Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma Nicholas J. Wanga,1, Zachary Sanbornb,1, Kelly L. Arnettc, Laura J. Baystonc, Wilson Liaod, Charlotte M. Probye, Irene M. Leighe, Eric A. Collissonf, Patricia B. Gordong, Lakshmi Jakkulaa, Sally Pennypackerd, Yong Zouh, Mimansa Sharmai, Jeffrey P. Northi, Swapna S. Vemulaj, Theodora M. Maurod, Isaac M. Neuhausd, Philip E. LeBoitj, Joe S. Hurk, Kyunghee Parkl, Nam Huhl, Pui-Yan Kwokd, Sarah T. Arrond, Pierre P. Massionh, Allen E. Baleg, David Hausslerb, James E. Cleaverd,2, Joe W. Graym, Paul T. Spellmann, Andrew P. Southe, Jon C. Asterc,3, Stephen C. Blacklowc,o,p,3, and Raymond J. Chod,2,3 aLife Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; bCenter for Biomolecular Science and Engineering, University of California, Santa Cruz, CA 95064; cDepartment of Pathology, Brigham and Women’s Hospital, Boston, MA 02115; Departments of dDermatology, fHematology and Oncology, and iPathology, University of California, San Francisco, CA 94143; eDivision of Cancer Research, Medical Research Institute, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom; gDepartment of Genetics, Yale University, CT 06520; hDivision of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University, TN 37232; jUniversity of California, San Francisco Dermatopathology Service, San Francisco, CA 94115; kSamsung Electronics Headquarters, Seocho-gu, Seoul 137-857, Korea; lEmerging Technology Research Center, Samsung Advanced Institute of Technology, Kyunggi-do 446-712, Korea; Departments of mBiomedical Engineering and nMolecular and Medical Genetics, Oregon Health Sciences University, Portland OR 97239; oDepartment of Cancer Biology, Dana–Farber Cancer Institute, and pDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 Contributed by James E. Cleaver, September 12, 2011 (sent for review August 1, 2011) Squamous cell carcinomas (SCCs) are one of the most frequent than 10% of head and neck SCCs and cSCCs carry EGFR and forms of human malignancy, but, other than TP53 mutations, few PIK3CA gain-of-function mutations (7–9), and the oncogenic causative somatic aberrations have been identified. We identified kinase DDR2 is activated in ∼5% of lung SCC primary tumors NOTCH1 or NOTCH2 mutations in ∼75% of cutaneous SCCs and and cell lines (10). In fact, most SCCs lack an identifiable clas- in a lesser fraction of lung SCCs, defining a spectrum for the most sical driver mutation, stalling deployment of targeted treatments. prevalent tumor suppressor specific to these epithelial malignan- Although virtually all SCCs harbor TP53 mutations, additional cies. Notch receptors normally transduce signals in response to tumor suppressors have proved elusive. Recent studies have ligands on neighboring cells, regulating metazoan lineage selec- implicated loss of function in NOTCH1, IRF6, and TP53 in head fi tion and developmental patterning. Our ndings therefore illus- and neck SCCs (11, 12). Several models suggest that inactivation trate a central role for disruption of microenvironmental com- of Smad proteins up-regulates TGF signaling, but somatic mu- NOTCH munication in cancer progression. aberrations include tations have not been detected in primary cancers (13). frameshift and nonsense mutations leading to receptor trunca- We recently integrated whole-exome DNA sequence and al- tions as well as point substitutions in key functional domains that lele-specific copy number data to examine somatically acquired abrogate signaling in cell-based assays. Oncogenic gain-of-func- genomic aberrations in eight primary cSCCs (14). Given the very tion mutations in NOTCH1 commonly occur in human T-cell lym- high mutation burdens in these cancers, we sought to increase phoblastic leukemia/lymphoma and B-cell chronic lymphocytic specificity for genes with a causative role in tumorigenesis. A leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and search for mutations accompanied by loss of the wild-type allele—a pattern shared by known tumor suppressors in our se- suggests that targeted inhibition of the Notch pathway may in- TP53 CDKN2A— fi duce squamous epithelial malignancies. ries, including and identi ed multiple muta- tions in Notch receptors. cancer genetics | genomic | cellular signaling Notch receptors participate in a highly conserved signal trans- duction pathway that regulates many aspects of development through context-dependent effects on cell fate determination (15, quamous cell carcinomas (SCCs) collectively are the most 16), growth (17), and survival (18). Cognate ligands expressed on common ectodermal cancers, resulting in >300,000 deaths S the surface of signal-sending cells bind the EGF-like repeats of per year (1, 2). SCCs arise from renewable squamous epithelial the Notch ectodomain on signal-receiving cells, initiating a series cells that serve to create an environmental barrier in the skin, of proteolytic cleavage events that allow the Notch intracellular esophagus, lung, and cervix. In normal squamous epithelia, basal domain (NICD) to translocate to the nucleus and form a tran- progenitors give rise to more superficial daughter cells that ter- MEDICAL SCIENCES minally differentiate into keratinized cells as they migrate toward the surface, coupling terminal differentiation with microanatomic Author contributions: N.J.W., Z.S., K.L.A., W.L., C.M.P., I.M.L., E.A.C., P.B.G., Y.Z., M.S., J.P.N., position. An early feature of squamous neoplasia of all types is S.S.V., T.M.M., I.M.N., P.E.L., J.S.H., K.P., N.H., P.-Y.K., S.T.A., P.P.M., A.E.B., J.E.C., P.T.S., disrupted differentiation to variable degrees, typically associated A.P.S., J.C.A., S.C.B., and R.J.C. designed research; N.J.W., Z.S., K.L.A., L.J.B., W.L., C.M.P., with thickening of the epithelium and increased proliferation. L.J., and R.J.C. performed research; K.P., N.H., D.H., and J.W.G. contributed new reagents/ Therefore, although SCCs from different sites demonstrate vary- analytic tools; N.J.W., Z.S., K.L.A., W.L., C.M.P., I.M.L., E.A.C., P.B.G., Y.Z., M.S., J.P.N., — S.S.V., T.M.M., I.M.N., P.E.L., J.S.H., K.P., P.-Y.K., S.T.A., P.P.M., A.E.B., D.H., P.T.S., J.C.A., ing epidemiologic associations UV radiation in skin cancers, S.C.B., and R.J.C. analyzed data; and N.J.W., P.B.G., S.P., M.S., J.P.N., T.M.M., J.E.C., A.P.S., alcohol and tobacco exposure in esophageal cancers, and human J.C.A., S.C.B., and R.J.C. wrote the paper. papillomavirus infection in cervical and head and neck cancers— The authors declare no conflict of interest. they likely share disruption of pathways that coordinate micro- Freely available online through the PNAS open access option. environment-dependent squamous differentiation (3). 1N.J.W. and Z.S. contributed equally to this work. Although emerging targeted therapies show promise in epi- 2To whom correspondence may be addressed. E-mail: [email protected] or chorj@ thelial cancers, few recurrent genetic aberrations have been derm.ucsf.edu. identified in lung SCCs or cutaneous SCCs (cSCCs) (4). Acti- 3J.C.A., S.C.B., and R.J.C. contributed equally to this work. HRAS vating mutations are highly prevalent in murine SCC-like This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. malignancies but rare in their human counterparts (5, 6). Fewer 1073/pnas.1114669108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114669108 PNAS | October 25, 2011 | vol. 108 | no. 43 | 17761–17766 Downloaded by guest on October 2, 2021 scription activation complex with the DNA binding factor RBPJ NOTCH1 or NOTCH2 (Table S1). No association with clinical (also known as CSL) and coactivators of the Mastermind-like subtype or TP53 status was noted (Table S2). (MAML) family (19). Although activating mutations in Notch1 Recent exome-sequencing data from 40 lung SCCs acquired occur in the majority of T-cell lymphoblastic leukemia/lym- by The Cancer Genome Atlas (TCGA) project were also ex- phoma (20) and are also often present in poor-prognosis B-cell amined. Three missense mutations and one splice site mutation chronic lymphocytic leukemia (21–23), loss of Notch activity can were identified in NOTCH1, as well as one missense and one also produce basal cell carcinoma-like cancers (24) or squamous nonsense mutation in NOTCH2—a combined mutation fre- cancers (25) in mice, suggesting that the consequence of specific quency of 5/40 samples or 12.5% (Table S1). Sanger sequencing somatic Notch aberrations in cancer is strongly influenced by for NOTCH1 and NOTCH2 in lung SCC cell lines SW900 and epigenetic context. Current models propose that Notch tran- HCC95, esophageal SCC lines TT and TE10, and lung adeno- scriptional down-regulation, possibly due to loss of p53 function, carcinoma lines H549 and A549 revealed a single heterozygous is a causative event in human epithelial malignancies (26), but nonsense NOTCH1 mutation in TE10. disabling somatic aberrations have not been reported in cSCCs or lung SCCs. Here, we describe and functionally characterize Analysis of Unique Notch1 Loss-of-Function Mutants. Amino acid frequent Notch loss-of-function mutations in these malignancies. (missense) changes were analyzed by PolyPhen-2 (http://genetics. bwh.harvard.edu/pph2/) for potential structural effects. Eighteen Results of 27 mutations were identified as “probably damaging” with Identification of Notch Mutations. Three additional cSCCs were the remainder being labeled “probably benign” (Table S3). Be- sequenced on the whole-exome level, in addition to the eight sides overt loss-of-function frameshift and nonsense mutations, originally reported (14).