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Cancer Genes and Genomics

Role of Neurofilament Light Polypeptide in Head and NeckCancer Chemoresistance

Baishen Chen1,3, Ju Chen2, Michael G. House1, Kevin J. Cullen4, Kenneth P. Nephew1,5, and Zhongmin Guo1,5

AbstractResistance to cisplatin-based chemotherapy is responsible for therapeutic failure of many common human

cancers including cancer of head and neck (HNC). Mechanisms underlying cisplatin resistance remain unclear. Inthis study, we identified neurofilament light polypeptide (NEFL) as a novel hypermethylated gene associated withresistance to cisplatin-based chemotherapy inHNC. Analysis of 14HNC cell lines revealed that downregulation ofNEFL expression significantly correlated with increased resistance to cisplatin. Hypermethylation of NEFLpromoter CpG islands was observed in cell lines as examined by bisulfiteDNA sequencing andmethylation-specificPCR (MSP) and tightly correlated with reduced NEFL mRNA and protein expression. Furthermore, in patientsamples withHNC (n¼ 51) analyzed by quantitativeMSP,NEFL promoter hypermethylation was associated withresistance to cisplatin-based chemotherapy [relative risk (RR), 3.045; 95% confidence interval (CI), 1.459–6.355;P ¼ 0.007] and predicted diminished overall and disease-free survival for patients treated with cisplatin-basedchemotherapy. Knockdown ofNEFL by siRNA in the highly cisplatin-sensitive cell line PCI13 increased (P< 0.01)resistance to cisplatin. In cisplatin-resistant O11 and SCC25cp cells, restored expression of NEFL significantlyincreased sensitivity to the drug. Furthermore, NEFL physically associated with tuberous sclerosis complex 1(TSC1), a known inhibitor of the mTOR pathway, and NEFL downregulation led to functional activation ofmTORpathway and consequentially conferred cisplatin resistance. This is the first study to show a role forNEFL inHNC chemoresistance. Our findings suggest that NEFL methylation is a novel mechanism for HNC chemore-sistance and may represent a candidate biomarker predictive of chemotherapeutic response and survival in patientswith HNC. Mol Cancer Res; 10(3); 305–15. �2012 AACR.

IntroductionAlthough platinum-based chemotherapeutic agents are

widely used to treat human malignancies, including headand neck cancer (HNC; ref. 1), intrinsic and acquiredresistance of cancer cells to cisplatin accounts for themajorityof chemotherapeutic failure in HNC (2). Resistance tocisplatin, reported in 15% to 30% of newly diagnosed and

more than 70% of relapsed patients, is an important con-tributing cause of the more than 12,000 deaths from HNCreported each year in the United States (3). Alternativetherapeutic strategies and predictive biomarkers for cisplat-in-resistant HNC are currently lacking.Mechanisms underlying the development of resistance to

platinum drugs are not fully understood (4). Extracellular andintracellular changes thatpromotedrugmetabolism,decreasedcellulardrugaccumulation,alteredexpressionofkeymoleculesin the apoptotic pathway, and increased repair of DNAadducts have been reported to contribute to drug resistance(4). InHNC, alterations of keymoleculesmediating cisplatin-induced apoptosis have been implicated in resistance (5), andmutated or overexpressed p53 and the Bcl-2 protein family,key players in regulating apoptotic pathways, were associatedwith HNC resistance to cisplatin-based chemotherapy (6). Inaddition, alteredmetabolism in reactive oxygen species (ROS),an essentialmolecule for cisplatin-induced cell killing,was alsoobserved inHNC (7).We have shown that amplification andoverexpression of glutathione S-transferase (GST), a moleculeimportant for preventing intracellular ROS accumulation,contributes to cisplatin resistance in HNC (8, 9).Altered DNA methylation is a hallmark of human cancer

and has been shown to contribute to drug resistant disease(10). Inactivation of tumor suppressor genes (TSG) by

Authors' Affiliations: 1Medical Sciences Program, Indiana UniversitySchool of Medicine, Bloomington, Indiana; 2Department of Surgery,Memorial Hospital, Sun Yat-Sen University School of Medicine, Guangz-hou, P. R. China; 3Department of Thoracic Surgery, the Third AffiliatedHospital of Southern Medical University, Guangzhou, P.R.China; 4Marleneand Stewart GreenebaumCancer Center, University of Maryland School ofMedicine, Baltimore,Maryland; and 5Department ofCellular and IntegrativePhysiology and Indiana University Simon Cancer Center, Indianapolis,Indiana

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

B. Chen and J. Chen contributed equally to this work.

Corresponding Author: Zhongmin Guo, Medical Sciences Program, Indi-ana University School of Medicine, Jordan Hall 104, 1001 E. Third St.,Bloomington, IN 47405. Phone: 812-856-2434; Fax: 812-855-4436; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-11-0300

�2012 American Association for Cancer Research.

MolecularCancer

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Published OnlineFirst January 13, 2012; DOI: 10.1158/1541-7786.MCR-11-0300

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promoter CpG island hypermethylation contributes totumor initiation and progression, and methylated DNA isa potential source of cancer-specific biomarkers for clinicalassessment (11). Studies using DNA methylation inhibitors[e.g., 5-aza-20-deoxycytidine (5-aza-dC)] to restore DNAdamage recognition, repair, and apoptosis pathways and thussensitivity to cisplatin-based chemotherapy further supportthe important role of DNA methylation in cancer (12, 13).Although epigenetic alterations in chemoresistant tumorshave been reported in ovarian, lung, colon, and testicularcancers (10, 14, 15), aberrant DNA methylation in HNCchemoresistance have only been reported in in vitro cell linemodels (16).To better understand the epigenetic mechanisms under-

lying cisplatin resistance in HNC and identify novel hyper-methylated genes as therapeutic targets and potential HNCbiomarkers, we carried out genome-wide DNAmethylationprofiling of isogenic, cisplatin-sensitive, and cisplatin-resis-tant HNC cell lines, using an approach that combines geneexpression microarray and pharmacologic unmasking (5-aza-dC treatment of cancer cells; ref. 17). This technique hasbeenwidely used inmethylation screening for different typesof cancers (16, 17). Among the identified candidates, neu-rofilament light polypeptide (NEFL) was selected for anal-ysis, due to its chromosome location of 8p21, a genetic locuspreviously reported to be frequently deleted in severalcommon cancers, includingHNC (18–20). TheNEFL geneencodes the light subunit of neurofilaments, which areessential for maintaining neuronal integrity and may playan essential role in intracellular transport to axons and

dendrites (21). In this study, we further examined NEFLmethylation and expression in a panel of HNC cell lines andpatient tumors with recorded responses to cisplatin-basedchemotherapy. We also investigated the functional role andpotential mechanisms of NEFL in HNC cisplatin response.Taken together, our results implicate that promoter hyper-methylation-mediated silencing of NEFL is a novel mech-anism for HNC chemoresistance, and methylated NEFLmay represent a candidate biomarker predictive of chemo-therapeutic response and survival for patients with HNC.

Materials and MethodsCell linesA total of 15 HNSCC cell lines, including 2 isogenic

cisplatin-sensitive/resistant lines (HN17B/HN17Bcp,SCC25/SCC25cp) were used in this study (Table 1). Inaddition, an in vitro immortalized keratinocyte cell lineHacat was used as a nonmalignant control. Cell linesO11, O12, O13, O22, O28, and O29 were obtained fromthe Department of Otolaryngology, Head and Neck CancerResearch Division, Johns Hopkins University School ofMedicine (Baltimore, MD) and have been described previ-ously (22). HN17Bcp cisplatin–resistant cells were devel-oped inDr. K. Cullen's laboratory by long-term treatment ofcisplatin-sensitive HN17B cells with increasing concentra-tions of cisplatin. The additional cell lines, sources, andculture conditions were described previously (8, 23). Cis-platin sensitivity of these cell lines was determined bytreating cells with various doses of cisplatin continuously

Table 1. Correlation of expression andmethylation ofNEFL gene with cisplatin sensitivity in HNC cell lines

Cell lines NEFL expression NEFL methylation Cisplatin IC50 value, mmol/L Cisplatin sensitivity

Hacat þ UM Not determined UnknownPCI13 þ UM 0.54 SO29 þ UM 1 SHN17B þ UM 1.4 SHN22A þ UM 1.6 SO12 þ UM 1.7 SHN38 � PM 2 MRO13 þ PM 2.25 MRSCC25 þ UM 2.5 MRO11 � M 2.65 MRO22 þ UM 4.7 MRPCI51 � M 6.5 RFadu � M 7 RSCC25cp � M 9.5 RHN17Bcp � PM 9.6 RO28 � M 18.45 R

NOTE: Expression of NEFL was detected by real-time quantitative reverse transcriptase PCR with GAPDH as a normalization control.Abundant expressionwas recordedasþandnoor reducedexpressionas�. NEFLmethylationwasanalyzedbyMSPandbisulfiteDNAsequencing. Cisplatin IC50 value was determined by 72-hour treatment of individual cell lines with cisplatin. Cisplatin sensitivity of thecell lines was categorized into sensitive (IC50 < 2 mmol/L), moderately resistant (IC50 ¼ 2–5 mmol/L), and resistant (IC50 > 5 mmol/L).Abbreviations: M, methylated; MR, moderately resistant; PM, partial methylation; R, resistant; S, sensitive; UM, unmethylated.

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Mol Cancer Res; 10(3) March 2012 Molecular Cancer Research306




for 72 hours and conducting MTT assays to measurecisplatin toxicity. IC50 values were determined with Prismsoftware (GraphPad Software).

Patient samplesFrozen tumor and matched normal tissues from patients

with HNC were also obtained from the Department ofOtolaryngology (Head and Neck Cancer Research Division,Johns Hopkins University School of Medicine). Fresh tissuessamples were collected and stored at�80�C. Archival tumortissues from an additional 51 patients withHNCwith clinicalinformation, including treatment histories and survival fol-low-up, were collected from Georgetown University Lom-bardi Cancer Center (Washington, DC). The patients fromthis cohort were selected on the basis of availability ofadequate tissues for analysis. After the initial biopsy, allpatients were treated with cisplatin-based chemotherapy fororgan preservation or unresectable disease. Determination ofchemotherapy responses and patient survival in this cohorthas beendescribed previously (24).Clinicopathologic featuresof all patients are described in Table 2. Sample and data

collection were approved by local Institutional ReviewBoards.

Real-time reverse transcriptase PCR: mRNA levels ofNEFL transcripts in HNC cell lines were quantitativelyassessed by real-time reverse transcriptase PCRFor cDNA synthesis, 2-mg RNA was used for reverse

transcription (First-Strand cDNA Synthesis kit; Invitrogen).Twenty microliters of cDNA reaction mixture were furtherdiluted to 100 mL, and then 3 of 100 mL cDNA for eachsample was mixed with SYBRGreenMaster mixture (RocheDiagnostics) and quantitatively amplified by real-time PCRin a LightCycler 480 instrument using a protocol suggestedby manufacturer (Roche Diagnostics). Primers for amplifi-cation of NEFL transcript were: forward: 50-GAAGAG-GAGGCAGCTGGAAGA and reverse: 50-AAGGAAAT-GGGGGTTCAATC. Quantitative amplification of glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) cDNAwas carried out using the same protocol and used as nor-malization control. Relative expression levels of NEFLtranscript in cell lines were calculated using the previouslypublished formula: 2�(Ct_NEFL � Ct_GAPDH) (25)

5-Aza-dC treatment and reverse transcriptase PCRHNC cell lines (PCI13, SCC25cp, PCI51, and O28)

were treated with 10 mmol/L 5-aza-dC for 3 days withreplacement of the drug and medium every 24 hours. RNAof cell lines was purified with TRIzol reagent following theinstruction from the manufacturer (Invitrogen). cDNAsynthesis was carried out as described earlier, and 3 mL ofthe diluted cDNA for each sample was amplified by PCRusing a protocol described in our previous study (17).Primers for amplification of NEFL transcript were the sameas for real-time PCR amplification of GAPDH. cDNA wasused as normalization control. PCR products (10 mL) wereresolved in 1% agarose gel and visualized by ethidiumbromide staining.

Methylation analysisMethylation analysis of 50 genomic DNA sequences was

conducted on bisulfite-converted DNA from cell lines andprimary tissues by an EZ DNA Methylation-Gold Kit(ZYMO Research) according to standard protocol from themanufacturer. Methylation status of individual CpG islandin 50 flanking genomic regions (þ269 to þ572) of NEFLgene in cell lines PCI13, SCC25cp, and PCI51 was deter-mined by plasmid cloning and bisulfite DNA sequencing.Bisulfite-treated DNA (2 mL) from each of above cell lineswere amplified in a 25 mL PCR reactions containing 1�PCR buffer, 1.5 mmol/LMgCl2, 200 mmol/L dNTP, 1 unitof PlatinumTaq polymerase (Invitrogen), and 0.4mmol/L ofeach primer. Primer sequences were: forward, GTAGT-TATTTTTTTAGTTTTGGAT and reverse, AAAATCT-CCTCCAACCCTTC. PCR amplification was carried outfor 45 cycles consisting of 95�C for 30 seconds, 56�C for 30seconds, and 72�C for 30 seconds, with 2 minutes at 95�Cfor initial denaturation and 7 minutes at 72�C for finalelongation. PCR products were verified by ethidium

Table 2. Correlation of NEFL promotermethylation with clinical characteristics ofpatients with HNC

NEFL methylation analysis

Clinical variables Methylated Unmethylated Pa

No. 25 26Age 55.0 � 19.37 59.0 � 16.23GenderM 18 21F 7 5 NS

LocationsOral cavity 13 13Others 12 13 NS

DifferentiationWell and moderate 17 17Poor 8 9 NS

Node statusN0 9 13>N1 16 13 NS

StagesEarly 11 13Late 14 13 NS

Chemoresponseb

Responsive 6 19Nonresponsive 19 7 0.007

Abbreviation: NS, not significant.aFisher exact test, 2-sided. (P > 0.05).bCases with chemotherapy responses included responsive(completely and partially responsive) and nonresponsive (noresponse and progressive diseases).

NEFL Methylation and Head and Neck Cancer Chemoresistance

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bromide staining, and 1 mL of the products was cloned into aTA cloning vector (QIAGEN Inc.). Ten clones from eachcell lines were selected for sequencing.Methylation-specific PCR (MSP) was used to analyze

methylation of NEFL gene promoter in cell lines and frozenprimary tissues. MSP primers were designed to detect themethylation status of CpG in the promoter regions analyzedby bisulfite DNA sequencing. MSP primers included meth-ylation-specific primers (forward, AACGATTTTAAGTT-TATTCGTACGT; reverse, CCTTATTCTACTCCAAC-TCGTA) and unmethylated primers (forward, AATGATT-TTAAGTTTATTTGTATGT; reverse, ACCTTATTC-TACTACTCCAACTAT). For amplification of both meth-ylated and unmethylated alleles, 2 mL of bisulfite convertedDNAwere amplified in a 25-mL PCR reaction following thesame protocol and cycling parameters as for bisulfite DNAsequencing. PCR products were resolved in agarosegel electrophoresis and visualized by ethidium bromidestaining.NEFL promoter methylation of archival HNC tissues was

quantitatively analyzed by real-time MSP. Real-time MSPwas carried out according to previously published protocol(26). Briefly, bisulfite-converted DNA (2 mL) was amplifiedin a 20 mL reaction containing 2.5 mmol/L MgCl2, 200mmol/L each dNTP, 1 unit Platinum Taq Polymerase, 1�standard reaction buffer (Invitrogen), 0.5 mmol/L each prim-er, 0.04 mmol/L ROX reference dye, and 0.25 mmol/Lfluorescence labeled probe. Primers and probe were designedto specifically detect the methylated NEFL promotersequences, including forward primer, AACGATTT-TAAGTTTATTCGTACGT; reverse primer, CCTTATT-CTACTCCAACTCGTA; and probe, (6-FAM)ACGCG-CTCGATAAAACTAACGAAACGAT(TAMRA). Amplifi-cation was carried out with a Roche 480 real-time thermalcycling analyzer (Roche) for 45 cycles consisting of 95�C 15seconds, 60�C 30 seconds, and 72�C 2 minutes, with 10minutes at 95�C for initial denaturation. Amplification ofunmethylated b-actin (ACTB) gene promoter was used as anormalization control using the same PCR protocol as forNEFL.To ensure the specificity of the real-timeMSP analysis,every 96-well plate contained wells with bisulfite-convertedDNA from patient tissues and in vitro–methylated lympho-cyte DNA or the methylation-positive DNA from PCI51 cellline as positive controls and normal lymphocyte DNA ormethylation-negative DNA from PCI13 cell line as negativecontrols. Relative methylation level was calculated from2�(cycle number of NEFL�cycle number of ACTB), as describedpreviously (27). To determine the optimal cutoff value ofrelative methylation level for discriminating methylated andunmethylated samples, we conducted real-time quantitativeMSP (qMSP) analysis on serial mixtures of in vitro–methyl-ated DNA and methylation-negative lymphocyte DNA. Wefound that a relative methylation level of value 1.5 was able todetect 25% of methylated DNA copies in 75% of methyl-ation-negative DNA background, and therefore, should besufficiently sensitive to determine the methylation status ofprimary tumors containing both cancer cells and somenormaltissues.

NEFL expression construct and siRNAFull-length NEFL coding sequences was PCR amplified

and cloned into a Topo TA pcDNA3.1 expression vectorwith an N-terminal HA tag (Invitrogen). DNA sequencingwas used to verify the constructs, and expression efficiencywas determined by Western blot analysis using HA tagmonoclonal antibody (Santa Cruz Biotechnology). NEFLsiRNA and scrambled control were purchased from Sigma-Aldrich.

MTT assayCell lines with silenced expression of NEFL (O11 and

SCC25cp) were seeded in 96-well plates at a density of 5 �103 cells per well. After overnight culture, cells were trans-fected with NEFL expression construct and controlpcDNA3.1 vector with a Lipofectamine 2000 reagent (Invi-trogen). At 48 hours after transfection, cells were treatedwithvarious concentrations of cisplatin (0, 2.5, 5, 10, 20, 40, and80 mmol/L). MTT assays were conducted after 24 hours ofcisplatin treatment. MTT assays were conducted intriplicate.The effect of NEFL knockdown was examined in the

cisplatin-sensitive cell line PCI13. Cells (5 � 103) wereseeded in 96-well plates. After overnight culture, cells weretransfected with NEFL siRNA and scrambled control (5pmol per well) with Lipofectamine 2000 reagent (Invitro-gen). After 48 hours of transfection, cells were treated withvarious concentrations of cisplatin (0, 2.5, 5, 10, 20, 40, and80 mmol/L), and MTT assay was applied to examinecytotoxicity of cisplatin after 24 hours of treatment.

Colony formation assayEffects of NEFL expression on restoration of cisplatin

sensitivity were also tested by colony formation assay. TheO11 and SCC25cp cell lines (5 � 105 cells per well) wereplaced in a 6-well plate and transfected with 1 mg of NEFLexpression construct or mock vector after overnight culture.After a 24-hour transfection period, cells from each well weresplit into three 10-cm petri dishes and cultured for anadditional 24 hours. After 48 hours of transfection, cellswere treated with 20 mmol/L cisplatin and cultured for up to2 weeks with replacement of fresh medium with selectiveagent hygromycin in every 3 to 4 days. Colonies were stainedwith crystal violet and counted.

Western blotting and immunoprecipitationProtein levels ofNEFL inHNSCC cell lines were detected

byWestern blot analysis using monoclonal antibody againstN-terminal of NEFL protein (Sigma-Aldrich). Phosphory-lation of ribosomal protein S6 kinase (p70S6k), whichindicates the functional status of mTOR pathway (28) wasdetected using polyclonal antibody against phosphorylatedp70S6k protein (Cell Signaling Technology). Protein pre-parations and Western blotting were carried out as we havedescribed (29).To investigate a physical interaction of NEFL and tuber-

ous sclerosis complex 1 (TSC1), immunoprecipitationanalysis was conducted using antibodies against NEFL

Chen et al.





(Sigma-Aldrich) and TSC1 (Cell Signaling Technology).Preparation of cell lysates and procedures of immunopre-cipitation followed the previously published protocols.Briefly, 1 � 107 PCI13 cells were lysed in RIPA bufferwith proteinase inhibitors. Immunoprecipitation was carriedout at 4�C overnight in 1 mL reaction mixture including500-mg protein, 1� RIPA buffer and 5-mg TSC1 antibody.Protein A agarose beads (50 mL; Upstate Biotechnology,Inc.) were added and incubated for 1 hour at room tem-perature. After washing 3 times inWestern lysis buffer, beadswere boiled for 5 minutes in 30 mL of 2� sample buffercontaining b-mercaptoethanol, incubated on ice (1minute),and then centrifuged to remove agarose beads prior toelectrophoresis.

Statistical analysisCorrelations of NEFL methylation with clinical charac-

teristics including chemotherapy responses of patients withHNCwere tested by the Fisher 2-sided exact test. Prognosticvalue of NEFLmethylation for patient survivals was assessedby Kaplain–Meier analysis. Differences of cisplatin cytotoxicresponses of cell lines (PCI13, O11, and SCC25cp) betweenNEFL expression or siRNA knockdown and mock transfec-tants and effects ofmTOR inhibitor on cisplatin cytotoxicityof HNC cell lines were examined by unpaired t test.

ResultsMethylation-mediated silencing of NEFL expressioncorrelates with cisplatin resistance in HNC cell linesTwo pairs of isogenic cisplatin-sensitive parental HNC

cell lines and their resistant subclones (HN17B/HN17Bcpand SCC25/SCC25cp) were used for initial screening ofnovel methylated candidate genes associated with cisplatinresistance using our previously established approach (17).TheHN17Bcp and SCC25cp cell lines were treated with thedemethylating agent 5-aza-dC (10 mol/L) for 3 days toreactivate the genes epigenetically silenced in cisplatin-resis-tant clones. Gene expression profiles of cisplatin-sensitiveHN17B and SCC25 cell lines and gene expression changesof cisplatin-resistant HN17Bcp and SCC25cp lines by 5-aza-dC treatment were analyzed using microarray chipscontaining 18,400 genes (Affymetrix U133A 2.0). Forty-one genes were significantly downregulated (>3-fold) inboth HN17Bcp and SCC25cp cisplatin-resistant subclonescompared with their sensitive parental cells HN17B andSCC25. After treatment with the demethylating agent (10mmol/L of 5-aza-dC), derepression of 19 genes of those 41downregulated genes was observed (upregulated >3-fold) inHN17Bcp and SCC25cp cells. This group may representfrequently methylated genes of candidates in cisplatin-resistant HNC cell lines and tumors. Among the 19derepressed genes, NEFL was of particular interest, dueto its genomic location at chromosome 8p21, a locusfrequently deleted in several common cancer types includ-ing HNC (18–20, 30). Allelic loss of 8p21 has beenreported to associate with clinical outcomes of breastcancer and patients with HNC (20, 31), implicating its

possible association with progressive phenotypes and per-haps chemoresistance of tumors.To show the role of methylation-mediated silencing of

NEFL expression inHNC cisplatin resistance, we proceededto examine both expression and promoter CpG islandhypermethylation of NEFL in a panel of 15 HNC cell lineswith known cisplatin sensitivities and the immortalizedkeratinocyte cell line, Hacat. NEFL expression was greatlyreduced in 47% of the HNC cell lines (7 of 15; Fig. 1Aand Table 1), and expression of NEFL in Hacat was high.Expression status of NEFL was correlated with cisplatin

sensitivity. As shown in Fig. 1A and Table 1, NEFLexpression was observed in all cisplatin-sensitive HNC celllines (IC50<2mmol/L; PCI13,HN17B,HN22A,O12, andO29), andNEFL expression was absent or greatly reduced inall 5 cell lines displaying the highest level of cisplatinresistance (IC50 > 5 mmol/L; HN17Bcp, SCC25cp, Fadu,PCI51, andO28), and in 2 of 5moderately resistant cell lines(IC50 ¼ 2–5 mmol/L; HN38 and O11). The other 3moderately sensitive lines (SCC25, O13, and O22) showedhigh NEFL expression. Interestingly, both highly resistantisogenic subclones HN17Bcp and SCC25cp displayedmarkedly reduced NEFL expression as compared with theirsensitive parental cells HN17B and SCC25 (Fig. 1A).Significant reduction of NEFL expression in resistant celllines was also confirmed at the protein level by Westernblotting (Fig. 1B).To examine the role of promoter CpG island hypermethy-

lation in downregulation of NEFL expression, bisulfiteDNA sequencing was carried out on a total of 28 CpGs ina 303-bp genomic region in exon 1 of NEFL (Fig. 1C). InHN17Bcp, SCC25cp and PCI51 cell lines, which displayedvery low or no expression of NEFL, the interrogated CpGswere heavily methylated (Fig. 1A and C). In PCI13 HN17Band SCC25 cells, which displayed abundant NEFL expres-sion, the CpGs assessed were mostly completely unmethy-lated (Fig. 1A and C). On the basis of the patterns ofmethylated CpGs identified from plasmid cloning andbisulfite DNA sequencing, an MSP assay was designed todetect NEFL promoter methylation in the other HNC celllines. As shown in Fig. 1D, no methylation was detected inan immortalizedHacat cell, and 7 of 14 of theHNCcell linesanalyzed by MSP were positive for NEFL methylation.Furthermore, DNA methylation was found to stronglycorrelate with the gene expression of NEFL in HNC celllines. NEFL methylation was detected in all 6 cell lines withreduced or no expression of NEFL, and of the 8 cell lineswith NEFL expression, only one (O13) displayed partialNEFLmethylation (Fig. 1A and D and Table 1). Moreover,as shown in Fig. 1F, treatment with a demethylating agent(5-aza-dC) dramatically restored NEFL expression inSCC25cp, PCI51, and O28 (positive for NEFL methyla-tion) and had no effect on NEFL expression in PCI13 cells(negative for NEFL methylation), further suggesting a crit-ical role for promoter methylation in regulating the expres-sion of NEFL in HNC cells.To determine whether NEFL methylation was present in

primary tumors, we used the MSP assay on a small cohort of






patient tumors with matched normal oral mucosa. NEFLmethylation was detected in 5 of 7 HNC primary tumors,whereas none of 5matched normal oral mucosal tissues werepositive for NEFL methylation (Fig. 1E), providing therationale for further analysis of an expanded cohort of clinicalsamples.

Promoter hypermethylation of NEFL is frequentlyobserved in primaryHNC tumors and strongly associateswith chemoresistance and poor prognosis for patientswith HNCOn the basis of the earlier results in cell lines and

tumors, we analyzed NEFL promoter methylation in 51HNC cases with recorded responses to cisplatin-basedchemotherapy and long-term clinical follow-up. NEFLpromoter CpG island methylation was quantitativelyassessed using qMSP. We determined that the relativemethylation level of 1.5 as an optimal cutoff value fordetecting NEFL methylation in primary tumor tissuesand the reliability of our established qMSP analysis was

further confirmed by direct bisulfite DNA sequencing(Supplementary Fig. S1). On the basis of predeterminedcutoff value, 49% (25 of 51) of cases were consideredpositive for NEFL hypermethylation (Table 2). Further-more, NEFL methylation was associated with responsesto cisplatin-based chemotherapy. For nonresponders (noresponse to chemotherapy and progressive disease), NEFLmethylation was detected in 73% (19 of 26) of cases,whereas NEFL methylation was observed in only 24%(6 of 25) of complete or partial responders (Table 2). Thedifference in NEFL methylation frequency betweenresponsive and nonresponsive cases was highly significant[relative risk (RR), 3.045; 95% confidence interval (CI),1.459–6.355; P ¼ 0.007, Fisher exact test, 2-tailed]. Nosignificant correlation was found between NEFL meth-ylation and other clinical parameters, including age,gender, tumor stage, node status, and histologic differ-entiation (Table 2).NEFL methylation was also strongly predictive of both

diminished overall survival and disease-free survival for

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Figure 1. Downregulation of NEFL expression correlates with its promoter hypermethylation in HNC. A, NEFL mRNA expression was examined by real-timereverse transcriptase PCR in 14 HNC cell lines and an immortalized epidermal keratinocyte Hacat using GAPDH as a normalization control. B, proteinexpression of NEFL tightly correlates with mRNA expression of the gene. C, methylated CpGs at 50 flanking genomic region (þ269 to þ572 bp) of NEFLgene by plasmid cloning and bisulfite DNA sequencing in NEFL-expressing PCI13, HN17B, and SCC25 cells and NEFL-negative cells HN17Bcp, SCC25cp,and PCI51. Unmethylated CpGs are indicated as unfilled squares and methylated as black squares. D and E, NEFL promoter methylation in Hacat,HNC cell lines, and primary tumors by MSP using primers specific for methylated (M) and unmethylated (U) sequences. In vitro methylated DNA (MD) andnormal human lymphocyte DNA (NL) served as positive and negative controls for the MSP assay, respectively. F, treatment with 5-aza-dC restoredNEFL expression in SCC25cp, PCI51, and O28 cells with NEFL promoter methylation. T, treated; N, nontreated.

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patients with HNC who received cisplatin-based chemo-therapy. Patients with NEFL methylation had mediansurvival of 29 months (95% CI, 15.6–32.4) for overallsurvival and 28 months (95% CI, 16.4–31.5) for disease-free survival, whereas patients negative for NEFL methyl-ation had median survival of 67 months (95% CI, 47.6–144.3) for overall and 58 months (95% CI, 37.4–82.5) fordisease-free survival, respectively (Fig. 2). Difference inboth overall and disease-free survival betweenNEFL-methy-lated and -unmethylated patients was highly significant(P < 0.001, log-rank test).

NEFL functionally associates with HNC cellularresponse to cisplatinHaving shown clinical relevance for NEFL inactivation by

promoter hypermethylation, it was next of interest to furtherinvestigate the functional relevance of aberrant NEFLexpression and cisplatin response in HNC. To do so, wealtered the expression of NEFL in HNC cell lines and thenexamined the effect on cisplatin sensitivity in vitro. Thecisplatin-sensitive cell line PCI13 was used to examine theeffect of NEFL knockdown on cisplatin response. Knock-down efficiency of siRNA on NEFL expression was verifiedby immunoblotting using NEFL-specific antibody (Fig. 3A,left). PCI13 cells were transfected with 10 mmol/L of NEFLsiRNA or scrambled control siRNA. After 48 hours ofsiRNA incubation, PCI13 cells were treated with variousdoses of cisplatin for 24 hours and cell viability wasmeasuredby MTT assay. As shown in Fig. 3A, downregulation ofNEFL protein level by siRNA significantly enhanced resis-tance of PCI13 cells to cisplatin treatment. The IC50 valuefor PCI13 transfected with NEFL siRNA was 8.5 mmol/Lcompared with a 5.2 mmol/L IC50 value in controls. Incontrast, restoring full expression of NEFL in O11 andSCC25cp cell lines dramatically increased cisplatin sensi-

tivity, as determined by MTT assay (Fig. 3B). Mock-transfected O11 cells displayed an cisplatin IC50 value of18.3 mmol/L compared with 2.3 mmol/L cisplatin IC50value for NEFL-transfected O11 cells (Fig. 3B, middle).Similar results were obtained in SCC25cp cells (60 vs. 32mmol/L cisplatin IC50 value for mock-transfectedSCC25cp vs. SCC25cp exogenously expressing NEFL,respectively; Fig. 3B, right). Essentially similar results wereobtained by colony formation assays. NEFL-transfectedO11 and SCC25cp cells showed substantially reducedcolony formation as compared with mock-transfectedcontrols (Fig. 3C).

NEFL inhibits mTOR pathway activity by physicallyinteracting with an mTOR suppressor TSC1To further investigate the mechanism underlying NEFL-

mediated cisplatin resistance, we examined potential path-ways modulated by NEFL. NEFL is a critical component ofthe cytoskeleton, interacting with multiple protein targetsessential for cell plasticity, proliferation, migration, apopto-sis, and molecule transporters (32, 33). Among these, wefurther investigated the TSC1 tumor suppressor. TSC1 hasbeen shown to function as a molecular inhibitor of themTOR oncogenic pathway, reported to be frequently con-stitutively activated in cisplatin-resistant cancers (28). NEFLhas been shown to bind TSC1 and stabilize the TSC1/2complex (33). Therefore, we hypothesized that inactivationof NEFL, leading to abnormal activation of mTOR path-way, confers cisplatin resistance.To test this hypothesis, we examined the status of the

mTOR pathway and the effects of altered NEFL expressionon mTOR activity in HNC cell lines with and withoutNEFL silencing. Immunoprecipitation assays revealed anassociation between NEFL and TSC1 in PCI13 cells withendogenous NEFL expression (Fig. 4A). Consistent withour hypothesis, mTOR pathway activity was correlated withNEFL expression. Phosphorylation of p70S6k, a criticaldownstream mTOR activation substrate and known indi-cator of an active mTOR pathway (28), was detected inO11and SCC25cp cells with methylation silencing of NEFLand absent in NEFL-expressing PCI13 cells (Fig. 4B).Restoration of NEFL expression using pcDNA3.1/NEFLvector suppressed p70S6k phosphorylation in both O11and SCC25cp cells (Fig. 4C, right), whereas knockdownofNEFL expression resulted in increased phosphorylation ofp70S6k in PCI13 cells (Fig. 4C, left).NEFL modulation of mTOR pathway activation indicat-

ed a role for mTOR activity in cellular cisplatin responsive-ness. To test this possibility, we examined the effects of anmTOR inhibitor, RAD001, on cisplatin sensitivity in HNCcell lines with or without NEFLmethylation. Cell lines werefirst treated with 10 nmol/L of RAD001 for 24 hours andthen incubated with 5 and 10 mmol/L of cisplatin or mockcontrols. MTT assays were conducted 24 hours after cis-platin treatment. Treatment with RAD001 significantlyinhibited SCC25cp, O11, and O28 cell growth (Fig.4D). No inhibitory effect of RAD001 on the NEFL-expres-sing cell line PCI13 was observed (Fig. 4D), although

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Figure 2. NEFL promoter hypermethylation predicts reduced survival ofpatientswithHNCwho received cisplatin-based chemotherapy. A total of51 cases of patients with HNC that received cisplatin-basedchemotherapy were included in the study. All tumor tissues werecollected prior to clinical treatment. NEFL methylation was detected byquantitative real-time MSP and predictive value of NEFL methylation inpatient survival was assessed byKaplan–Meier survival curve. Differenceof patient survival between methylation-positive (Methylated) and-negative (Unmethylated) groups was statistically analyzed by log-ranktest.






increased cell growth was seen at the lower dose. For PCI51cells (hypermethylated and silenced NEFL), there was atrend for cell growth inhibition byRAD001, but this was notstatistically significant.

DiscussionNEFL gene encodes type IV intermediate filament hetero-

polymers that functionallymaintain the neuronal caliber andplay an important role in intracellular transport of neuro-transmitters to axons and dendrites (34). Germline muta-tions of NEFL gene are known to cause Charcot-Marie-Tooth disease types 1F, disorders of the peripheral nervoussystem that are characterized by distinct neuropathies (35).Although in cancer, a role for NEFL has not been previouslyshown; several lines of evidence implicate NEFL as apotential TSG. First, NEFL is located at chromosome

8p21, which has been identified as one of the genetic locifrequently affected by both heterozygous and homozygousdeletions in a variety of common human cancers, includingcancers of prostate and breast (18, 30). In HNC, loss ofheterozygosity at the NEFL locus was reported in more than40% of cases (20); moreover, deletions of the 8p21 NEFLlocus have been shown to predict poor prognosis in patientswith HNC (20). Second, neurofilament heavy peptide(NEFH), a functional partner of NEFL and a subunit ofneurofilament heteropolymers, has been recently shown tobe a putative TSG frequently inactivated by promoterhypermethylation in esophageal cancers (36). Third, NEFLhas been shown to interact with a number of functionalmolecular targets found in several critical cancer-associatedpathways. These NEFL-interacting targets include TSC1(33), a potent inhibitor of mTOR pathway and glutamatereceptor, ionotropic, N-methyl-D-aspartate 1 (NMDAR1,

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Figure 3. NEFL functionally associates with cisplatin responses in HNC cells. A, knockdown of NEFL conferred cisplatin resistance in HNC cells. PCI13 cellswere transfected with 10 mmol/L NEFL siRNA (NEFL-si) or scrambled control (Control-sc) for 48 hours and treatment of the transfected cells withvarious concentrations of cisplatin was continued for additional 24 hours.MTT assaywas conducted tomeasure cell growth 72 hours later. NEFL knockdownby transfected siRNA in PCI13 cells was verified by Western blotting using NEFL-specific antibody. NEFL knockdown significantly increases cisplatinresistance in PCI13 cells (unpaired t test, �,P < 0.05; ��,P < 0.01). B, enforced overexpression of NEFL resensitizedNEFL-negative cells to cisplatin treatment.O11 and SCC25cp cells were transfected with pcDNA3.1/NEFL expression construct or mock (empty vector) for 48 hours and further treated withvarious doses of cisplatin for 24 hours. Cell growth was measured by MTT 72 hours after drug treatment. Efficacy of pcDNA3.1/NEFL expression constructwas verified by transient expression of the construct in HEK293 cells and then examined by Western blotting. Restored expression of NEFL significantlyenhances cisplatin sensitivity in both O11 and SCC25cp cells (�, P < 0.05; ��, P < 0.01). C, enforced overexpression of NEFL reduced colony formationin O11 andSCC25cp cells. O11 andSCC25cp cells were transfectedwith pcDNA3.1/NEFL expression construct andmock vector for 48 hours and incubatedwith 10mmol/L of cisplatin for an additional 24hours.Cellswere further cultured inG418selectivemedium for 2weeks to allow the formationof visible colonies.Difference in numbers of colonies between NEFL and mock-transfected cells in both O11 and SCC25cp cells was significant (��, P < 0.01, unpaired t test).

Chen et al.





Grin1; ref. 37), a newly identified TSG in human esophagealcarcinoma (26). Taken together, these studies stronglysuggest that NEFL, and an intact neurofilament structure,may play a critical role in suppressing cancer initiationand/or progression.In the current study, we show that downregulation of

NEFL expression is seen in about 50%ofHNCcell lines andcorrelates with NEFL hypermethylation (Fig. 1). In addi-tion, NEFL CpG island promoter methylation was detectedin 49% ofHNC primary tumors (Table 2) but was absent inmatched normal oral mucosa tissues (Fig. 1D). Our studyprovides the first direct evidence that NEFL is epigeneticallysilenced in a human cancer and supports the possibility thatNEFL may be the putative TSG candidate located atchromosome 8p21, which may have important implicationsfor a broad range of common cancers with frequent 8p21deletions, including prostate, breast, bladder, lung, and livercancers (18, 30).

Drug resistance is a major limitation for the clinicalapplication of cisplatin and its analogues in HNC chemo-therapy and contributes significantly to morbidity andmortality from HNC (38). Despite intensive efforts forseveral decades, the mechanism(s) underlying resistance tocisplatin in patients with HNC remains unclear (39). Pre-vious studies have shown that p53 mutations, leading toinactivation of p53 protein and defects of apoptoticresponses to chemotherapeutic agents, are strongly associ-ated with cisplatin resistance in HNC (40). In addition, wehave reported a strong association between chromosome11q13 amplification, resulting in overexpression of GST pi,which detoxifies cisplatin-induced ROS, and decreasedchemotherapy responses in patients with HNC (8). In thisstudy, we show that NEFL is silenced by DNA methylationin cisplatin-resistant but not in cisplatin-sensitive HNC celllines and our functional studies further show a requirementfor NEFL in cisplatin-induced growth inhibitory responses.

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Figure 4. NEFLphysically interactswithmTORsuppressor TSC1, and functional inactivation ofNEFL results inmTORpathwayactivation inHNCcells. A, directbinding of NEFL and TSC1 in PCI13 cells was determined by immunoprecipitation (IP) using TSC1 antibody for IP and NEFL antibody for Western blotting(see Materials and Methods). B, activation of mTOR pathway was assessed by Western blot analysis detection of phosphorylation of p70S6k, a substrate ofmTOR pathway, using a specific antibody against phosphorylated p70S6k (pp70S6k). Phosphorylated p70S6k was detected in O11 and SCC25cp celllines with NEFL inactivation whereas absent in PCI13 cell with normal NEFL expression. C, NEFL expression, restored by transfecting an NEFL expressionvector, dramatically reduced the level of phosphorylated p70S6k inO11 andSCC25cp cells as comparedwithmock vector (left). In contrast, downregulation ofNEFL by siRNA (NEFL-si) in PCI13 cells induced p70S6k phosphorylation as compared with scrambled siRNA transfection (Control-sc). D, inhibition ofmTORactivity resensitizedcell lineswithNEFLmethylation to cisplatin. NEFL-negative cell lines (O11, SCC25cp, PCI51, andO28) and -positive cell linePCI13were treated with mTOR inhibitor RAD001 (10 nmol/L) for 24 hours and further incubated with 5 and 10 mm of cisplatin for additional 24 hours. Cellgrowth was measured by MTT assay. � indicates the difference in cell growth between RAD001 treatment and control groups (P < 0.05; unpaired t test).






Clinically, NEFL methylation was strongly associated inpatient responses to cisplatin-based chemotherapy. This isthe first report to identify downregulation of NEFL expres-sion by promoter methylation as a novel and importantmechanism for HNC chemoresistance.Predictive and prognostic markers for chemotherapy

responses and patient outcomes are highly valuable forguiding therapeutic strategies and improving survival ofpatients with cancer (41). Lack of such biomarkers forclinical use has had a substantial influence on the successfulmanagement of patients with HNC, as HNCmorbidity hasremained essentially unchanged for several decades (42).Weand others, using immunohistochemical analysis have iden-tified protein biomarkers that significantly correlated withchemotherapy responses and patient prognosis with HNC(43). In this study, patients with HNC with positive NEFLpromoter methylation were nearly 3 times more likely to beresistant to cisplatin-based chemotherapy than those with-out NEFL methylation, regardless of other clinical para-meters of the patients (Table 2). NEFL methylation alsopredicted reduced overall and disease-free survival forpatients with HNC who received cisplatin-based chemo-therapy (Fig. 2). These findings suggest that NEFL meth-ylation is a candidate independent predictive marker forresponse to cisplatin-based chemotherapy and that methyl-atedNEFLmay predict amoremalignant clinical phenotypein patients with HNC.We recognize that a limitation of thecurrent study is the small number of patients and that ouranalysis is not able to account for possible effects of otherreported prognostic factors, such as human papilloma virus(HPV) status and the combined radiation therapy.However,the strong correlation of NEFL methylation with chemo-therapy responses and patient outcomes, as well as ourfindings in vitro functional experiments, support furtheranalysis of a larger patient cohort.mTOR is a key downstream protein kinase of the phos-

phatidylinositol 3-kinase (PI3K)/AKT signaling pathwayand has reported to play a central role in controlling cancercell growth (44). AKT and mTOR function as "switch"proteins to modulate metabolism, the cell cycle, and apo-ptosis in cancer cells (45, 46). Constitutive activation ofmTOR has been found in common human malignancies,

including HNC (44) and has been shown to associate withresistance to several chemotherapeutic agents, includingcisplatin (28). In cultured cortical neurons,NEFLwas foundto physically bind to the putative tumor suppressor TSC1(33), amolecular inhibitor ofmTORactivity.We conducteda series of functional studies to show that NEFL is requiredfor suppression of mTOR activity in HNC cells. Further-more, downregulation of NEFL leads to mTOR activation,conferring cisplatin resistance. We further show that NEFLphysically associates with TSC1 in NEFL-expressing PCI13cells and modulates mTOR activity. Altered mTOR path-way activation also associates with NEFL-negative HNC.These findings provide convincing evidence that activationofmTORpathway, a consequence ofNEFL downregulationby promoter hypermethylation, is one of the mechanismsunderlying cisplatin resistance in HNC. However, we rec-ognize that inhibition of mTOR activity by RAD001 alonewas not able to restore cisplatin sensitivity in all cell lines withNEFL methylation (e.g., PCI51), suggesting that othermechanisms may be involved in NEFL-associated responsesto cisplatin. NEFL has been found to interact with a numberof target proteins that participate in various importantcellular processes. Among these, glutamate receptor, iono-tropic, NMDAR1, a subunit of NMDAR, has recently beenshown to be a novel TSG in esophageal carcinoma (26). Acrucial role for NMDAR in response to stress signals,including cisplatin in neurologic system (47), has also beenrecently reported. As the interaction of NEFL and NMDARmay represent an additional novel mechanism for NEFL-associated cisplatin responses in cancer cells, we are currentlyinvestigating this potential mechanism.

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

Grant SupportThis work was supported by awards #062545 from the Flight Attendant Medical

Research Institute (to Z.Guo) and by theNational Cancer Institute Integrative CancerBiology Program U54CA113001 (to K.P. Nephew).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received June 23, 2011; revisedDecember 13, 2011; acceptedDecember 29, 2011;published OnlineFirst January 13, 2012.

References1. Specenier PM, Vermorken JB. Current concepts for the manage-

ment of head and neck cancer: chemotherapy. Oral Oncol 2009;45:409–15.

2. Seve P, Dumontet C. Chemoresistance in non-small cell lung cancer.Curr Med Chem Anticancer Agents 2005;5:73–88.

3. Colevas AD. Chemotherapy options for patients with metastatic orrecurrent squamous cell carcinoma of the head and neck. J Clin Oncol2006;24:2644–52.

4. Borst P, Rottenberg S, Jonkers J. How do real tumors becomeresistant to cisplatin? Cell Cycle 2008;7:1353–9.

5. Higuchi E, Oridate N, Furuta Y, Suzuki S, Hatakeyama H, Sawa H,et al. Differentially expressed genes associated with CIS-diammi-nedichloroplatinum (II) resistance in head and neck cancer usingdifferential display and CDNA microarray. Head Neck 2003;25:187–93.

6. Andrews GA, Xi S, Pomerantz RG, Lin CJ, Gooding WE, Wentzel AL,et al. Mutation of p53 in head and neck squamous cell carcinomacorrelates with Bcl-2 expression and increased susceptibility to cis-platin-induced apoptosis. Head Neck 2004;26:870–7.

7. Parise O Jr, Janot F, Luboinski B, Massaad L, Albin N, Toussaint C,et al. Thymidylate synthase activity, folates, and glutathione system inhead and neck carcinoma and adjacent tissues. Head Neck1994;16:158–64.

8. Cullen KJ, Newkirk KA, Schumaker LM, Aldosari N, Rone JD, HaddadBR. Glutathione S-transferase pi amplification is associated withcisplatin resistance in head and neck squamous cell carcinoma celllines and primary tumors. Cancer Res 2003;63:8097–102.

9. Rudin CM, Yang Z, Schumaker LM, VanderWeele DJ, Newkirk K,Egorin MJ, et al. Inhibition of glutathione synthesis reverses Bcl-2-mediated cisplatin resistance. Cancer Res 2003;63:312–8.

Chen et al.





10. Shen L, Kondo Y, Ahmed S, Boumber Y, Konishi K, Guo Y, et al. Drugsensitivity prediction by CpG island methylation profile in the NCI-60cancer cell line panel. Cancer Res 2007;67:11335–43.

11. Baylin SB, Chen WY. Aberrant gene silencing in tumor progression:implications for control of cancer. Cold Spring Harb Symp Quant Biol2005;70:427–33.

12. Fang F, Balch C, Schilder J, Breen T, Zhang S, Shen C, et al. A phase 1and pharmacodynamic study of decitabine in combination with car-boplatin in patients with recurrent, platinum-resistant, epithelial ovar-ian cancer. Cancer 2010;116:4043–53.

13. Fang X, Zheng C, Liu Z, Ekman P, Xu D. Enhanced sensitivity ofprostate cancerDU145cells to cisplatinumby5-aza-20-deoxycytidine.Oncol Rep 2004;12:523–6.

14. Wei SH, Balch C, Paik HH, Kim YS, Baldwin RL, Liyanarachchi S, et al.PrognosticDNAmethylationbiomarkers in ovarian cancer.ClinCancerRes 2006;12:2788–94.

15. Olasz J, Mandoky L, Geczi L, Bodrogi I, Csuka O, Bak M. Influence ofhMLH1 methylation, mismatch repair deficiency and microsatelliteinstability on chemoresistance of testicular germ-cell tumors. Anti-cancer Res 2005;25:4319–24.

16. Chang X, Monitto CL, Demokan S, KimMS, Chang SS, Zhong X, et al.Identification of hypermethylated genes associated with cisplatinresistance in human cancers. Cancer Res 2010;70:2870–9.

17. Wu G, Guo Z, Chang X, Kim MS, Nagpal JK, Liu J, et al. LOXL1 andLOXL4 are epigenetically silenced and can inhibit ras/extracellularsignal-regulated kinase signaling pathway in human bladder cancer.Cancer Res 2007;67:4123–9.

18. KnowlesMA, ShawME, Proctor AJ. Deletionmapping of chromosome8 in cancers of the urinary bladder using restriction fragment lengthpolymorphisms and microsatellite polymorphisms. Oncogene1993;8:1357–64.

19. Lerebours F, Olschwang S, Thuille B, Schmitz A, Fouchet P, BuecherB, et al. Fine deletion mapping of chromosome 8p in non-small-celllung carcinoma. Int J Cancer 1999;81:854–8.

20. CoonSW,SaveraAT, ZarboRJ, BenningerMS,ChaseGA,Rybicki BA,et al. Prognostic implications of loss of heterozygosity at 8p21 and9p21 in head and neck squamous cell carcinoma. Int J Cancer2004;111:206–12.

21. Previtali SC, Zerega B, ShermanDL, Brophy PJ, Dina G, King RH, et al.Myotubularin-related 2 protein phosphatase and neurofilament lightchain protein, bothmutated inCMTneuropathies, interact in peripheralnerve. Hum Mol Genet 2003;12:1713–23.

22. Tokumaru Y, Yamashita K, Osada M, Nomoto S, Sun DI, Xiao Y, et al.Inverse correlation between cyclin A1 hypermethylation and p53mutation in head and neck cancer identified by reversal of epigeneticsilencing. Cancer Res 2004;64:5982–7.

23. Newkirk K, Heffern J, Sloman-Moll E, Sessions RB, Rasmussen AA,Andrews PA, et al. Glutathione content but not gamma glutamylcysteine synthetase mRNA expression predicts cisplatin resistancein head and neck cancer cell lines. Cancer Chemother Pharmacol1997;40:75–80.

24. Shiga H, Heath EI, Rasmussen AA, Trock B, Johnston PG, ForastiereAA, et al. Prognostic value of p53, glutathione S-transferase pi, andthymidylate synthase for neoadjuvant cisplatin-based chemotherapyin head and neck cancer. Clin Cancer Res 1999;5:4097–104.

25. Monney L, Sabatos CA, Gaglia JL, Ryu A, Waldner H, Chernova T,et al. Th1-specific cell surface protein Tim-3 regulates macrophageactivation and severity of an autoimmune disease. Nature 2002;415:536–41.

26. KimMS, Yamashita K, Baek JH, Park HL, Carvalho AL, OsadaM, et al.N-methyl-D-aspartate receptor type 2B is epigenetically inactivatedand exhibits tumor-suppressive activity in human esophageal cancer.Cancer Res 2006;66:3409–18.

27. Yang H, Kadia T, Xiao L, Bueso-Ramos CE, Hoshino K, Thomas DA,et al. Residual DNA methylation at remission is prognostic in adultPhiladelphia chromosome-negative acute lymphocytic leukemia.Blood 2009;113:1892–8.

28. Mabuchi S, Kawase C, Altomare DA, Morishige K, Sawada K, HayashiM, et al. mTOR is a promising therapeutic target both in cisplatin-sensitive and cisplatin-resistant clear cell carcinoma of the ovary. ClinCancer Res 2009;15:5404–13.

29. Fan M, Bigsby RM, Nephew KP. The NEDD8 pathway is required forproteasome-mediated degradation of human estrogen receptor (ER)-alpha and essential for the antiproliferative activity of ICI 182,780 inERalpha-positive breast cancer cells.Mol Endocrinol 2003;17:356–65.

30. Macoska JA, Trybus TM,BensonPD, SakrWA,GrignonDJ,WojnoKD,et al. Evidence for three tumor suppressor gene loci on chromosome8p in human prostate cancer. Cancer Res 1995;55:5390–5.

31. Seitz S, Werner S, Fischer J, Nothnagel A, Schlag PM, Scherneck S.Refined deletion mapping in sporadic breast cancer at chromosomalregion 8p12-p21 and association with clinicopathological parameters.Eur J Cancer 2000;36:1507–13.

32. Goehler H, Lalowski M, Stelzl U, Waelter S, Stroedicke M, Worm U,et al. A protein interaction network links GIT1, an enhancer ofhuntingtin aggregation, to Huntington's disease. Mol Cell 2004;15:853–65.

33. Haddad LA, SmithN, BowserM,Niida Y,Murthy V,Gonzalez-Agosti C,et al. The TSC1 tumor suppressor hamartin interacts with neurofila-ment-L and possibly functions as a novel integrator of the neuronalcytoskeleton. J Biol Chem 2002;277:44180–6.

34. Leermakers FA, Zhulina EB. How the projection domains of NF-L andalpha-internexin determine the conformations of NF-M and NF-H inneurofilaments. Eur Biophys J 2010;39:1323–34.

35. Houlden H, Reilly MM. Molecular genetics of autosomal-dominantdemyelinating Charcot-Marie-Tooth disease. Neuromolecular Med2006;8:43–62.

36. Kim MS, Chang X, LeBron C, Nagpal JK, Lee J, Huang Y, et al.Neurofilament heavy polypeptide regulates the Akt-beta-catenin path-way in human esophageal squamous cell carcinoma. PLoS One2010;5:e9003.

37. Ehlers MD, Fung ET, O'Brien RJ, Huganir RL. Splice variant-specificinteraction of the NMDA receptor subunit NR1 with neuronal interme-diate filaments. J Neurosci 1998;18:720–30.

38. Kelland L. The resurgence of platinum-based cancer chemotherapy.Nat Rev Cancer 2007;7:573–84.

39. Le Tourneau C, Chen EX. Molecularly targeted agents in the treatmentof recurrent or metastatic squamous cell carcinomas of the head andneck. Hematol Oncol Clin North Am 2008;22:1209–20, ix.

40. Schumaker L, Nikitakis N, Goloubeva O, Tan M, Taylor R, Cullen KJ.Elevated expression of glutathione S-transferase pi and p53 conferspoor prognosis in head and neck cancer patients treated with che-moradiotherapy but not radiotherapy alone. Clin Cancer Res2008;14:5877–83.

41. Zalcman G, Bergot E, Creveuil C, Levallet G, Lechapt E. Integratingbiomarkers into clinical trials: methodological issues for a new para-digm in nonsmall cell lung cancer. Curr Opin Oncol 2011;23:106–11.

42. Brockstein BE, Vokes EE. Head and neck cancer in 2010: maximizingsurvival and minimizing toxicity. Nat Rev Clin Oncol 2011;8:72–4.

43. Cullen KJ, Schumaker L, Nikitakis N, Goloubeva O, Tan M, Sarlis NJ,et al. beta-Tubulin-II expression strongly predicts outcome in patientsreceiving induction chemotherapy for locally advanced squamouscarcinoma of the head and neck: a companion analysis of the TAX324 trial. J Clin Oncol 2009;27:6222–8.

44. ZoncuR, EfeyanA, Sabatini DM.mTOR: fromgrowth signal integrationto cancer, diabetes and ageing. Nat Rev Mol Cell Biol 2011;12:21–35.

45. Foster DA. Phosphatidic acid signaling to mTOR: signals for thesurvival of human cancer cells. Biochim Biophys Acta 2009;1791:949–55.

46. Castedo M, Ferri KF, Kroemer G. Mammalian target of rapamycin(mTOR): pro- and anti-apoptotic. Cell Death Differ 2002;9:99–100.

47. Gozdz A, Vashishta A, Kalita K, Szatmari E, Zheng JJ, Tamiya S, et al.Cisplatin-mediated activation of extracellular signal-regulated kinases1/2 (ERK1/2) by inhibition of ERK1/2 phosphatases. J Neurochem2008;106:2056–67.






2012;10:305-315. Published OnlineFirst January 13, 2012.Mol Cancer Res Baishen Chen, Ju Chen, Michael G. House, et al. ChemoresistanceRole of Neurofilament Light Polypeptide in Head and Neck Cancer

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