c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions.
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.
Ma
Patrick C
PC
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.
Kijima
Takashi
T
Maulik
Gautam
G
Fox
Edward A
EA
Sattler
Martin
M
Griffin
James D
JD
Johnson
Bruce E
BE
Salgia
Ravi
R
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
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