Role Of Cmet Ckit And Egfr

c-Met signaling (Met-HGF/SF) has been well studied in various forms of human cancers. c-Met has been found to have a number of biological effects related to the cytoskeleton, for example scattering, cell motility, invasion, migration, and finally metastasis. HGF/SF-Met signaling plays an important role in lung cancer (solid tumor) and can invade and metastasize using this signaling pathway (Figure 5.2).24 SCF/HGF-Met signaling significantly increases the motility of epithelial cells, which play a key role in physiology and various disease processes.23

Autophosphorylation-mediated activation of c-Met triggers multiple signal transducing inter-mediates such as Grb2, the p85 subunit of PI3-kinase, Stat-3, and Gabi.60 The PI3-kinase pathway is assumed to control a number of cellular processes, including cytoskeletal organization, cell growth, and survival.61-63 Significant c-Met phosphorylation was observed on several unique tyrosines in response to HGF in SCLC (Figure 5.3).64

Various phospho-specific c-Met antibodies (anti-phospho-tyrosine [pY]1003, pY1313, pY1349, pY1365, and pY 1230/1234/1235-c-Met) were used to judge the functionality of the HGF/c-Met signaling pathway, as shown in Figure 5.4A.64 H69 cell (lysates) exposed once to HGF (40 ng/ml, 7.5 min) immuno-precipitated with c-Met antibody. Membranes were immunoblotted with the particular phospho-specific c-Met and c-Met antibodies. Increased phosphorylation of tyrosine 1003 (c-Cb1 binding site), 1313 (PI3K binding site), 1230/1234/1235 (major ligand-induced autophosphorylation site), 1349 (SHC, Src, and Gab 1 binding site), and 1365 (site known to inhibit cell morphogenesis) were observed in response to HGF. The quantity of c-Met immunoprecipitate was found to be the same with or without HGF (Figure 5.4B).64

In the same study, examination of cell motility with time-lapse video microscopy documented that activation of c-Met leads to increased cell motility, as observed by the rate of formation of cellular clusters (Figure 5.5).64 Cell motility is found to be regulated also by the focal adhesion molecules. It has been demonstrated that certain components of the focal adhesion, such as paxillin, p125FAK, and PYK2, are phosphorylated at specific sites due to HGF (Figure 5.6). Paxillin, a key focal adhesion cytoskeletal protein, was phosphorylated in R988C Met and T10101 Met, two different c-Met missense mutations in the juxtamembrane (JM) domain (R988C found in NCI-H69 and H 249 cell lines, and T10101 in SCLC tumor sample T31). This finding strongly supports a unique role of JM mutations in modulating SCLC cytoskeletal signaling and function.26 JM domain alternative splicing is an important mechanism to modulate c-Met signaling, which includes at least three 8-kb c-Met mRNA variants generated by alternative splicing.65

HGF/SF-Met protects cells from DNA damage involving PI3K to c-Akt, resulting in enhanced DNA repair66 and reduced apoptosis. In SCLC, PI3K is chronically activated because it was constitutively expressed. In MDA-MB-231 cancer cells, c-Met signaling has been found to increase

FIGURE 5.2 Mechanism of c-Met signaling and metastasis in a solid tumor. The role of c-Me/HGF and integrins signaling in the transformation and metastasis in solidtumor lung cancer cells is shown here schematically. Increased motility, scattering, and migration allow tumor cells (such as lung cancer) to proceed eventually to invasion of the ECM (extracellular matrix). Metastasis of the circulating tumor cells to various

FIGURE 5.2 Mechanism of c-Met signaling and metastasis in a solid tumor. The role of c-Me/HGF and integrins signaling in the transformation and metastasis in solidtumor lung cancer cells is shown here schematically. Increased motility, scattering, and migration allow tumor cells (such as lung cancer) to proceed eventually to invasion of the ECM (extracellular matrix). Metastasis of the circulating tumor cells to various distant organs (brain, liver, bone, and bone marrow shown here) is represented. Microenvironment of the tumor-host involves secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) by the tumor cells as well as secretion of HGF, uPA, and MMPs by the host stromal cells. (From Maulik, G. et al., Cytokine Growth Factors Rev., 13, 41, 2002. With permission.)

cell adhesion on laminins 1 and 5 fibronectin and vitronectin through a PI3-kinase-dependent mechanism. Increased adhesion was found to increase invasiveness through reconstituted basement membranes. PI3K activation may also occur via integrin signaling. Integrin clustering is generally triggered by HGF-c-Met activation, at the actin-rich adhesive sites and lamellipodia, without interrupting the integrin expression level. Therefore, HGF/c-Met signaling promotes cell adhesion/ invasion by triggering the integrin-signaling pathway.67 Recently, it was demonstrated that PI3K is associated with SCF and SDF-1a, the natural ligands for c-kit and CXCR4, respectively.

Both c-Crk and CRKL members of the Crk family of adapter proteins are important candidates for HGF/c-Met signaling pathway. Several studies have implicated Gab1 (multisubstrate signaling adapter) as an essential mediator of HGF/SF-induced branching tubulogenesis of MDCK epithelial cells.68,69

The c-Kit that encodes the receptor for stem cell factor plays a significant role in signal transduction as well as in metastasis. More than 70% of SCLC cell lines and tumors coexpress the c-kit receptor and its ligand stem cell factor, compared with 40% in NSCLC. In SCLC, kit signaling promotes autocrine growth regulation and thus accelerates tumor proliferation. c-Kit is also found

FIGURE 5.3. (A) Dose-response HGF stimulation on tyrosine phosphorylation in the H69 SCLC cell line. Cell lysates for the H69 cell line were applied on a 7.5% SDS-PAGE gel, transferred to an Immobilon-P

membrane, and probed with the anti-phospho tyrosine, anti-phospho Akt, and ß actin. Tyrosine phosphorylated proteins were detected in cellular lysates by immunoblotting using an anti-phospho tyrosine antibody (upper panel) and an anti-phospho-Akt antibody (middle panel). The ß-actin control of the same membrane shows the similar protein loading (bottom panel); (B) Kinetics study of HGF stimulation on tyrosine phosphorylation in the H69 SCLC cell line. Tyrosine phosphorylated proteins were detected in cellular lysates by immunoblotting using an anti-phospho tyrosine antibody (upper panel) and an anti-phospho-Akt antibody (middle panel). The ß-actin control of the same membrane shows the similar protein loading (bottom panel). (From Maulik, G. et al., J. Cell. Mol. Med., 6, 539, 2002. With permission.)

to be responsible for the cellular cluster movement and the individual cell membrane ruffling/ blebbing documented in SCLC cell lines.37'70'71 c-Kit demonstrates its downstream signaling via several intermediates, including that of cytoskeleton, in response to SCF binding. The binding leads to the phosphorylation of molecules involved in cell survival, such as PI3K, Akt, and S6K. The p85 PI3K subunit phosphorylation activates the p110 catalytic subunit of PI3K, which activates its downstream targets Akt, p70 ribosomal s6kinase. Akt has been found to play a significant role in

FIGURE 5.4 (A) Predicted functional domains of c-Met. Depicted here are the Sema domain (semaphorin-like),

the PSI domain (found in plexins, semaphorins, and integrins), the IPT-repeat domains (found in Ig-like regions, plexins, and transcription factors), and the TK domain (tyrosine kinase located intracellularly). The various amino acid residues with regulatory functions for the c-Met/HGF pathway are illustrated here; (B) Phosphorylation of various tyrosine residues of c-Met in response to HGF in SCLC. Cell lysates for the H69 were treated with or without HGF (40ng/ml; 7.5min) and were immunoprecipitated with anti c-Met. The whole cell lysate and immunoprecipitations were immunoblotted with anti-phospho tyrosine pY1003, pY1313, 1230/1234/1235, pY1349, and pY1365 of c-Met and anti-c-Met antibody. (From Maulik, G. et al., J. Cell. Mol. Med., 6, 539, 2002. With permission.)

FIGURE 5.5 (A) SCLC morphology in response to HGF and PI3K inhibition. H69 cells were starved overnight and treated with or without HGF (100ng/ml) and with or without LY294002 (50|M). Shown are representative pictures before and after 4h with the conditions; (B) SCLC cellular speed in response to HGF and PI3K inhibition. Time-lapse video

FIGURE 5.5 (A) SCLC morphology in response to HGF and PI3K inhibition. H69 cells were starved overnight and treated with or without HGF (100ng/ml) and with or without LY294002 (50|M). Shown are representative pictures before and after 4h with the conditions; (B) SCLC cellular speed in response to HGF and PI3K inhibition. Time-lapse video microscopy analysis in terms of speed of H69 cells in response to (-) or (+) HGF (100 ng/ml) and (-) or (+) LY294002 (50|M). Serum-starved H69 cells were visualized by time-lapse video microscopy and recorded for 4h with or without HGF and with or without LY294002. These images were then analyzed by NIH image analysis, and each cell/cluster in every frame was traced every 5 min. The position of the cell centroid was measured, and the corresponding X-and 7-axis values were noted. The distance traversed by each cell/cluster was calculated using these values, and from this the speed was determined for each cell/cluster. These data are represented for each set of experiments, and the corresponding average speed has also been shown.

SCLC cellular clusters in response to HGF and PI3K inhibition. Time-lapse video microscopy analysis in terms of the percentage change of total number of clusters of H69 cells in response to (-) or (+) HGF (100 ng/ml) and (-) or (+) LY294002 (50|iM) over a period of 4h. Serum-starved H69 cells were visualized by time-lapse video microscopy and recorded for 4h with or without HGF and with or without LY294002 (50|iM). These images were then analyzed by NIH image analysis, and each cell/cluster in every frame was traced every 5 min. The coming together of the smaller clusters to form larger ones was followed over a period of 4h. These data have been plotted as the percent change in the total number of clusters formed for each of the four sets of experiments. (From Maulik, G. et al., J. Cell. Mol. Med, 6, 539, 2002. With permission.)

mediating the balance between survival and apoptosis. Previous studies demonstrated that wild-type c-kit could promote multiple signal transduction pathways, including Src family members such as JAK/STAT pathway, MAPKinase cascade apart from PI3K.

c-Kit-mediated metastasis depends on the expression of chemokine CXCR4 receptor. CXCR4 is a seven-transmembrane G-protein coupled receptor and a co-receptor for HIV. CXCR4 and c-kit were found to induce morphological changes in the NCI-H69 SCLC cell line.72 This study also demonstrated that SCF and SDF-1a are able to induce proliferation of the NCI-H69 SCLC line, and SDF-1a itself increased motility and adhesion.72

EGFR is expressed on the cell membrane of various malignant cells. Activation of EGFR leads to phosphorylation of c-terminus. Proteins in the SH-2 (Src homology) domain have been found to bind to the phosphotyrosine residues of the receptor. The binding of EGF or TGF-a to EGFR stimulates the formation of dimeric or oligomeric structures involving several receptors. The activated receptor initiates a series of signal-transduction events through the tyrosine phosphorylation of interacting proteins that belong to the SH-2 family. This results in a sequence of responses that are involved in the mitogenic signal-transduction pathways of cells, which leads to DNA replication and cell division.73'74 Besides tyrosine phosphorylation, the EGF-activated receptors are phosphorylated in vivo on serine/threonine, a mechanism by which receptor functions are reduced. Protein kinase C (PKC), MAP kinase, p34 cdk2kinase, and casein kinase II are considered to be involved in this serine/ threonine phosphorylation and receptor attenuation.75 The activated EGF receptor/ligand complex is

FIGURE 5.6 Phosphorylation of p125FAK, PYK2, and paxillin on specific amino acid residues on the stimulation of HGF in H69 cells. Shown are specific phosphorylations of p125FAK on tyrosines 397 and 861, PYK2 on tyrosines 402 and 881, and paxillin on tyrosine 31. Shown are WCLs of H69 cells with (+) or without (-) stimulation with HGF (7.5min, 40ng/ml) separated on SDS-PAGE gel, applied to membrane, and immunoblotted with various antibodies: (A) expression and specific phosphorylation of p125FAK, anti-p125FAK, and anti-phospho-FAK (tyrosine residues 397 or 861); (B) expression and specific phosphorylation of PYK2, anti-PYK2, and anti-phospho-PYK2 (tyrosine residues 402 or 881); (C) expression and specific phosphorylation of paxillin, antipaxillin clone 5H11, and anti-phospho-paxillin (tyrosine residues 31, 118, or 181). (From Maulik, G. et al., Clin. Cancer Res., 8, 620, 2002. With permission.)

internalized and is eventually degraded in lysosomes or dissociated for EGF-receptor recycling. Several studies revealed that intrinsic tyrosine kinase activity and auto-phosphorylation sites of the EGFR are not required for all signaling pathways activated by EGF.

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