Using high-throughput analyses and the TRANSFAC database, we characterized TF signatures of head and neck squamous cell carcinoma (HNSCC) subgroups by inferential analysis of target gene expression, correcting for the effects of DNA methylation and copy number. Using this discovery pipeline, we determined that human papillomavirus-related (HPV+) and HPV- HNSCC differed significantly based on the activity levels of key TFs including AP1, STATs, NF-κB and p53. Immunohistochemical analysis confirmed that HPV- HNSCC is characterized by co-activated STAT3 and NF-κB pathways and functional studies demonstrate that this phenotype can be effectively targeted with combined anti-NF-κB and anti-STAT therapies. These discoveries correlate strongly with previous findings connecting STATs, NF-κB and AP1 in HNSCC. We identified five top-scoring pair biomarkers from STATs, NF-κB and AP1 pathways that distinguish HPV+ from HPV- HNSCC based on TF activity and validated these biomarkers on TCGA and on independent validation cohorts. We conclude that a novel approach to TF pathway analysis can provide insight into therapeutic targeting of patient subgroup for heterogeneous disease such as HNSCC.

Department of Biostatistics and Computational Biology Dana Farber Cancer Institute Boston MA

Department of Mathematics and Statistics The College of New Jersey Ewing NJ

Department of Oncology Johns Hopkins Medical Institutions Baltimore MD

Department of Otolaryngology Head and Neck Surgery Johns Hopkins Medical Institutions Baltimore MD

Department of Pathology Johns Hopkins Medical Institutions Baltimore MD

Division of Oncology Biostatistics Johns Hopkins Medical Institutions Baltimore MD

Laboratory of Molecular Pathology Institute of Molecular and Translational Medicine Palacky University Olomouc Czech Republic

Mid Michigan Ear Nose and Throat East Lansing MI

Milton J Dance Head and Neck Center Greater Baltimore Medical Center Baltimore MD

University of Virginia Charlottesville VA

See more in PubMed

Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, 2014. CA: a cancer journal for clinicians. 2014;64:9–29. PubMed

Psyrri A, Boutati E, Karageorgopoulou S. Human papillomavirus in head and neck cancers: biology, prognosis, hope of treatment, and vaccines. Anticancer Drugs. 2011;22:586–90. PubMed

Chaturvedi AK, Engels EA, Pfeiffer RM, Hernandez BY, Xiao W, Kim E, Jiang B, Goodman MT, Sibug-Saber M, Cozen W, Liu L, Lynch CF, et al. Human papillomavirus and rising oropharyngeal cancer incidence in the United States. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2011;29:4294–301. PubMed PMC

Smith IM, Mydlarz WK, Mithani SK, Califano JA. DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage. International journal of cancer Journal international du cancer. 2007;121:1724–8. PubMed

Bonner JA, Harari PM, Giralt J, Cohen RB, Jones CU, Sur RK, Raben D, Baselga J, Spencer SA, Zhu J, Youssoufian H, Rowinsky EK, et al. Radiotherapy plus cetuximab for locoregionally advanced head and neck cancer: 5-year survival data from a phase 3 randomised trial, and relation between cetuximab-induced rash and survival. The lancet oncology. 2010;11:21–8. PubMed

Lui VW, Hedberg ML, Li H, Vangara BS, Pendleton K, Zeng Y, Lu Y, Zhang Q, Du Y, Gilbert BR, Freilino M, Sauerwein S, et al. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer discovery. 2013;3:761–9. PubMed PMC

Agrawal N, Frederick MJ, Pickering CR, Bettegowda C, Chang K, Li RJ, Fakhry C, Xie TX, Zhang J, Wang J, Zhang N, El-Naggar AK, et al. Exome sequencing of head and neck squamous cell carcinoma reveals inactivating mutations in NOTCH1. Science. 2011;333:1154–7. PubMed PMC

Sun W, Gaykalova DA, Ochs MF, Mambo E, Arnaoutakis D, Liu Y, Loyo M, Agrawal N, Howard J, Li R, Ahn S, Fertig E, et al. Activation of the NOTCH pathway in head and neck cancer. Cancer research. 2014;74:1091–104. PubMed PMC

Fertig EJ, Markovic A, Danilova LV, Gaykalova DA, Cope L, Chung CH, Ochs MF, Califano JA. Preferential activation of the hedgehog pathway by epigenetic modulations in HPV negative HNSCC identified with meta-pathway analysis. PloS one. 2013;8:e78127. PubMed PMC

Chung CH, Parker JS, Karaca G, Wu J, Funkhouser WK, Moore D, Butterfoss D, Xiang D, Zanation A, Yin X, Shockley WW, Weissler MC, et al. Molecular classification of head and neck squamous cell carcinomas using patterns of gene expression. Cancer cell. 2004;5:489–500. PubMed

Tootle TL, Rebay I. Post-translational modifications influence transcription factor activity: a view from the ETS superfamily. Bioessays. 2005;27:285–98. PubMed

Allen C, Saigal K, Nottingham L, Arun P, Chen Z, Van Waes C. Bortezomib-induced apoptosis with limited clinical response is accompanied by inhibition of canonical but not alternative nuclear factor-{kappa}B subunits in head and neck cancer. Clinical cancer research : an official journal of the American Association for Cancer Research. 2008;14:4175–85. PubMed

Gill JS, Zhu X, Moore MJ, Lu L, Yaszemski MJ, Windebank AJ. Effects of NFkappaB decoy oligonucleotides released from biodegradable polymer microparticles on a glioblastoma cell line. Biomaterials. 2002;23:2773–81. PubMed

Sen M, Paul K, Freilino ML, Li H, Li C, Johnson DE, Wang L, Eiseman J, Grandis JR. Systemic Administration of a Cyclic Signal Transducer and Activator of Transcription 3 (STAT3) Decoy Oligonucleotide Inhibits Tumor Growth without Inducing Toxicological Effects. Molecular medicine. 2014;20:46–56. PubMed PMC

Rathi KS, Gaykalova DA, Hennessey P, Califano JA, Ochs MF. Correcting Transcription Factor Gene Sets for Copy Number and Promoter Methylation Variations. Drug Dev Res. 2014 PubMed PMC

Carvalho AL, Henrique R, Jeronimo C, Nayak CS, Reddy AN, Hoque MO, Chang S, Brait M, Jiang WW, Kim MM, Claybourne Q, Goldenberg D, et al. Detection of promoter hypermethylation in salivary rinses as a biomarker for head and neck squamous cell carcinoma surveillance. Clinical cancer research : an official journal of the American Association for Cancer Research. 2011;17:4782–9. PubMed PMC

Singhi AD, Califano J, Westra WH. High-risk human papillomavirus in nasopharyngeal carcinoma. Head Neck. 2012;34:213–8. PubMed

Matys V, Fricke E, Geffers R, Gossling E, Haubrock M, Hehl R, Hornischer K, Karas D, Kel AE, Kel-Margoulis OV, Kloos DU, Land S, et al. TRANSFAC: transcriptional regulation, from patterns to profiles. Nucleic Acids Res. 2003;31:374–8. PubMed PMC

Scharpf RB, Irizarry RA, Ritchie ME, Carvalho B, Ruczinski I. Using the R Package crlmm for Genotyping and Copy Number Estimation. J Stat Softw. 2011;40:1–32. PubMed PMC

Nottingham LK, Yan CH, Yang X, Si H, Coupar J, Bian Y, Cheng TF, Allen C, Arun P, Gius D, Dang L, Van Waes C, et al. Aberrant IKKalpha and IKKbeta cooperatively activate NF-kappaB and induce EGFR/AP1 signaling to promote survival and migration of head and neck cancer. Oncogene. 2014;33:1135–47. PubMed PMC

Rampias T, Sasaki C, Weinberger P, Psyrri A. E6 and E7 gene silencing and transformed phenotype of human papillomavirus 16-positive oropharyngeal cancer cells. J Natl Cancer Inst. 2009;101:412–23. PubMed

Haraf DJ, Nodzenski E, Brachman D, Mick R, Montag A, Graves D, Vokes EE, Weichselbaum RR. Human papilloma virus and p53 in head and neck cancer: clinical correlates and survival. Clinical cancer research : an official journal of the American Association for Cancer Research. 1996;2:755–62. PubMed

Poeta ML, Manola J, Goldwasser MA, Forastiere A, Benoit N, Califano JA, Ridge JA, Goodwin J, Kenady D, Saunders J, Westra W, Sidransky D, et al. TP53 mutations and survival in squamous-cell carcinoma of the head and neck. N Engl J Med. 2007;357:2552–61. PubMed PMC

Geman D, d'Avignon C, Naiman DQ, Winslow RL. Classifying gene expression profiles from pairwise mRNA comparisons. Stat Appl Genet Mol Biol. 2004;3 PubMed PMC

Leek JT. The tspair package for finding top scoring pair classifiers in R. Bioinformatics. 2009;25:1203–4. PubMed PMC

Hatakeyama H, Cheng H, Wirth P, Counsell A, Marcrom SR, Wood CB, Pohlmann PR, Gilbert J, Murphy B, Yarbrough WG, Wheeler DL, Harari PM, et al. Regulation of heparin-binding EGF-like growth factor by miR-212 and acquired cetuximab-resistance in head and neck squamous cell carcinoma. PloS one. 2010;5:e12702. PubMed PMC

Lee TL, Yang XP, Yan B, Friedman J, Duggal P, Bagain L, Dong G, Yeh NT, Wang J, Zhou J, Elkahloun A, Van Waes C, et al. A novel nuclear factor-kappaB gene signature is differentially expressed in head and neck squamous cell carcinomas in association with TP53 status. Clinical cancer research : an official journal of the American Association for Cancer Research. 2007;13:5680–91. PubMed

Wang Q, Huber N, Noel G, Haar L, Shan Y, Pritts TA, Ogle CK. NF-kappaBeta inhibition is ineffective in blocking cytokine-induced IL-8 production but P38 and STAT1 inhibitors are effective. Inflamm Res. 2012;61:977–85. PubMed

Sun J, Blaskovich MA, Jove R, Livingston SK, Coppola D, Sebti SM. Cucurbitacin Q: a selective STAT3 activation inhibitor with potent antitumor activity. Oncogene. 2005;24:3236–45. PubMed

Haftchenary S, Luchman HA, Jouk AO, Veloso AJ, Page BD, Cheng XR, Dawson SS, Grinshtein N, Shahani VM, Kerman K, Kaplan DR, Griffin C, et al. Potent Targeting of the STAT3 Protein in Brain Cancer Stem Cells: A Promising Route for Treating Glioblastoma. ACS medicinal chemistry letters. 2013;4:1102–7. PubMed PMC

Weinberger PM, Yu Z, Haffty BG, Kowalski D, Harigopal M, Brandsma J, Sasaki C, Joe J, Camp RL, Rimm DL, Psyrri A. Molecular classification identifies a subset of human papillomavirus--associated oropharyngeal cancers with favorable prognosis. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2006;24:736–47. PubMed

Yan B, Chen G, Saigal K, Yang X, Jensen ST, Van Waes C, Stoeckert CJ, Chen Z. Systems biology-defined NF-kappaB regulons, interacting signal pathways and networks are implicated in the malignant phenotype of head and neck cancer cell lines differing in p53 status. Genome biology. 2008;9:R53. PubMed PMC

Yan B, Yang X, Lee TL, Friedman J, Tang J, Van Waes C, Chen Z. Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-kappaB and other signal transcription factors in head and neck squamous cell carcinoma. Genome biology. 2007;8:R78. PubMed PMC

Squarize CH, Castilho RM, Sriuranpong V, Pinto DS, Jr., Gutkind JS. Molecular cross-talk between the NFkappaB and STAT3 signaling pathways in head and neck squamous cell carcinoma. Neoplasia. 2006;8:733–46. PubMed PMC

Li Q, Verma IM. NF-kappaB regulation in the immune system. Nat Rev Immunol. 2002;2:725–34. PubMed

Dauer DJ, Ferraro B, Song L, Yu B, Mora L, Buettner R, Enkemann S, Jove R, Haura EB. Stat3 regulates genes common to both wound healing and cancer. Oncogene. 2005;24:3397–408. PubMed

Cho HJ, Kang JH, Kwak JY, Lee TS, Lee IS, Park NG, Nakajima H, Magae J, Chang YC. Ascofuranone suppresses PMA-mediated matrix metalloproteinase-9 gene activation through the Ras/Raf/MEK/ERK- and Ap1-dependent mechanisms. Carcinogenesis. 2007;28:1104–10. PubMed

Ondrey FG, Dong G, Sunwoo J, Chen Z, Wolf JS, Crowl-Bancroft CV, Mukaida N, Van Waes C. Constitutive activation of transcription factors NF-(kappa)B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines. Molecular carcinogenesis. 1999;26:119–29. PubMed

Song JI, Grandis JR. STAT signaling in head and neck cancer. Oncogene. 2000;19:2489–95. PubMed

Vandermark ER, Deluca KA, Gardner CR, Marker DF, Schreiner CN, Strickland DA, Wilton KM, Mondal S, Woodworth CD. Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization. Virology. 2012;425:53–60. PubMed PMC

Sato O, Kuriki C, Fukui Y, Motojima K. Dual promoter structure of mouse and human fatty acid translocase/CD36 genes and unique transcriptional activation by peroxisome proliferator-activated receptor alpha and gamma ligands. J Biol Chem. 2002;277:15703–11. PubMed

Bancroft CC, Chen Z, Yeh J, Sunwoo JB, Yeh NT, Jackson S, Jackson C, Van Waes C. Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-kappaB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines. International journal of cancer Journal international du cancer. 2002;99:538–48. PubMed

Lee SJ, Qin H, Benveniste EN. Simvastatin inhibits IFN-gamma-induced CD40 gene expression by suppressing STAT-1alpha. J Leukoc Biol. 2007;82:436–47. PubMed

McArdle MA, Finucane OM, Connaughton RM, McMorrow AM, Roche HM. Mechanisms of obesity-induced inflammation and insulin resistance: insights into the emerging role of nutritional strategies. Frontiers in endocrinology (Lausanne) 2013;4:52. PubMed PMC

Lee TL, Yeh J, Friedman J, Yan B, Yang X, Yeh NT, Van Waes C, Chen Z. A signal network involving coactivated NF-kappaB and STAT3 and altered p53 modulates BAX/BCL-XL expression and promotes cell survival of head and neck squamous cell carcinomas. International journal of cancer Journal international du cancer. 2008;122:1987–98. PubMed

Gaykalova DA, Mambo E, Choudhary A, Houghton J, Buddavarapu K, Sanford T, Darden W, Adai A, Hadd A, Latham G, Danilova LV, Bishop J, et al. Novel insight into mutational landscape of head and neck squamous cell carcinoma. PloS one. 2014;9:e93102. PubMed PMC

Freed-Pastor WA, Prives C. Mutant p53: one name, many proteins. Genes & development. 2012;26:1268–86. PubMed PMC

Redmond AM, Carroll JS. Defining and targeting transcription factors in cancer. Genome biology. 200910:311. PubMed PMC

Yeh JE, Toniolo PA, Frank DA. Targeting transcription factors: promising new strategies for cancer therapy. Curr Opin Oncol. 2013;25:652–8. PubMed

Turkson J, Kim JS, Zhang S, Yuan J, Huang M, Glenn M, Haura E, Sebti S, Hamilton AD, Jove R. Novel peptidomimetic inhibitors of signal transducer and activator of transcription 3 dimerization and biological activity. Mol Cancer Ther. 2004;3:261–9. PubMed

Sen M, Thomas SM, Kim S, Yeh JI, Ferris RL, Johnson JT, Duvvuri U, Lee J, Sahu N, Joyce S, Freilino ML, Shi H, et al. First-in-human trial of a STAT3 decoy oligonucleotide in head and neck tumors: implications for cancer therapy. Cancer discovery. 2012;2:694–705. PubMed PMC