Hepatocyte growth factor receptor (Met) plays an important role in the progression of multiple cancer types. The overexpression of Met in DLD-1 colon carcinoma cells with kirsten rat sarcoma oncogene ...homolog (KRAS) oncogene activation resulted in enhanced subcutaneous and orthotopic tumor growth rate and increased metastatic potential. To elucidate the mechanism of this effect, we stably expressed kinase-inactive MetK1110A, Src homology 2 (SH2)-binding domain-inactive MetY1349/1356F, growth factor receptor-bound protein 2 (Grb2) non-binding MetN1358H and mutant receptors with ability to selectively recruit signaling proteins Grb2, src homology domain c-terminal adaptor homolog (Shc), phospholipase c-gamma (PLCγ) and p85 phosphatidyl inositol 3 kinase. As subcutaneous implants, DLD-1 cells that expressed the majority of these receptor constructs failed to recapitulate the tumor growth-enhancing effect of the wild-type Met receptor. The Grb2- and Shc-recruiting Met mutants demonstrated slight but consistent tumor-suppressive activity, whereas the expression of N1358H mutant stimulated tumor growth rate comparable with the wild-type receptor. This suggests that direct Grb2/Shc binding does not contribute to the tumor progression activity of Met receptor. The tumors expressing Grb2- and Shc-recruiting Met receptors demonstrated a marked loss in Grb2-associated adaptor protein 1 (Gab1) protein levels, which was not observed in the cell lines, consistent with a post-translationally regulated process. Moreover, a moderate level of Gab1 overexpression stimulated tumor growth. The findings suggest a delicate balance for intact Y1349/1356 SH2-binding domain to mediate the tumor progression activity of the coactivated Met–rat sarcoma oncogene homolog (RAS) pathways. Selectivity for specific adaptor protein involvement may be the key that determines the tissue- and cell-type specificity of Met-mediated tumorigenicity in human cancers.
Endobronchial implantation of NCI-H460 cells into the nude rat generates a primary lung tumor with mediastinal lymph node spread, but rarely systemic metastases. We isolated tumor cells from ...mediastinal nodes, orthotopically reimplanted the cells into nude rats and repeated this four times to derive a cell line, designated H460SM, that spontaneously metastasizes to bone, kidney, brain, soft tissue and contralateral lung. H460SM cells demonstrated higher invasive activity in vitro than parental NCI-H460 cells. Spectral karyotyping revealed a new inversion within 17q and loss of an extra normal copy of chromosome 14 present in parental NCI-H460 cells. Expression profiling of orthotopic primary tumors revealed differential expression of 360 genes. Of these, 173 were represented in the probe set of a 19.2K OCI cDNA microarray previously used to profile the gene expression of surgically resected lung cancer specimens. We have computationally validated clinical importance of these genes by using in silico analysis of 18 cases of pulmonary adenocarcinoma, which were split into two patient groups with markedly different clinical outcome. The model identifies additional novel candidate genes for the progression of lung cancer to systemic metastases and poor prognosis.
Complete resection of early-stage non-small cell lung cancer (NSCLC) is potentially curative, yet approximately 50% of patients are at risk for developing metastatic recurrence. Met, the receptor for ...hepatocyte growth factor (HGF) is a receptor tyrosine kinase with demonstrated roles in regulating cellular proliferation, motility, morphogenesis, and apoptosis. Met receptor and its ligand, HGF, are commonly overexpressed in NSCLC, and their overexpression has been associated with poor prognosis, which could potentially involve a paracrine and/or autocrine activation loop. However, there is as yet no direct evidence that HGF-Met signaling directly promotes metastasis in NSCLC cells. Using retroviral transduction, we overexpressed the human
c-met
and
hgf
complementary DNA, alone or in combination in the NCI-H460 human large cell carcinoma cell line. The HGF/Met co-overexpressing (H460-HGF/Met) cells demonstrated enhanced tumorigenicity in xenograft
SCID
mice. When these cells are implanted orthotopically into the lungs of nude rats, only the H460-HGF/Met cells showed higher spontaneous metastases to distant organs including bone, brain, and kidney. These results provide evidence that autocrine overactivation of the Met- HGF loop enhances systemic metastases in NSCLC. Targeted interference of this loop may potentially be an effective adjuvant therapy to improve survival of early-stage NSCLC patients.
Hepatocyte growth factor (HGF), the ligand for the Met tyrosine kinase receptor, is a multifunctional cytokine with a role in cell survival, migration, invasion, morphogenesis, angiogenesis, and ...cellular transformation. Deregulation of HGF/Met signaling has a role in many different types of cancer, including colon carcinoma. Ki-ras proto-oncogene belongs to a family of small, membrane-associated GTPases. Dominant activating mutations in Ki- ras are associated with tumorigenic transformation in many tissue types. Both Ki-ras mutation and Hepatocyte Growth Factor (HGF) receptor Met overexpression occur at high frequency in colon cancer. The model system used in these studies included the DLD-1 colon cancer cell line with a mutated Ki-ras allele, and the DKO-4 cell line generated from DLD-1, with its mutant Ki-ras allele inactivated by targeted disruption. These cell lines were transduced with cDNAs Met receptor. By increasing the duration of mitogen activated protein kinase (MAPK) activation and decreasing the frequency of apoptosis, Ki-ras mutations and HGF/Met signaling co-operate to enhance colon xenograft tumor growth rates in vivo. Microarray global transcriptional profiling data demonstrate that transcriptional changes induced by Met receptor activation overlap with those induced by Ki-ras oncogene alone, indicating that the Ki-ras oncogene is the key transcriptional regulator in these colon carcinoma cells. Novel transcriptional targets of HGF/Met and Ki- ras signaling were identified by overlaying microarray and protein-protein interaction data in order to generate novel molecular interaction networks. Expression of a panel of modified Met receptors in DLD-1 cells demonstrates that Met downstream adaptor proteins Grb2 and Shc can play a role in tumor growth suppression through decreasing Gab1 protein levels. Gab1 is a large adaptor molecule that is crucial in regulating numerous biological processes downstream from the Met receptor. The regulation of Gab1 protein levels by Met signaling presents a novel mechanism for the regulation of tumor growth rate by HGF/Met signaling. Overall, the work presented here provides new insight into the interactions among different Met signaling pathways in the context of colon cancer progression.
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