Saracatinib impairs the peritoneal dissemination of diffuse-type gastric carcinoma cells resistant to Met and fibroblast growth factor receptor inhibitors.

Diffuse-type gastric carcinomas (DGC) exhibit more aggressive progression and poorer prognosis than intestinal-type and other gastric carcinomas. To identify potential therapeutic targets, we examined protein tyrosine phosphorylation in a panel of DGC and other gastric cancer cell lines. Protein tyrosine phosphorylation was significantly enhanced or altered in DGC cell lines compared with that in other gastric cancer cell lines. Affinity purification and mass spectrometry analysis of tyrosine-phosphorylated proteins identified Met as a protein that is preferentially expressed and phosphorylated in DGC cell lines. Unexpectedly, Met inhibitors blocked cell growth, Met downstream signaling and peritoneal dissemination in vivo in only a subset of cell lines that exhibited remarkable overexpression of Met. Likewise, only cell lines with overexpression of fibroblast growth factor receptor 2 (FGFR2) or phosphorylation of FRS2 were sensitive to an FGFR2 inhibitor. A Src inhibitor saracatinib impaired growth in cell lines that are insensitive to both Met and FGFR2 inhibitors. Saracatinib also effectively impaired peritoneal dissemination of Met-independent and FGFR2-independent SGC cells. Moreover, DGC cell lines exhibited nearly mutually exclusive susceptibility to Met, FGFR and Src inhibitors. These results suggest that DGC have distinct sensitivities to molecular target drugs and that targeting Src is beneficial in the treatment of DGC insensitive to Met and FGFR inhibition.

Stromal fibroblasts mediate extracellular matrix remodeling and invasion of scirrhous gastric carcinoma cells.

Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.

Phosphoinositide 3-kinase signaling pathway mediated by p110α regulates invadopodia formation.

Invadopodia are extracellular matrix-degrading protrusions formed by invasive cancer cells that are thought to function in cancer invasion. Although many invadopodia components have been identified, signaling pathways that link extracellular stimuli to invadopodia formation remain largely unknown. We investigate the role of phosphoinositide 3-kinase (PI3K) signaling during invadopodia formation. We find that in human breast cancer cells, both invadopodia formation and degradation of a gelatin matrix were blocked by treatment with PI3K inhibitors or sequestration of D-3 phosphoinositides. Functional analyses revealed that among the PI3K family proteins, the class I PI3K catalytic subunit p110α, a frequently mutated gene product in human cancers, was selectively involved in invadopodia formation. The expression of p110α with cancerous mutations promoted invadopodia-mediated invasive activity. Furthermore, knockdown or inhibition of PDK1 and Akt, downstream effectors of PI3K signaling, suppressed invadopodia formation induced by p110α mutants. These data suggest that PI3K signaling via p110α regulates invadopodia-mediated invasion of breast cancer cells.