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Activated c-Met signals through PI3K with dramatic effects on cytoskeletal functions in small cell lung cancer
G Maulik1, P Madhiwala, S Brooks
1Department of Adult Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Abstract:
Small cell lung cancer (SCLC) is an aggressive illness with early metastases. There are several receptor tyrosine kinases (RTKs) overexpressed in SCLC, including c-Met. c-Met contains an external semaphorin-like domain, a cytoplasmic juxtamembrane domain, tyrosine kinase domain and multiple tyrosines that bind to adapter molecules. We have previously reported that c-Met is abundantly expressed in the NCI-H69 SCLC cell line and now have determined the downstream effects of stimulating c-Met via its ligand hepatocyte growth factor (HGF). Utilizing unique phospho-specific antibodies generated against various tyrosines of c-Met, we show that Y1003 (binding site for c-Cbl and a negative regulatory site), Y1313 (binding site for PI3K), Y1230/Y1234/Y1235 (autophosphorylation site), Y1349 (binding site for Grb2), Y1365 (important in cell morphogenesis) are phosphorylated in response to HGF (40 ng/ml, 7.5 min) in H69 cells. Since multiple biological and biochemical effects are transduced through the PI3K pathway, we determine the role of PI3K in the c-Met/HGF stimulation pathway. We initially determined that by inhibiting PI3K with LY294002 (50 microM over 72 hours), there was at least a 55% decrease in viability of H69 cells. Since H69 SCLC cells form clusters in cell culture, we determined the effects of HGF and LY294002 on cell motility of the clusters by time-lapse video microscopy. In response to HGF, SCLC moved much faster and formed more clusters, and this was inhibited by LY294002. Finally, we determined the downstream signal transduction of HGF stimulation of c-Met with and without inhibition of c-Met (with geldanamycin, an anisamycin antibiotic that inhibits c-Met in SCLC) or PI3K (with LY294002). We show that association of c-Met with PI3K and GAB2 is diminished by inhibiting c-Met. In summary, activation of the c-Met pathway targets the PI3K pathway in SCLC and this may be an important therapeutic target.
Insights
Activation of the c-Met pathway in small cell lung cancer (SCLC) stimulates the PI3K pathway, impacting cell viability and motility. This suggests targeting the c-Met/PI3K pathway could be a promising therapeutic strategy for SCLC.
Area of Science:
- Oncology
- Molecular Biology
- Cell Signaling
Background:
- Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis.
- Overexpression of receptor tyrosine kinases (RTKs), such as c-Met, is observed in SCLC.
- c-Met signaling plays a crucial role in various cellular processes relevant to cancer progression.
Purpose of the Study:
- To investigate the downstream effects of hepatocyte growth factor (HGF) stimulation on c-Met signaling in SCLC cells.
- To elucidate the role of the phosphoinositide 3-kinase (PI3K) pathway in mediating c-Met/HGF signaling in SCLC.
- To evaluate the therapeutic potential of targeting the c-Met/PI3K axis in SCLC.
Main Methods:
- Utilized phospho-specific antibodies to detect c-Met tyrosine phosphorylation in response to HGF in NCI-H69 SCLC cells.
- Assessed cell viability using PI3K inhibition with LY294002.
- Analyzed cell motility and cluster formation using time-lapse video microscopy.
- Investigated downstream signaling pathways by inhibiting c-Met or PI3K.
Main Results:
- HGF stimulation led to phosphorylation of multiple c-Met tyrosines, including Y1313, a PI3K binding site.
- Inhibition of PI3K significantly decreased H69 cell viability and reduced HGF-induced cell motility and cluster formation.
- HGF-induced association of c-Met with PI3K and GAB2 was diminished upon c-Met inhibition.
Conclusions:
- Activation of the c-Met pathway in SCLC cells directly targets and activates the PI3K pathway.
- The c-Met/PI3K signaling axis is critical for SCLC cell viability and motility.
- Targeting the c-Met/PI3K pathway represents a potential therapeutic strategy for small cell lung cancer.