surviving patientswas 154 months (range 82–185). No significant differ-ence in LRR was found between patients with wild-typeTP53 and any mutant TP53 (Fig. 1A). However, classifi-cation of TP53 as disruptive, nondisruptive, or wild-type,showed that disruptive TP53 is an independent predictorof LRR on multivariate analysis (P ¼ 0.022). Patients withdisruptive TP53 had a 5-year freedom from LRR of 41%compared with 64% and 76% in patients with wild-typeand nondisruptive TP53, respectively (P ¼ 0.03; Fig. 1B).No significant difference in LRR was seen betweenpatients with wild-type and nondisruptive TP53 (P ¼0.48; Fig. 1B). Similarly, the OS rates for patients in thisstudy were predicted by TP53 classification. Specifically,5-year OS rate for patients with disruptive TP53 muta-tions was 19% compared with 52% in patients with

wild-type TP53 and 41% in patients with nondisruptiveTP53 (P ¼ 0.031).

Disruptive TP53 mutations are associated withp53-mediated radioresistance

The finding that disruptive TP53mutations are associatedwith a high rate of LRR resulted in the hypothesis thatHNSCC tumor cells with disruptive TP53 are intrinsicallymore radioresistant. To test this hypothesis, we evaluatedthe radiosensitivity of a panel of 38 HNSCC cell lines ofknown TP53 status (Supplementary Table S3) and foundthat cell lines harboring disruptive TP53 mutations weresignificantly more radioresistant than those with eithernondisruptive or wild-type TP53 (Fig. 2A).

To determine whether this observed correlation was dueto p53 expression, p53 was silenced using stably expressedshRNA (shp53) in the following HNSCC cell lines:(i) UMSCC1 17A and HN 30 (wild-type TP53); (ii) Detroit(R175H, nondisruptive TP53); and (iii) HN 31 (C176F)

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and FADU (R248L; disruptive TP53). Inhibition ofp53 expression in cell lines expressing wild-type or nondis-ruptive TP53 rendered cells more radioresistant (Fig. 2B;Supplementary Table S2). However, inhibition of p53expression in cell lines expressing disruptive TP53 renderedthese cells more radiosensitive (Fig. 2D; SupplementaryTable S2). Conversely, UMSCC1 cells, which have noendogenous p53, were engineered to express: (i) wild-typeTP53; (ii) nondisruptive TP53 (R175H and R282W); or (iii)disruptive TP53 (C176F and E336X). Disruptive TP53-expressing UMSCC1 cells were found to be radioresistantrelative to wild-type TP53- and nondisruptive TP53-expres-sing cells (Fig. 2C; Supplementary Table S2).

Alteration of p53 expression does not affect radiation-induced mitotic death or apoptosis

To explore themeans by which disruptive TP53mutationconfers radioresistance in HNSCC cells, we evaluated sev-eral modes of cellular response to radiation. Initially, weexamined mitotic death, thought to be one of the primarymodes of cell death following radiation (19).Mitotic death,as measured by the presence of micronuclei, was significantfollowing radiation, with all HNSCC cell lines tested exhi-biting mitotic death in 20% to 30% of irradiated cells.However, there was no correlation between micronucleiformation, TP53 expression, and relative radiosensitivity(Supplementary Fig. S2).

Furthermore, when either annexin V or sub-G1 stainingwere analyzed, neither of these measures of apoptosis wererelated to TP53 status (Supplementary Fig. S3). In fact, only

a very small proportion of apoptotic cells were observed(�1%–5%), including in those cells shown to be radiosen-sitive in the clonogenic survival assay. As radiation-inducedapoptosis is thought to be caspase-dependent (32), we alsotreated cells with a pan-caspase inhibitor (z-vad-fmk; BDPharmingen) to determine the role of apoptosis in thismodel. Inhibition of caspase activity had almost no effecton clonogenic survival following radiation (SupplementaryFig. S3), supporting the hypothesis that apoptosis does notplay a major role in the radiation response.

Radiation robustly induces SA-b-gal activity in HNSCCcells which is strongly associated with TP53 status andcorrelates with clonogenic survival

In cells with wild-type or nondisruptive TP53, treatmentwith radiation resulted in adecrease in theproportionof cellsinSphase anda less prominentG1arrest (Supplementary Fig.S4). Furthermore, inhibition of p53 expression in HN 30(wild-type TP53) cells partially abrogated the observedinability to progress through S phase. Because this phenom-enon has been previously linked to cellular senescence, wehypothesized a role for altered senescence in the observeddifferences in radiosensitivity. To evaluate cellular senes-cence, cells were treated with radiation and stained for theSA-b-gal. As shown in Fig. 3, radiation treatment resulted insignificant levels of SA-b-gal activity in cell lines expressingeither wild-type (HN 30) or nondisruptive mutant TP53(Detroit, R175H). Furthermore, in these cells with SA-b-galstaining, morphologic characteristics of cellular senescencewere observed, specifically a large, flattened cell with the

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Figure 2. In vitro radiosensitivity ismodulated by p53 expressiondepending on TP53 classification.A, a panel of 38 HNSCC cell lineswas sequenced for TP53 statusand evaluated for radiosensitivityusing standard clonogenic assays.Each cell line was then grouped byTP53 status, and the averageclonogenic survival data for eachgroup are shown. B, the effect ofstable inhibition of wild-type p53expression (shp53) onradiosensitivity in HN 30 andUMSCC1 17A cells. C, the effect offorced expression of wild-type,R175H, R282W, or C176F TP53 inp53-null UMSCC1 cells onradiosensitivity. D, the effect ofstable inhibition of p53 expression(shp53) on radiosensitivity in FADUand HN 31 cells (disruptive TP53).

Skinner et al.





classic "fried egg" appearance on microscopy. Inhibition ofp53expression inbothof thesecell lines significantly reducedlevels of radiation-induced SA-b-gal activity.However, inHN31 (C176F, disruptive TP53) cells, which have disruptiveTP53 and are otherwise isogenic with the HN 30 (wild-typeTP53) cell line, inhibition of p53 expression was associatedwith elevated levels of radiation-induced SA-b-gal activity.Forced expression of wild-type, as well as 2 nondisruptive,TP53 mutations (R175H and R282W) in p53-null cells,increased radiation-induced SA-b-gal activity; conversely,expression of disruptive TP53 mutations (C176F andE336X) led to decreased SA-b-gal activity compared withempty vector control (Fig. 3B). Furthermore, in experiments

comparing micronuclei formation and SA-b-gal staining inthe same cell, the main differences between cells of differentTP53 statuses appeared tobewithin the cells stainingpositivefor SA-b-gal alone (Fig. 3D).

The effects of TP53 status on radiation-induced SA-b-gal activity are correlated with p21 expression andmediated by ROS production

In addition to SA-b-gal activity, senescence has beenpreviously linked to induction of p21 expression and ROSproduction, both of which are believed to be necessary formaintenance of the senescent phenotype (33–36). To deter-mine whether radiation-induced p21 expression correlates

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Figure 3. TP53modulates radiation-induced senescence, which correlates with radiosensitivity. A, representative light microscopy showing SA-gal staining.B, percentage of SA-gal–positive cells per total number of cells in a high-power field (hpf) after 4 Gy of radiation for the times indicated in UMSCC1cells expressing representative wild-type and mutant TP53 constructs and HN 30 (WT), Detroit (R175H), and HN 31 (C176F) where p53 is inhibited. C,representative microscopy showing SA-gal staining, DAPI fluorescence, and GFP. D, percentage of cells SA-gal–positive (S alone), exhibiting micronuclei(M alone), or both at 4 days after 4Gy of radiation inHN30 andHN31 cells where p53 expression is inhibited andUMSCC1 cells are expressing representativewild-type andmutant TP53 constructs. �, significantly elevated over baseline (P < 0.05);þ, significantly different from null at the indicated time point (P < 0.05);and #, significantly different from HN 30 control. XRT, X-ray therapy.






with TP53 classification, we assayed p21 protein levelsfollowing radiation in cell lines expressing representativeTP53 mutations. In cells expressing wild-type TP53 or anondisruptive TP53 mutation, p21 protein and reporteractivity were induced by radiation treatment (Fig. 4A). Incontrast, cells expressing disruptive TP53 (C176F) had noinduction of p21.

In addition, ROS play a key role in senescence andradiation effect. In the present study, radiation-inducedROS levels correlated with p21 expression, SA-b-gal activity,and relative radiosensitivity. Specifically, ROS were mosthighly induced in cells whichhad greater levels of radiation-induced SA-b-gal activity (wild type and R175H), whereaslittle or no ROS induction was seen in cells with disruptive

TP53 (C176F; Fig. 4B). Inhibition of ROS using NAC 2hours following radiation treatment, designed to inhibitsecondary ROS production following the initial radiationinsult, dramatically decreased senescence in cell lines withnormally high levels of radiation-induced SA-b-gal, but hadno effect on cells with disruptive TP53 (Fig. 4C). Theseresults closely correlated with the observed effects of NACon clonogenic survival following radiation (Fig. 4D).

Metformin preferentially potentiates the effects ofradiation in HNSCC cells partially via increasedsenescence

On thebasis of the combinedmorphologic appearance ofirradiated cells, the presence of SA-b-gal staining, the

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Figure 4. Radiation-induced senescence is associated with p21 expression and dependent on prolonged ROS production. A, p21 protein expression and p21luciferase reporter activity in UMSCC1 cells expressing the indicated TP53 constructs treated with the indicated doses of radiation for 24 hours unlessotherwise stated. B,ROSproductionmeasuredas indicated in theMaterials andMethods section after 2Gyof radiation.C, percentageof cells SA-Gal positive(S alone), exhibiting micronuclei (MC alone), or both at 4 days after 4 Gy of radiation. Cells were treated with the indicated dose of NAC starting 2 hours afterradiation and treatment was continued overnight. D, clonogenic survival after 2 Gy of radiation. Cells were treated with NAC in an identical manner to (C). �,significantly increased over unirradiated control (P < 0.05);þ, significantly different from UMSCC1 wild-type in the same group (P < 0.05); and #, significantlyincreased compared with non–NAC-treated group (P < 0.001). hpf, high-power field; XRT, X-ray therapy.

Skinner et al.





characteristic induction of p21 and ROS, and the lack ofapoptosis, cells with either wild-type or nondisruptive TP53mutation appear to be undergoing radiation-induced senes-cence, whereas cells with a disruptive TP53 mutation arenot. Because the induction of senescence may be clinicallybeneficial, we intended to investigate drug-based therapiesdesigned to this end. As there are no specific senescence-inducing agents currently available, we examined metfor-min, an antidiabetic agent with minimal clinical toxicity,that has been shown to both induce ROS in some cellularcontexts (37) as well as preferentially target cell lineswith mutant TP53 (38). In the present study, concurrentradiation and metformin treatment was active againstHNSCC cell lines that harbored disruptive TP53 (Supple-

mentary Fig. S5). Conversely, no effect of metformin wasseen in cells expressingwild-type TP53 (HN30 andMCF-7).Furthermore, the addition of metformin dramaticallyincreased ROS in HN 31 cells, which have a disruptive(C176F) TP53 mutation, but had much less effect inHN 30 cells, their wild-type TP53 isogenic counterpart(Supplementary Fig. S5). Similarly, metformin decreasedclonogenic survival following radiation and increased radi-ation-induced SA-b-gal activity in HN 31 cells (C176F,disruptive TP53), but had little effect in HN 30 cells(wild-type TP53; Fig. 5A and B). Furthermore, after inhibi-tion of wild-type p53 expression, metformin was found topotentiate SA-b-gal activity and decrease clonogenic surviv-al (Fig. 5A and B).

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Figure 5. Metformin selectively radiosensitizes cells with disruptive TP53mutations, partially due to altered senescence. A, clonogenic survival after treatmentwith radiationat the indicateddoses togetherwithmetformin for 24hours inHN30andHN31cells inwhichp53expression is inhibited usingshRNA (shp53).B,percentage of SA-b-gal–positive cells per total number of cells in a high-power field (hpf) 4 days following 4 Gy of radiation and the indicated doses ofmetformin. �, significantly different from unirradiated control (P < 0.05); þ, significantly changed compared with no metformin treatment (P < 0.05). C, tumorvolume inmicewith orthotopic tumors derived fromHN31cells (C176F) after treatmentwith radiation (6Gy),metformin (250mg/kg, intraperitoneal), or both. �,significantly different from radiation alone (P < 0.05);þ, significantly different from metformin treatment alone (P < 0.05). D, LRR in patients taking metforminduring treatment comparedwith the remainder of the studypopulation aswell aspatientsmatched for tumor andnodal stage, surgicalmargin status, andTP53status to the metformin-treated group (P ¼ 0.04).






Given the above findings, we examined the effect ofmetformin treatment on tumor response to radiation in anorthotopic model of HNSCC described previously (26, 31).As seen in Fig. 5C, the addition of metformin to radiationdramatically decreased tumor growth compared with eitherradiation alone (P¼ 0.015) ormetformin alone (P¼ 0.008).

To further investigate the clinical use of metformin, acohort of patients treated with postoperative radiationtherapy (PORT) for HNSCC was identified and the impactof concurrent metformin use was investigated. In additionto thepatients in the current study, an additional 30patientswith similar disease characteristics and treatment as well asknown TP53 status were evaluated. Although only 10patients were taking metformin at the time of radiation,these patients had a dramatically lower LRR rate thancontrols matched for tumor and nodal stage, surgical mar-gin status, and TP53 status (P ¼ 0.04; Fig. 5D). On multi-variate analysis controlling for TP53 classification, tumorstage, and surgical margin positivity, metformin use wassignificantly associated with decreased LRR (P ¼ 0.04) aswell as improved OS (P¼ 0.01). Specifically, 5-year OS ratewas 87% in the patients taking metformin compared with41% in the remaining patients (P ¼ 0.04).

Discussion

In HNSCC, the majority of patient deaths result fromLRR, with up to 60% to 75%dying of their disease (39). Theprimary method of treating advanced HNSCC involvesradiation, either in the definitive or postoperative setting.To date, there are few clinically useful predictive indicatorsof LRR following radiotherapy. In the current study, weexamined LRR in HNSCC and, for the first time, showedthat the disruptive classification of TP53 is predictive ofpoor response to a specific therapy, namely PORT. Inter-estingly, mutant TP53 alone was not predictive for LRR,arguing for the use of a TP53mutation classification schemethat can show differential function between TP53 mutantswith regard to radioresistance.

Because the goal of PORT is to eradicate microscopicresidual disease, we hypothesized that LRRwould primarilyresult from intrinsically radioresistant cells. To investigatethis, we used an in vitro approach, combining silencing ofendogenous p53 expression with genetically engineeredexpression of different TP53 mutations; this approachshowed that HNSCC cells with disruptive TP53 exhibiteda radioprotective effect of p53 expression, arguing for a"gain of function" (GOF) with regard to radiosensitivity.These GOFmutations in TP53 have been shown previouslyfor other cellular processes; however, this report is one ofthe first to do so for in vitro radiosensitivity. Furthermore, wefound that 2 types of cell death most commonly associatedwith radiation, namely apoptosis and mitotic death, wereeither not present and/or unaffected by TP53 status. In fact,the main response to radiation affected by TP53 status inthis model was cellular senescence.

Senescence is a form of cell arrest in which cells remainmetabolically active, but lack replicative potential. Several

recent studies have linked response to chemotherapy andradiation to this process; however, the exact role of senes-cence in the response to radiation is far from clear (17,18, 40). In the present study, we found radiation-inducedsenescence correlated strongly with in vitro radiosensitivityand was inhibited in the presence of a disruptive TP53mutant, again arguing for a GOF by at least a subset ofTP53 mutations within the disruptive category, drivingresistance to radiotherapy.

Induction of both p21 and ROS appear to be criticalmediators of cellular senescence. Induction of p21 alonecan lead to increased ROS production (34), and ROSinhibition can reverse senescence in this context. Converse-ly, ROS have been linked to induction of p21, and afeedback loop between ROS and p21 is proposed to berequired for DNA damage-induced senescence (35, 41).

In the proposedmodel, we found radiation-induced ROSand p21 only in HNSCC cells with wild-type and nondis-ruptive TP53. Specifically, p21 expression was increased inresponse to radiation in the context of the nondisruptiveTP53mutation R175H. Previously, R175H has been shownto have little remnant p53-driven transcription (42, 43).Furthermore, in one report, the disruptive mutation,C176F, has been shown to have some p53-dependenttranscriptional activity in a yeast-based assay (43); however,this has not been observed in other studies (44, 45).Nonetheless, in the current model, both protein expressionand transcription of p21 is induced by radiation in cellsexpressing either wild-type or R175H TP53. This could beattributable to p53 transcription independent induction ofp21 similar to that seen in other models (46, 47) or it couldresult from residual p53 transcriptional activity. It has beenshown recently that only a small subset of p53 transcrip-tional targets are necessary for induction of senescence;thus, even a small remnant of transcriptional function maybe sufficient (48). Further studies are in progress to discernthe mechanism behind this phenomenon.

Furthermore, radiation-induced ROS production wasinhibited by disruptive TP53. In our study, inhibition ofsecondary ROS production several hours after the initialcellular insult by radiation had a dramatic effect on cellularsenescence. This argues for prolonged ROS productionfollowing radiation playing a key role in the maintenanceof the senescent phenotype. These ROS are most likelyderived from the mitochondria and, in fact, we have pre-viously observed decreased baseline mitochondrial com-plex activity in cells expressing C176F compared with wild-type TP53 (27). This argues for a suppressive effect on themitochondria of disruptive TP53, which is relieved byinhibition of p53 expression. This offers a possible linkbetween disruptive TP53mutationsGOF and the inhibitionof radiation-induced ROS and senescence. Theoretically,disruptive p53 could interact with known targets of wild-type p53 involved in mitochondrial function, exerting aninhibitory function as opposed to the stimulation seenwithwild-type or some nondisruptive forms of TP53.

Our data strongly indicate that disruptive TP53 portendspoor clinical outcome in HNSCC associated with increased

Skinner et al.





radioresistance. Parallel work in our laboratory has showedthat cells with disruptive TP53 exhibit a therapeuticallyexploitable decrease inmetabolic flexibility (27).One agentthat can target this process is metformin. Recently, it hasbeen shown that patients taking metformin have improvedresponses following neoadjuvant chemotherapy (49) andthat metformin is a radiosensitizer in vitro (50). Metforminis hypothesized to affect mitochondrial function, and sev-eral studies have shown that it induces ROS in certaincellular backgrounds (37). In addition, it has been hypoth-esized that metformin may induce senescence in cells thatare "senescence prone" (51). Interestingly, when we exam-ined the effects of metformin in vitro, radiation-inducedsenescence and toxicity were potentiated only in cellsexpressing disruptive TP53, an effect that was modulatedby altering p53 expression. This dramatic effect on theefficacy of radiation was also seen in our in vivo orthotopicmodel, as well as in patients takingmetformin at the time ofPORT. Although this only included 10 patients in themetformin group, the reduction in LRR was profound andargues for the possibility of a dramatic clinical benefit withtargeting of radiation-induced senescence.The present study is limited by its retrospective nature,

although the patient cohort includes a homogenous groupof patients treated uniformly with surgery and PORT. Fur-thermore, the in vitro studies are limited by the analysis of asubset of disruptive and nondisruptive TP53 mutations,and the possibility exists that other disruptive TP53 muta-tions may behave differently. However, examination of alarge panel of HNSCC cell lines confirmed the in vitropredictive value of the disruptive classification. In addition,clinical data showingdramatically increased LRR inpatientswith disruptive TP53 mutations supports the overallhypothesis. Although the disruptive category is empiric innature, the fact that senescence appears to be stronglyinhibited by these mutations allows further refinement.Specifically, testing of TP53 mutants’ ability to induce

senescence could be carried out, refining an already clini-cally predictive model. Finally, although the clinical dataregarding the benefit of metformin is compelling, only asmall number of patients were treated with the drug at thetime of radiation. Despite the intriguing in vitro, in vivo, andclinical finding of radiation potentiation by metformin,these findings do not provide a definitive conclusion, butrather are hypothesis-generating and require additionalvalidation.

Despite these limitations,wehave shown for the first timethat disruptive TP53mutation predicts both LRR followingPORT and in vitro response to radiation in HNSCC. Inaddition, we have shown that this effect is due to alteredsenescence and can be overcome using metformin, whichappears to have dramatic clinical benefit.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Mei Zhou and Samar Jasser for their assistance withbasic laboratory work and logistics. The authors also thank Christina Dodgefor her assistance with the in vivo experiments.

Grant Support

This work was partially supported by an American Society of RadiationOncologyResident SeedAward; theUniversity of TexasMDAndersonCancerCenter PANTHEON program; NIH Specialized Program of Research Excel-lence (grant P50CA097007; RO1DE14613NIH); National Research ScienceAward Institutional Research Training (grant T32CA60374); and NIH Pro-gram Project (grant CA06294) and Cancer Center Support (grantCA016672).

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 September 1, 2011; revisedNovember 3, 2011; acceptedNovem-ber 5, 2011; published OnlineFirst November 16, 2011.

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2012;18:290-300. Published OnlineFirst November 16, 2011.Clin Cancer Res Heath D. Skinner, Vlad C. Sandulache, Thomas J. Ow, et al. SenescenceTreatment Failure through Inhibition of Radiation-Induced

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