title: c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions.
abstract: Small cell lung cancer (SCLC) is an aggressive cancer, and most patients present with cancer already spread beyond the lung. The receptor tyrosine kinase (RTK) c-MET has been implicated in various solid tumors, including SCLC, and is involved in mediating tumorigenesis, cell motility, scattering, invasion and metastasis. Mutations of c-Met have been described in renal papillary carcinoma and gastrointestinal cancers including hepatocellular carcinoma. The sequence of c-MET was examined for possible mutations in the 10 SCLC cell lines and 32 paired-SCLC/normal tissues. Novel c-MET alterations were identified among 3 of 10 separate SCLC cell lines and in 4 of 32 SCLC tumor tissue samples. These include two different c-MET missense mutations in the juxtamembrane (JM) domain (R988C found in NCI-H69 and H249 cell lines; and T1010I in SCLC tumor sample T31). Also, there are one Sema domain missense mutation (E168D in SCLC tumor sample T5), two-base-pair insertional mutations (IVS13- (52-53)insCT in both SCLC tumor samples T26 and T27) within the pre-JM intron 13, as well as an alternative transcript involving exon 10 (H128 cell line). c-MET receptors are expressed at various levels among the 10 SCLC cell lines studied (high expression: H69, H345, H510, and H526; medium-expression: H128 and H146; and low/no-expression: H82, H209, H249, and H446). The level of c-MET expression does not have any apparent correlation with presence or absence of mutations of c-MET in the cell lines. We show that the two identified JM mutations (R988C and T1010I), when introduced into the interleukin-3 (IL-3)-dependent BaF3 cell line, regulated cell proliferation resulting in a small but significant growth factor independence. When introduced into a SCLC cell line (H446, with minimal endogenous wild-type c-MET expression), the JM mutations also regulated cell morphology and adhesion, as well as causing enhanced tumorigenicity by both increases in focus-formation and soft-agar colony-formation assays. Both of the JM mutations also increased cell motility and migration evident in wound healing assay and time-lapse video-microscopy speed analysis. The JM mutations also altered the c-MET RTK signaling, resulting in preferentially increased constitutive tyrosine phosphorylation of various cellular proteins, including the key focal adhesion protein paxillin on tyrosine residue Y31 (first CRKL-binding site), correlating with increased motility. These results suggest a novel and unique role of the JM domain in c-MET signaling in SCLC with significant implications in cytoskeletal functions and metastatic potential. The novel JM gain-of-function somatic mutations described are the first to be reported in SCLC, and may be associated with a more aggressive phenotype. It would now be useful to study the inhibition of c-MET as a therapeutic target against SCLC.
authors.affiliation: Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Patrick C
authors.initials: PC
authors.lastname: Ma
authors.forename: Takashi
authors.initials: T
authors.lastname: Kijima
authors.forename: Gautam
authors.initials: G
authors.lastname: Maulik
authors.forename: Edward A
authors.initials: EA
authors.lastname: Fox
authors.forename: Martin
authors.initials: M
authors.lastname: Sattler
authors.forename: James D
authors.initials: JD
authors.lastname: Griffin
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Johnson
authors.forename: Ravi
authors.initials: R
authors.lastname: Salgia
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D018288:Carcinoma, Small Cell / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002448:Cell Adhesion / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics* / Q000502:physiology; D004252:DNA Mutational Analysis; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D020125:Mutation, Missense; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D019859:Proto-Oncogene Proteins c-met / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D015398:Signal Transduction / Q000235:genetics; D014162:Transfection
chemical_list: D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D020033:Protein Isoforms; C496062:Pxn protein, mouse; C496063:Pxn protein, rat; D019859:Proto-Oncogene Proteins c-met