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Cancer Biology and Signal Transduction

MEK Inhibition Overcomes Cisplatin ResistanceConferred by SOS/MAPK Pathway Activation inSquamous Cell CarcinomaLi Ren Kong1, Kian Ngiap Chua1,Wen Jing Sim2, Hsien Chun Ng2, Chonglei Bi1,Jingshan Ho3,4, Min En Nga5,Yin Huei Pang5,Weijie Richard Ong6, Ross Andrew Soo1,3,4,Hung Huynh6,Wee Joo Chng1,3,4, Jean-Paul Thiery1,2,7, and Boon Cher Goh1,3,4

Abstract

Genomic analyses of squamous cell carcinoma (SCC) have yetto yield significant strategies against pathway activation toimprove treatment. Platinum-based chemotherapy remains themainstay of treatment for SCC of different histotypes either as asingle-agent or alongside other chemotherapeutic drugs or radio-therapy; however, resistance inevitably emerges, which limits theduration of treatment response. To elucidate mechanisms thatmediate resistance to cisplatin, we compared drug-induced per-turbations to gene and protein expression between cisplatin-sensitive and -resistant SCC cells, and identified MAPK–ERKpathway upregulation and activation in drug-resistant cells.

ERK-induced resistance appeared to be activated by Son ofSevenless (SOS) upstream, and mediated through Bim degra-dation downstream. Clinically, elevated p-ERK expression wasassociated with shorter disease-free survival in patients withlocally advanced head and neck SCC treated with concurrentchemoradiation. Inhibition of MEK/ERK, but not that of EGFRor RAF, augmented cisplatin sensitivity in vitro and demon-strated efficacy and tolerability in vivo. Collectively, these find-ings suggest that inhibition of the activated SOS–MAPK–ERKpathway may augment patient responses to cisplatin treatment.Mol Cancer Ther; 14(7); 1–11. �2015 AACR.

IntroductionLung cancer remains a leading cause of cancer-related mor-

tality, accounting for an estimated 1.4 million deaths in 2010(1). Squamous cell carcinoma (SCC) arises primarily fromcigarette smoking and is the second most common lung cancersubtype. Major advances have been made in the identificationof oncogenic driver mutations in adenocarcinoma of the lung,but these efforts have been less successful in SCC. Althoughtargeting EGFR mutations in the tyrosine kinase domain, ROS1and EML4-ALK rearrangements have yielded impressive gainsin therapeutic efficacy in adenocarcinoma of the lung, as havethe use of bevacizumab and pemetrexed chemotherapy fornon-SCC histologies (2–5); for SCC histologies, however,

cetuximab remains as the only approved noncytotoxic agentto date (6).

Aberrant activations in oncogenic pathways by EGFR muta-tions and ALK fusions are rare in lung SCC (7). It is generallyconsidered that SCC is associated with few genetic aberrationsthat are actionable with targeted small-molecule compounds orantibodies. Hence, platinum-based chemotherapy remains thestandard-of-care treatment for metastatic SCC (8), usually in thecombination with a second agent, such as gemcitabine or pacli-taxel (9, 10). Cisplatin-based chemotherapy has a response rate of30% to 40% in patients with SCC; however, platinum resistanceinevitably develops, limiting the success of treatment (11). Unlikenon-SCC histologies, second-line agents are limited. Accordingly,there is an urgent need to develop novel combinations for thetreatment of SCC that leverage on understanding the pathwaysunderpinning cisplatin resistance.

In this study, we performed exome sequencing on lung SCCtumors to define the mutational profile of SCC. Using severalgenomics and proteomics-based technologies, we conductedparallel investigations to (i) elucidate the pathways associatedwith cisplatin resistance in lung SCC and (ii) dissect potentialchemosensitizing mechanism(s) that enhance cisplatin cyto-toxicity. These two approaches led us to the discovery thatactivation of the MAPK–ERK pathway under cisplatin treatmentpressure could confer cisplatin resistance. Importantly, weshowed that disruption of the ERK signaling pathway viapharmacologic inhibition improved both in vivo and in vitroefficacy of cisplatin treatment. As SCC of the lung sharescommon etiology, demographics, and molecular biology withSCC of the head and neck (HNSCC), we extended these

1Cancer Science Institute of Singapore, National University of Singa-pore, Singapore. 2Institute of Molecular and Cell Biology, A�STAR,Singapore. 3Department of Hematology-Oncology, National Univer-sity Hospital, Singapore. 4National University Cancer Institute, Singa-pore. 5Department of Pathology, National University Hospital, Singa-pore. 6National Cancer Centre, Singapore. 7Department of Biochem-istry, Yong Loo Lin School of Medicine, National University of Singa-pore, Singapore.

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

Corresponding Author: Boon Cher Goh, National University Cancer Institute,Singapore, 7th floor, NUHS Tower Block, 1E Lower Kent Road, Singapore 119228,Singapore. Phone: 65-6772-4617; Fax: 65-6777-5545; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-15-0062

�2015 American Association for Cancer Research.

MolecularCancerTherapeutics

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investigations to include HNSCC. Finally, our study also pro-vided conclusive evidence for the potential role of ERK phos-phorylation as a predictive biomarker of clinical outcomes forcisplatin-treated SCC patients.

Materials and MethodsCell lines

All lung cell lines were purchased from the ATCC. Lung SCClines H226, H2170, H520, H596, and ChaGo-k-1 were culturedin RMPI-1640, Calu-1 in McCoy's 5a, SK-MES-1 in DMEM,SW900 in Leibovitz', H1869 in ACL-4 and H2066 in HITESmedium. Lung normal fibroblast cells MRC-5, IMR-90, and WI-38 were cultured in EMEM medium. Head and neck SCC(HNSCC) cell lines were provided by Dr Timothy A. Chan fromMemorial Sloan –Kettering Cancer Center. HNSCC cell linesUMSCC1, SCC13, and 93-VU-147 were cultured in DMEM. Allmedia were supplemented with 10% FBS, 2 mmol/L L-gluta-mine, 100 mg/mL streptomycin and 100 U/mL penicillin. ACL-4and HITES were further supplemented with other nutrients asrecommended by the ATCC. All cell lines were authenticated byshort tandem repeat analysis using GenePrint 10 System (Pro-mega) in 2013.

Cell viability assayCells were cultured (2,000–4,000 cells/well) on 96-well plate

and coincubated with cisplatin for 72 hours. At the end of thetreatment, 20mL/well of CellTiter 96AQueousOne Solution (MTS)solution (Promega)was added and incubated at 37�C for 3 hours.Absorbance was measured at 490 nm. All data points were set upwith four replicates for each experiment. The IC50 was calculatedby GraphPad Prism software (GraphPad Software).

Anchorage-independent soft agar assaySoft agar was mixed with culture media to form agar layers

with different concentration: A bottom layer with 0.6% agar; amiddle layer comprised of 5,000 to 10,000 cells suspended in0.36% agar; a top layer with complete media containing com-pound or compound combinations at various doses. Colonieswere allowed to form for 2 to 4 weeks. On the final day of assay,40 mL of MTT solutions (5 mg/mL in PBS) were added andincubated at 37�C for 4 hours. Images of each well wereacquired with Epson V330 Photo scanner. The number andsize of the colonies were analyzed and quantified using ImageJ(NIH). The percentage of cell colony formation was calculatedrelative to vehicle control treated cells.

MicroarrayTotal RNA from cell lines were harvested with the RNeasy Mini

Kit (Qiagen) 8 hours post-cisplatin treatment to capture cisplatin-induced transcriptional changes before activation of apoptosis.Gene-expression profiling was conducted in technical duplicateswith AffymetrixHumanGeneChip 1.0ST array (Affymetrix) as perthe manufacturer's instructions. Data normalization and analysiswere performed with GeneSpring Software V12. Genes with anabsolute fold change >1.5 across the comparison groups weredenoted as commonly altered genes (Fig. 1C; SupplementaryTable S1). Pathway analysis was conducted with the publiclyavailable Database for Annotation, Visualization and IntegratedDiscovery (DAVID) Bioinformatics Resources 6.7 Software (12).The gene-expression data have been submitted to GEO repository(GEO accession number: GSE66549)

Proteome profiler antibody arrayWhole-cell lysates from cells treated with cisplatin or vehicle

controls were prepared 48 hours after drug incubation andnormalized according to protein concentrations. Cell lysates(250 mg) were mixed with biotinylated detection antibodies andsubjected to Proteome Profiler Phospho-MAPK arrays (R&DSystems). Signal detection was performed with Chemilumines-cence detection system (GE Healthcare) and densitometric datawas analyzed with ImageJ. The analyses were performed bysubtracting the background density signal, and normalizing withpositive controls on each membrane. Comparisons between thetreatment groups where then made using normalized values, andexpressed in fold change to the vehicle control group.

Total protein extraction and Western blottingCultured cells were rinsed with PBS and lysed with Cell Lytic

buffer (Sigma; 150 mmol/L NaCl, 0.41% bicine, 2% EDTA)supplemented with complete protease inhibitor cocktail (Roche)and phosphatase inhibitor cocktail (Roche). Protein concentra-tion was quantified and normalized samples were resolved withBio-Rad SDSPAGE system on 8% to 12% protein gels. Signaldetection was performed with chemiluminescence detection sys-tem (GE Healthcare). Blotting was performed with the followingantibodies: PARP, Caspase-3, p-ERK1/2 (Thr202/Tyr204), totalERK1/2, p-p38 (Thr180/Tyr182), total p38, p-BRAF (Ser445),p-MEK1/2 (Ser217/221), total MEK1/2, SOS1, p-JNK (Thr183/Tyr185), total JNK, p-Src (Tyr416), total Src, b-actin, and HRP-conjugated anti-rabbit from Cell Signaling Technology; SOS2from Abcam.

Immunofluorescent staining and image analysisCells were seeded and treated with cisplatin at the indicated

doses for 48 hours. At the assay endpoint, cells were fixedwith 4%paraformaldehyde andpermeabilizedwith 0.3%Triton-X prior tostaining. Samples were then immunostained with p-ERK1/2 anti-body (Cell Signaling Technology; 1:500), followed byAlexa-Fluor488 secondary antibody (Life Technologies; 1:1,000), andHoechst 33342 nuclear counterstain (Sigma Aldrich; 1:2,000).Protocols for image acquisition and analysis were adapted fromimage cytometry methods as described previously (13).

RNA interferenceFor gene knockdown, MEK1 siRNA (sequence: 50-

GUGAAUAAAUGCUUAAUAATT-30), MEK2 siRNA (sequence:50-GCAUUUGCAUGGAACACAUTT-30), ERK1 siRNA (sequence:50-CGUCUAAUAUAUAA-AUAUATT-30), ERK2 siRNA (sequence:50-GUUCGAGUAGCUAUCAAGATT-30), SOS1 siRNA (sequence:50-GGAGGUCCUAGGUUAUAAATT-30), SOS2 siRNA (sequence:50-UCAUUAUCGUAGUACUCUATT-30), Bim siRNA (sequence:50-GAGACGAGUUUAACGCUUATT-30), Src siRNA (sequence: 50-AAGCAGTGCCTGCCTATGAAA-30), and AllStar negative controlsiRNA were obtained from Qiagen. Transfection (50 nmol/LsiRNA for each target in each reaction) was conducted withJetPRIME reagent (Polyplus Transfection). For gene overexpres-sion, MAP2K1 (D67N) and empty vector (pCMV-entry) plasmidconstructs were obtained from OriGene Technologies. Plasmidtransfection was conducted with ViaFect reagent (Promega).

Clinicopathologic and immunohistochemistry analysesImmunohistochemistry was performed on formalin-fixed, par-

affin-embedded (FFPE) sections of HNSCC and xenograft tumors

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as described in the Supplementary Methods. Preparation andscoring of clinical samples was performed by two pathologistsin a blinded manner based on the staining intensity in thecytoplasm and/or the nucleus (0, negative; 1, weak; 2, moder-ate; and 3, strong) and the fraction of stained tumor cells(0%–100%). The total immunoreactive score (IRS) was deter-mined by multiplying the intensity score with the fraction ofstained cells (0–300). Survival outcomes were defined bydisease-free survival (DFS). Photomicrograph images acquiredfrom xenograft tumors for each of the treatment groups wereprocessed with an adapted image processing workflow usingImageJ as described previously (14).

Xenograft studiesPatient-derived xenograft (PDX) model SCC10-0508 was

established and maintained in 9- to 10-week-old SCID mice asdescribed previously (15). In brief, SCC10-0508 tumors wereexcised and minced into small 1 to 2 mm3 dices before beingimplanted s.c. into left and right flanks of SCID mice. Mice wereassigned treatment in to four stratified groups based on averagetumor volume from the left and right flanks of themouse: Vehicle(30% w/v Captisol), PD0325901, Cisplatin, PD0325901 þ Cis-platin (n ¼ 5 animals/group). Cisplatin was provided in 0.9%NaCl and given at 2 mg/kg by i.p., once weekly for 3 weeks.PD0325901was formulated in 30% (w/v) Captisol (CyDex) at 10mg/kg and given orally, once daily until mice were terminated.

Mice were treated 9 days after tumor implantation (tumorvolume �200 mm3). Tumor growth (length � width � width �3.14159/6) was monitored. Bodyweight at sacrifice and tumorsamples were collected when mice were sacrificed 26 days aftertreatment commenced. Animals were housed in accordance toIACUC guidelines.

Statistical analysisAll experiments were conducted three times unless stated

otherwise. Results are expressed asmean� SD. Statistical analysisfor the comparison between two groups was conducted using theStudent t test, whereas comparisons betweenmultiple groups wasconducted usingANOVA. TheGehan–Breslow testwas performedto test the statistical differences in DFS. All tests were two-sidedand the significance level was set at P < 0.05.

ResultsPaucity of somatic DNA alterations and presence of cisplatin-resistant subtypes in lung SCC tumors

To look for oncogenic mutations in SCC tumors among Asianpatients that would be responsive tomolecular targeting, targetedexome sequencing were performed on 45 lung SCC samples afterpathway-specific PCR-based exon enrichment. Consistent withthe findings by The Cancer Genome Atlas (TCGA; ref. 16), com-mon oncogenic lesions in lung adenocarcinoma were rarely

Figure 1.Cisplatin resistance in SCC cells isassociated with activation of MAPK/ERK pathway. A, lung SCC cells andnormal lung fibroblast cell lines weretreated with cisplatin (CDDP) for 72hours and cell viabilitywas determinedwith CellTiter assay. B, H226, H520,H2170, H520 and ChaGo-k-1 cells weregrown in soft agar to assess cisplatinsensitivity under anchorage-independent conditions. The numberof colonies formed under increasingcisplatin concentrations weredetermined by MTT staining. Data in Aand B are represented as mean IC50 �SD (n¼ 3). C, Venn diagram representsthe number of altered genes (foldchange > 1.5) identified by AffymetrixMicroarrays among the cisplatin-sensitive (H596, H1869, H226, andChaGo-k-1) and cisplatin-resistant(Calu-1, H2170, SW900, and H2066)cell lines after treatment with cisplatinat respective IC50. D, Calu-1 and H596cells were treated with cisplatin at10 mmol/L and 5 mmol/L, respectively,for 48 hours with the protein lysatesharvested for phospho-kinaseprofiling. Dots representing p-ERK1/2are highlighted in red, and p-p38 inblue. E, immunoblotting wasperformed in lung SCC cell lines aftercisplatin treatment to evaluate thechanges in phosphorylated proteinlevels of the kinases identified in D.

ERK as Predictive and Therapeutic Marker for SCC

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found in SCC tumors. Despite observing EGFR mutations in82% of patient samples (37/45), these alterations were almostexclusively codon-silent events (30/37); whereas nonsynon-ymous mutations in KRAS, BRAF, AKT1, and PIK3CA were rare(<10%; Supplementary Fig. S1). Moreover, oncogenic muta-tions were rare among the 10 lung SCC cell lines used in thisstudy as curated from the Sanger Catalogue of Somatic Muta-tions in Cancer (COSMIC) Database, with only two cell lines(Calu-1 and SW900) harboring mutated KRAS and none withmutated EGFR.

Using 10 tumor-derived and three normal lung fibroblast-derived cell lines as representative study models, we determinedthe IC50 of cisplatin via cell proliferation and anchorage-inde-pendent assays.Whereas lungfibroblast cell lineswere sensitive tocisplatin (IC50 < 5 mmol/L), lung SCC cell lines displayed differ-ential cisplatin sensitivity, with a 6-fold difference between themost sensitive andmost resistant cells. Four SCC cell lines (H596,H1869, H226, and ChaGo-k-1) had IC50 < 10 mmol/L (cisplatinsensitive) and six cell lines (H2066, SW900, H2170, Calu-1,H520, and SK-MES-1) had IC50 > 10 mmol/L (cisplatin-resistant; Fig. 1A). Consistent with these observations, cisplatinstrongly inhibited colony formationofH226 andChaGo-k-1 cells(IC50 < 100 nmol/L), whereas H520, H2170 and Calu-1 weremore resistant to cisplatin (IC50 > 200 nmol/L; Fig. 1B; Supple-mentary Fig. S2).

ERK phosphorylation is associated with cisplatin resistanceGene-expression profiling was performed to identify putative

genes and cellular pathways that may be associated with cellularresponse to cisplatin. Cell lines were grouped according to theircisplatin IC50 (H596, H1869, H226, and ChaGo-k-1 as sensitive;Calu-1, H2170, SW900, and H2066 as resistant). Upon exposureto cisplatin, a total of 150 and 382 genes were found to be alteredin the cisplatin-resistant and -sensitive study sets, respectively(fold change > 1.5; P < 0.05). Of these genes, 123 genes werecommonlymodulated in both study sets (Fig. 1C). To identify keyfactors that regulate cellular sensitivities to cisplatin in lung SCC,we compared themutually exclusive genes that were differentiallyregulated between the resistant (27) and sensitive (259) pheno-types (Fig. 1C; Supplementary Table S1) by using Kyoto Ency-clopedia of Genes and Genomes (KEGG) pathway analysis (P <0.05). Cisplatin treatment downregulated genes associated toErbB, MAPK, and phosphatidylinositol pathways within thecisplatin-sensitive cell lines (Table 1). Conversely, such alterationswere not observed in the resistant cell lines.

To verify the pathways identified from gene-expression profil-ing in terms of phosphorylated protein regulation, we performedparallel phospho-kinase arrays covering the MAPK and phospha-tidylinositol pathways on Calu-1 (resistant) andH596 (sensitive)cells in the presence or absence of cisplatin (Fig. 1D). The signalintensities of each pair of duplicate spots were quantified (Sup-plementary Fig. S3A and B), and the perturbations to proteinexpression profiles of the two cells lines were compared. Bycontrast, p-ERK1/2 showed largest increase in expression (1.87-fold) in cisplatin-resistant cells (Calu-1) after cisplatin treatment,whereas p-p38a showed themost apparent decrease in expression(0.14-fold) in cisplatin-sensitive cells (H596) (SupplementaryFig. S3C). Conversely, cisplatin suppressed Akt phosphorylationin both cell lines (Supplementary Fig. S3A and S3B).

The observed changes in kinase phosphorylation were nextvalidated by Western blot analyses on independent cell lysates ofcisplatin-sensitive (H596 and H226) and -resistant (Calu-1,H2170, and H520) cells. Interestingly, ERK1/2 but not p38, wereconsistently phosphorylated in resistant cells, but dephosphory-lated in sensitive cells with increasing cisplatin concentrations(Fig. 1E).Moreover, cisplatin treatment triggered PARP cleavage atlower doses in cisplatin-sensitive cells (H596 and H226) ascompared with that in cisplatin-resistant cells (Calu-1 andH520; Fig. 1E). Collectively, these data indicate that p-ERK ispositively associated with cisplatin resistance in SCC cells.

MAPK/ERK upregulation in cisplatin-treated cells is mediatedthrough SOS and leads to Bim degradation

The RAS–RAF–MEK–ERK signaling axis is a complex networkthat triggers cell growth and/or cell death through downstreamstimulation of transcription factors. Upon activation, RAS bindsto RAF and the resulting complex phosphorylates MAPK/ERKkinase (MEK1/2), which, in turn, mediates activation and nuclearlocalization of ERK1/2 (17, 18). Here, we showed that the phos-phorylation patterns of both MEK and BRAF were consistent withthat of p-ERK1/2 in both cisplatin-resistant and -sensitive cells(Fig. 2A). Furthermore, cisplatin treatment induced the cyto-plasmic accumulation and nuclear translocation of p-ERK1/2 inthe cisplatin-resistant Calu-1 cells, but reduced p-ERK1/2 in thecisplatin-sensitive H596 cells (Fig. 2B and C).

Thus far, our data have suggested a correlation between ERKand cellular response to cisplatin, but the factor(s) leading to thedifferential regulation of p-ERK remains unclear. Importantly, thephosphorylation of EGFR was not affected by cisplatin treatmentin SCC cells (data not shown). To identify the alternative factor(s)

Table 1. Pathways downregulated by cisplatin in cisplatin-sensitive lung SCC cell lines identified by KEGG pathway analysis (P < 0.05)

Pathways Regulation Terms Genes

KEGG (P < 0.05) Down Pathways in cancer PRKCA, PLD1, CBLB, MSH3, GSK3B, SOS2, SMAD3, CDK6, GLI3, AKT3, DAPK1T-cell receptor signaling pathway CBLB, GSK3B, SOS2, VAV2, AKT3, TECChronic myeloid leukemia CBLB, SOS2, SMAD3, CDK6, AKT3Colorectal cancer MSH3, GSK3B, SOS2, SMAD3, AKT3Tight junction PRKCA, MAGI3, INADL, ASH1L, PRKCH, AKT3ErbB signaling pathway PRKCA, CBLB, GSK3B, SOS2, AKT3Non–small cell lung cancer PRKCA, SOS2, CDK6, AKT3MAPK signaling pathway PRKCA, MAP4K3, NF1, SOS2, RAPGEF2, STK3, AKT3, MAP2K5Glioma PRKCA, SOS2, CDK6, AKT3Pancreatic cancer PLD1, SMAD3, CDK6, AKT3Phosphatidylinositol signaling system PRKCA, INPP4B, PIP4K2A, ITPR2B-cell receptor signaling pathway GSK3B, SOS2, VAV2, AKT3Axon guidance EPHA4, GSK3B, SEMA3A, SLIT2, SRGAP2Fc epsilon RI signaling pathway PRKCA, SOS2, VAV2, AKT3

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that modulated MAPK/ERK signaling, we focused on the sup-pressed genes in cisplatin-treated H596 cells that were associatedwith MAPK signaling (Table 1). Interesting, the genes that wereprofoundly suppressed included Son of Sevenless homolog 2(SOS2), a key regulator of the MAPK signaling cascade. SOS is aguanine nucleotide exchange factor that facilitates the GDP toGTP exchange of RAS, thereby allowing it to interact with RAF andstimulate theMAPK–ERK signaling cascade (19, 20). Accordingly,we hypothesized that SOS positively modulates p-ERK activity inSCC cells. Cisplatin treatment upregulated both SOS1 and SOS2in the resistant cell lines that displayed strong ERK activation(H520 and Calu-1) but their expression was either reduced(H596) or unchanged (H226) in sensitive cell lines (Fig. 2D).Mechanistically, we showed that siRNA silencing of SOS1 andSOS2 was associated with decreased ERK1/2 phosphorylation,with the combined knockdown of both SOS molecules demon-strating the most significant effects (Fig. 2E). Moreover, depletionof SOS abrogated cisplatin-associated ERK phosphorylation inCalu-1 cells (Fig. 2F).

Activation of ERK1/2 is known to promote phosphorylationand degradation of proapoptotic Bim, and eventually confercisplatin resistance (21, 22). Consistent with these findings, basal

p-ERK1/2 led to Bim phosphorylation in Calu-1 and H520 cellstransfected with control siRNA; while inhibition of ERK by SOSsilencing reduced Bim phosphorylation and increased Bim accu-mulation (Fig. 2E). Notably, silencing of either Erk1/2 orMEK1/2reducedmobility shift and increased stabilization of Bim in Calu-1 cells (Supplementary Fig. S4D). Taken together, we postulate acisplatin-induced signal transduction pathway involving SOSupregulation, activation of the MAPK–ERK axis, and Bim degra-dation that renders cisplatin resistance in SCC cells throughprevention of Bim-induced apoptosis.

Activation of the MAPK–ERK pathway confers survivaladvantage to SCC cells treated with cisplatin

To address the functional role of the MAPK–MEK–ERKpathway in cisplatin resistance, we found that silencingERK1/2 increased cisplatin sensitivity in a time- and concen-tration-dependent manner (Fig. 3A; Supplementary Fig. S4A),with a significant reduction in the cisplatin IC50 by more than2-fold in all three cisplatin-resistant lung SCC cell lines (Calu-1,H2170, and H520; Fig. 3B; Supplementary Fig. S4B and S4C).Consistent with our findings of upstream activation, siRNAsilencing of MEK1/2 abrogated ERK1/2 phosphorylation and

Figure 2.Cisplatin regulates MAPK/ERKactivity in lung SCC cells through SOSupstream and Bim downstream. A,lysates from Fig. 1E were evaluated byimmunoblotting to determine thechanges in upstream kinases of MEK/ERK. B, immunofluorescent stainingwas performed on p-ERK1/2 in Calu-1and H596 cells after cisplatin (CDDP,10 mmol/L and 3 mmol/L, respectively)treatment for 48 hours. C, high-content analyses of p-ERK1/2fluorescence intensity in thecytoplasm and nuclear fractions wereperformed, and are presented asmean � SD (n ¼ 3). D, lysatesfrom Fig. 1E were evaluated byimmunoblotting to investigate theexpression of SOS1 and SOS2 aftercisplatin treatment. E, immunoblotsshowing the effects of SOS silencingon ERK1/2 and Bim. 50 nmol/L siRNAwas used per transfection for eachtarget. F, effects of silencing SOS1 andSOS2 on ERK phosphorylation wereevaluated after cisplatin treatment.50 nmol/L siRNA was used pertransfection.






reduced cisplatin IC50 in Calu-1 cells (Fig. 3C). Conversely, ascompared with empty vector (EV) control, overexpression ofD67N MEK1 mutant constitutively induced p-MEK1 andp-ERK1/2 in H596 cells (Fig. 3D), and this abrogated Bim-induced apoptosis, as indicated by the reduced cleavage ofPARP and caspase-3 (Fig. 3D). By generating a H596 cells thatrendered cisplatin resistance (CR-H596; 3-fold higher cisplatinIC50 as compared with parental H596) through chronic low-dose exposure to cisplatin (Supplementary Fig. S5A), we dem-onstrated that acquired resistance was mediated throughMAPK/ERK signaling. In comparison with parental H596 cells,p-ERK1/2 and SOS2 were highly expressed in CR-H596 cells,whereas Bim-induced apoptosis was attenuated upon cisplatintreatment (Supplementary Fig. S5B). Together, these observa-

tions indicate the critical role of the MAPK pathway in render-ing cisplatin resistance.

ERK1/2phosphorylation correlateswith shorterDFS inHNSCCpatients treated with adjuvant chemotherapy

To support the hypothesis that strong ERK activity is respon-sible for cisplatin resistance in a patient population, we analyzedERK phosphorylation levels in patients with locally advancedSCC. However, the lung SCC patients at NUH are not treated withsingle-agent cisplatin, and the correlation of p-ERK expressionand patient survival may be confounded by the addition of asecond chemotherapeutic agent. Therefore, clinicopathologicanalysis was conducted on HNSCC instead where concurrentsingle-agent cisplatin treatment with radiotherapy is the standard

Figure 3.High p-ERK1/2 modulates cisplatinsensitivity in lung SCC cells, andcorrelates to poor DFS in patientswithHNSCC following cisplatin treatment.A, cell viability of Calu-1 cells uponcisplatin (CDDP) treatment (24 and48 hours) after ERK1/2 knockdownwas assessed with CellTiter assay.Values represent the change inviable cell (%) relative to the controlgroup � SD (n ¼ 4). Immunoblotsvalidate ERK1/2 siRNA knockdown inCalu-1 cells. Cisplatin IC50� SD (n¼ 3)of Calu-1 cells after silencing of ERK1/2(B) and MEK1/2 (C) was determinedwith CellTiter assay after 72 hours ofcisplatin treatment. Immunoblotsvalidate ERK/MEK siRNA knockdownin Calu-1 cells. D, immunoblots showthe effects of MAP2K1 (D67N)overexpression on affecting cisplatin-induced apoptosis in H596 cells.50 nmol/L siRNA or 1 mg of plasmidwas used per transfection. E,immunohistochemical analysis forintracellular expression of p-ERK1/2 inHNSCC tumors (IRS: 2 and 3, left toright) and the corresponding H&Estaining. Scale bar, 200 mm. Tumorsamples were categorized into highp-ERK1/2 (IRS < 50; n ¼ 17) and lowp-ERK1/2 (IRS > 50; n¼ 28). F, KaplanMeier curves for DFS are plotted forthe high and low p-ERK1/2populations. Tick marks indicatepatients for whom data was censored(Data cutoff point: June 2014).� , P < 0.05.

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first-line regime. It is generally accepted that HNSCC sharesimilar etiology and histopathologic characteristics of SCC ofthe lung. Among three HNSCC cell lines, we identifiedUMSCC1 and 93-VU-147 cells to be more tolerable to cisplatintreatment (Supplementary Fig. S6A). Consistent with ourhypothesis on lung SCC, cisplatin-sensitive SCC13 cells dem-onstrated downregulation of SOS2, p-MEK1/2 and p-ERK1/2upon increasing doses of cisplatin, together with the cleavage ofPARP; whereas cisplatin-resistant UMSCC1 cells had sustainedactivation of p-MEK1/2 and p-ERK1/2 with minimal PARPcleavage (Supplementary Fig. S6B).

The characteristics of the selected HNSCC patient populationwere presented in Supplementary Table S2. ERK activation wasanalyzed by immunohistochemistry (Fig. 3E). The DFS—definedas the interval between the chemoradiation completion to thefirsthistologically or radiologically confirmed tumor recurrence local-ly in the head and neck region or distantmetastases—was definedin 45 HNSCC patients. Significantly, shorter DFS was determinedfor patients with higher expression of p-ERK (log-rank test, P ¼0.02; Fig. 3F), suggesting that activation of the MAPK–ERK path-way is predictive of cisplatin efficacy in the treatment of SCC.Human papillomavirus (HPV) is a known cause of HNSCC and isassociated with better prognosis (23). Using p16 as a surrogatebiomarker forHPV infection, four patients fromeach of the p-ERKhigh and low groups were defined as HPV-positive (Supplemen-tary Table S2). Therefore, the HPV status is not likely a confound-ing variable in the association of p-ERK and DFS among theHNSCC cases.

MEK inhibition enhances cisplatin cytotoxicity throughupregulation of Bim

Our data so far suggest that ERK activation and its down-stream signaling events are crucial in mediating resistance tocisplatin. Consequently, the inhibition of ERK signaling maytherefore be a potential therapeutic approach in patients withresistant SCC. To investigate this possibility further, Calu-1 cellswere treated with several pharmacologic inhibitors of theEGFR–MAPK pathway [i.e., EGFR antibody (cetuximab), RAFinhibitor (GDC-0879), and MEK inhibitors (PD0325901,RDEA119, and GSK1120212)] at the determined sub-lethaldoses in combination with cisplatin. Despite inhibiting EGFRand BRAF, cetuximab and GDC-0879 had limited effects on p-ERK in Calu-1 cells (Supplementary Fig. S7A). In contrast, MEKinhibitors abrogated p-ERK expression while simultaneouslyinducing the accumulation of p-MEK1/2 (Supplementary Fig.S7A). Coincubation of cisplatin and MEK inhibitors, but notcetuximab and GDC-0879, significantly lowered cisplatin IC50

in Calu-1 cells (Fig. 4A; Supplementary Fig. S7B). The failure ofcetuximab and GDC-0879 to suppress p-ERK possibly accountsfor the lack of chemosensitizing effects in cisplatin-resistantSCC cells that are highly-dependent on MAPK–ERK activity.Consistently, effective MEK–ERK inhibition increased cisplatinsensitivity in H520 cells (Fig. 4B; Supplementary Fig. S7C).Mechanistically, abrogation of p-ERK by MEK inhibitors abol-ished cisplatin-induced Bim phosphorylation, which preventedmobility shift and increased accumulation of Bim in Calu-1cells, therefore resulting in the activation of caspase-3 andcleavage of PARP (Fig. 4C). In concordance with these observa-tions, siRNA-silencing of Bim effectively rescued cell deathinduced by MEK inhibition in cisplatin-treated Calu-1 cells(Fig. 4D). These findings confirm that pharmacologic inhibi-

tion of ERK could successfully enhance cisplatin-induced apo-ptosis in cisplatin-resistant SCC cells.

MEK inhibition augments the antiproliferative activity ofcisplatin in xenograft models

Under in vitro anchorage-independent condition, combinedtreatment of GSK1120212 with cisplatin further suppressed col-ony formation inH520 cell line (IC50¼132nmol/L) as comparedwith cisplatin treatment alone (IC50 ¼ 193 nmol/L; Supplemen-tary Fig. S8B); whereas GSK1120212 alone effectively inhibitedcolony formation (at a dose as low as 0.01 mmol/L) in Calu-1 cells(Supplementary Fig. S8A).

Given the improved in vitro efficacy of MEK inhibitor–cisplatincombination, we next used two xenograft models to test thecombinatorial effect of theMEK–ERK coinhibition with cisplatin,using doses previously documented to result in therapeuticallyrelevant concentrations (24, 25). Tumor-bearing mice were ran-domized to treatment with vehicle, the MEK inhibitorPD0325901, cisplatin, or the combination of PD0325901 andcisplatin. In H520-derived xenografts, PD0325901 and cisplatinattenuated tumor growth as a single agent monotherapy, with thecombined treatment demonstrating the most pronounced inhib-itory effect (Supplementary Fig. S9A–S9C). The efficacy of thistreatment regime was next investigated in a patient-derivedHNSCC xenograft model (PDX, SCC10-0508) that harborswild-type EGFR/RAS/BRAF and expresses high basal p-ERK. Rel-ative to vehicle-treated controls, cisplatin treatment alone hadlittle effect on suppressing tumor growth (Fig. 5A–C). In contrast,treatment with PD0325901 alone dramatically inhibited tumorgrowth; whereas the combination treatment showed similartumor inhibition, with one tumor showing regression (Fig.5A–C).Minimal weight loss was observed inmice in all treatmentgroups (data not shown). Sustained ERK activationwere observedin both xenograft models after cisplatin treatment, whereasPD0325901 treatment suppressed p-ERK and upregulated Bimin tumor tissues (Fig. 5D and E; Supplementary Fig. S9D).Together, these data strongly support the therapeutic potentialof combining cisplatin with MEK inhibitors in cisplatin-resistantSCC tumors, including those with wild-type BRAF and RAS.

DiscussionSCC of the lung and head and neck, when recurrent or

metastatic, carries a poor prognosis and has limited therapeuticoptions. Cisplatin chemotherapy is the backbone of treatmentagainst these cancers (8), but patients will eventually developplatinum resistance and very limited or no effective second linetreatment exists. To date, only the use of cetuximab, a mono-clonal antibody against EGFR, in combination with platinum-based chemotherapy, has been reported to improve their treat-ment outlook (26, 27). The use of small-molecule inhibitorsand monoclonal antibodies have not achieved desirable clin-ical outcome in patients with lung or HNSCCs. Some recentdata have highlighted DDR1 and FGFR1 mutations in SCClung, but these mutations are still considered rare (28, 29). Ourdata also confirm the findings of the TCGA group that there is alow incidence of oncogenic mutations in lung SCC despite thedetection of a high mutation frequency (16). As such, com-pared with adenocarcinoma of the lungs, it remains difficult forindividualized targeted therapies to be applied for the treat-ment of SCC.






One likely mechanism of resistance to chemotherapy is thecapacity of cancer cells to upregulate signaling pathways such asthe MAPK pathway to provide additional survival and prolifer-ative signals. Cisplatin resistance has previously not been knownto be associated with any specific signaling pathways directly, andour data provide fresh evidence that whereas both MEK and ERKare rarely activated by mutation in cancers, activation of SOS/MAPK appears to be a drug-associated characteristic of SCCsubtypes that are more resistant to cisplatin. In addition, upre-gulation of MAPK/ERK is seemingly a possible mechanism foracquired resistance to cisplatin. Despite so, contradicting reportshavedemonstrated both theprosurvival andproapoptotic roles ofERK signaling in cisplatin-treated cells, which are dependent onthe downstream components activated by p-ERK (30–32). Here,we found that MEK/ERK silencing and pharmacologic inhibition

of MEK in clinically relevant concentrations could act synergisti-cally with cisplatin in cisplatin-resistant cell lines. In contrast,inhibitors targeting components upstream of MEK, such as RAFkinase,werenot able to achieve similar synergypossibly due to theactivation of alternative pathways that conversely sustained ERKphosphorylation (33, 34). It has been reported that Src kinasecould directly regulate MAPK cassettes through RAS (35, 36), butin our study, Src phosphorylation did not seem to correlate withERK in several lung SCC cell lines (Supplementary Fig. S10A). Inaddition, inhibition of Src signaling with either Src-specific inhib-itor, AZD0530, or Src siRNA did not affect ERK activation andPARP cleavage in Calu-1 cells (Supplementary Fig. S10A andS10B).

In terms of downstream mechanisms, we found that inhi-bition of MEK–ERK signaling induced Bim expression and

Figure 4.Combined treatment of MEKinhibitors and cisplatin suppresses p-ERK1/2 and increases Bim-inducedapoptosis. Calu-1 (A) and H520 (B)cells were treated with cisplatin(CDDP) in combination with DMSO orMEK inhibitors (PD0325901, RDEA119and GSK1120212). Cisplatin IC50 wasassessed 72 hours after drugtreatment with CellTiter assay. Dataare presented asmean�SD (n¼ 3). C,Calu-1 cells were treated with DMSO,MEK inhibitors (1 mmol/L), cisplatin(10 mmol/L), or a combination of MEKinhibitor and cisplatin for 48 hours,and indicated targets were evaluatedby immunoblotting. D, the effects ofMEK inhibition after silencing of Bim inCalu-1 treatedwithDMSO, cisplatin (10mmol/L), GSK1120212 (1 mmol/L), or acombination of GSK1120212 andcisplatin were assessed with Westernblotting. 50 nmol/L siRNA was usedper transfection. � , P < 0.05.

Kong et al.





enhanced cisplatin cytotoxicity; whereas constitutive stimula-tion of ERK activity through ectopic expression of MEK1 abro-gated Bim-induced apoptosis. This suggest a mechanism fordysregulation of cisplatin cytotoxicity via ERK activation in SCCcells, which is consistent with previous reports in ovariancancer cell lines where Bim degradation was associated tocisplatin resistance (21), and this process could be reversed byMEK inhibition (30, 31). It is widely accepted that MEKinhibitors exert a cytostatic, rather that cytotoxic, effect whenused in cancer cells (37). Concordantly, we showed thatGSK1120212 alone predominantly induced G1 arrest in SCCcells, but this effect was attenuated in the presence of cisplatin(Supplementary Fig. S11A–S11D). Furthermore, apoptosis wastriggered in GSK1120212/cisplatin-treated cells, suggesting thatMEK inhibition leads to cytotoxic instead of cytostatic effect inthe presence of cisplatin.

SCC head and neck are similar to lung SCC in etiology, histol-ogy, and mutational landscape (38–40). Thus, to determinewhetherMEK/ERK activation occurs in patient tumor samples andcould potentially reduce the antitumor effect of cisplatin, weconducted a clinicopathologic analysis on samples from patientswith newly diagnosed chemo-naive HNSCC, where the primarytreatment included concurrent standard radiotherapy with single-agent cisplatin chemotherapy. There was wide inter-individualvariability of p-ERK expression in tumors, and DFS was shown tobe shorter for the high ERK-expressing patients, suggesting p-ERKmaybean important predictor of response to cisplatin treatment inHNSCC.Given there is lackingofdrivermutations toguide therapyin lung SCC, we propose ERK as a potential therapeutic candidatefor treatment of relapsed and cisplatin-refractory tumors.

Though the RAF–MEK–ERK pathway may be inhibited atseveral levels by pharmacologic perturbations, the specific level

Figure 5.Therapeutic efficacy of MEK inhibitorin xenograft models that is non-responsive to cisplatin treatment.Patient-derived HNSCC xenografts(PDX SCC10-0508) were treated withvehicle, PD0325901 (PD, 10 mg/kgdaily), cisplatin (CDDP, 2 mg/kgweekly), or a combination ofPD0325901 and cisplatin for 26 days.A, change in tumor volume ispresented asmean� SD (n > 5 tumorsper group). B, waterfall plots showingchange in tumor volume (relative toinitial tumor volumeat treatment-startday) for each individual tumor inrespective treatment group. C, tumorsharvested on treatment-end day weredisplayed. D, immunoblots showinglevels of SOS2, p-MEK1/2, p-ERK1/2,and Bim in 3 independent tumortissues from C. E, tumor tissues from Cwere evaluated for intracellularexpression of p-ERK1/2 and Bim byIHC. Scale bar, 100 mm. Bar chartsshow percentage of positive cells ineach treatment group, data presentedas mean percentage � SD (n ¼ 3 pertreatment group). � , P < 0.05.






of inhibition may be critical. For instance, tumors with wild-typeBRAF, including those with mutant RAS, circumvent RAF inhibi-tion through paradoxical stimulation of ERK activity (33). Incontrast, observations in several findings have indicated thatMEKinhibitors are clinically active in tumors with BRAF mutation assingle agent (41), whereas the data from our xenograft modelssuggest that this class of inhibitors could also inhibit growth ofwild-type BRAF tumor that has high ERK activity, and showspromising combinatorial effect with cisplatin. Importantly, thisdrug combination appeared to be tolerated by the treated ani-mals, without exacerbating the toxicities of cisplatin as no appar-ent weight loss was observed. Deployed as a single agent, it islikely thatMEK inhibitors have limited efficacy in SCC as noted inclinical observations fromaphase II study that reported the lack ofefficacy of PD0325901 in six patients with lung SCC (42). Ourdata suggest that, through selection of patients with SCC whosetumors express high p-ERK, treatment with cisplatin and MEKinhibitor may be a promising combination strategy.

In summary, we have found that upregulation of MEK/ERK isassociated to cisplatin resistance. Although there are otherknown mechanisms of cisplatin resistance, including reducedinflux transport via the copper transporter, conjugation byglutathione and methionine, and enhanced DNA repair (43),the relative importance of each of these pathways is still notdefined in SCC. More crucially, our results suggest a role for thecombined use of cisplatin with the MEK inhibitor for patientswith upregulated MAPK/ERK signaling in SCC of the head andneck and lung. Several MEK inhibitors currently undergoingclinical development have shown promising endpoint resultsin various trials on NSCLC (44–46). Furthermore, MEK inhi-bitors have demonstrated favorable tolerability profiles andside effects consisting mainly of diarrhea, rash, and centralserous chorioretinopathy (47). Cisplatin is well established andhas a nonoverlapping toxicity profile, which lends its potentialto be combined with an MEK inhibitor. Indeed, there is evi-dence for potential renal tubular protection from cisplatin-induced renal tubulopathy by MEK inhibition (48). For a

proof-of-concept, it will be critical to study the combinationof cisplatin and an MEK inhibitor in patients whose tumorsdemonstrate evidence of ERK phosphorylation. Such studiesare currently being planned.

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

Authors' ContributionsConception and design: L.R. Kong, K.N. Chua, H.C. Ng, W.J. Chng, B.C. GohDevelopment of methodology: L.R. Kong, K.N. Chua, H.C. Ng, W.J. Chng,B.C. GohAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.R. Kong, W.J. Sim, H.C. Ng, C. Bi, M.E. Nga,Y.H. Pang, W.R. Ong, R.A. Soo, H. Huynh, B.C. GohAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L.R. Kong, K.N. Chua, H.C. Ng, C. Bi, M.E. Nga,Y.H. Pang, H. Huynh, J.-P. ThieryWriting, review, and/or revision of the manuscript: L.R. Kong, K.N. Chua,R.A. Soo, W.J. Chng, J.-P. Thiery, B.C. GohAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L.R. Kong, K.N. Chua, W.J. Sim, J. Ho, B.C. GohStudy supervision: B.C. Goh

AcknowledgmentsThe authors thank Lingzhi Wang, Jianbiao Zhou, and Azhar bin Ali for their

critical comments; E.Y. Foo for preparation of tissue sections; and the Centrefor Translational Research and Diagnostics for STR analysis on cell lineauthentication.

Grant SupportThiswork is supportedby grants from theNationalMedical ResearchCouncil

(NPRC10/NM14O; to B.C. Goh) and the Cancer Science Institute, Singapore(R-713-001-011-271; to B.C. Goh).

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

Received January 22, 2015; revised March 30, 2015; accepted April 26, 2015;published OnlineFirst May 4, 2015.

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Published OnlineFirst May 4, 2015.Mol Cancer Ther Li Ren Kong, Kian Ngiap Chua, Wen Jing Sim, et al. Carcinomaby SOS/MAPK Pathway Activation in Squamous Cell MEK Inhibition Overcomes Cisplatin Resistance Conferred

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