BackgroundStat3 is a cytokine- and growth factor-inducible transcription factor that regulates cell motility, migration, and invasion under normal and pathological situations, making it a promising target for cancer therapeutics. The hepatocyte growth factor (HGF)/c-met receptor tyrosine kinase signaling pathway is responsible for stimulation of cell motility and invasion, and Stat3 is responsible for at least part of the c-met signal.MethodsWe have stably transfected a human squamous cell carcinoma (SCC) cell line (SRB12-p9) to force the expression of a dominant negative form of Stat3 (S3DN), which we have previously shown to suppress Stat3 activity. The in vitro and in vivo malignant behavior of the S3DN cells was compared to parental and vector transfected controls.ResultsSuppression of Stat3 activity impaired the ability of the S3DN cells to scatter upon stimulation with HGF (c-met ligand), enhanced their adhesion, and diminished their capacity to invade in vitro and in vivo. Surprisingly, S3DN cells also showed suppressed HGF-induced activation of c-met, and had nearly undetectable basal c-met activity, as revealed by a phospho-specific c-met antibody. In addition, we showed that there is a strong membrane specific localization of phospho-Stat3 in the wild type (WT) and vector transfected control (NEO4) SRB12-p9 cells, which is lost in the S3DN cells. Finally, co-immunoprecipitation experiments revealed that S3DN interfered with Stat3/c-met interaction.ConclusionThese studies are the first confirm that interference with the HGF/c-met/Stat3 signaling pathway can block tumor cell invasion in an in vivo model. We also provide novel evidence for a possible positive feedback loop whereby Stat3 can activate c-met, and we correlate membrane localization of phospho-Stat3 with invasion in vivo.

Cancer cell invasion and induction of the metastatic cascade are often the consequence of deregulated cell adhesion and migration. There is substantial evidence for the involvement of Stat3 in cell motility, migration, and invasion under normal and pathological situations. Stat3 activation is required for induction of genes encoding MMP-1, MMP-2, and MMP-9, key components of tumor invasion. Through binding to its receptor, cmet, hepatocyte growth factor (HGF) can regulate cell survival, growth, migration, and angiogenesis. cmet is often constitutively active in human tumors and HGF/cmet signaling is at least partially mediated by Stat3. Overall, these observations suggest that positive feedback signals in the HGF/cmet/Stat3 signaling pathway contribute to tumorigenesis. To explore the role of Stat3 in skin cell malignancy in a human cell culture model, our lab has overexpressed a dominant negative form of Stat3β in the tumorigenic SCC cell line, SRB12-p9. We established SRB12-p9 cell clones expressing FLAG-tagged Stat3β-Y705F (S3DN). Suppression of Stat3 activity impaired the ability of these cells to scatter upon stimulation with HGF, diminished their capacity to invade, and enhanced adhesion in vitro. S3DN cells also showed suppressed HGF-induced activation of cmet, as revealed by a phospho-specific cmet antibody. We next assessed the role of Stat3 in tumor invasion using in vivo mouse tumorigenicity experiments. When injected subcutaneously into immune-deficient mice, S3DN cells produced tumors that were less locally invasive, though WT tumors were more differentiated as indicated by K10 immunostaining. Upon further examination of these tumors, we found that those arising from WT cells had more intense membrane staining for total cmet and phospho-cmet than S3DN cells, indicating stronger cmet activity in these tumors. This pattern of cmet staining correlated with increased phospho-Stat3 membrane staining in the WT tumors. Western blotting revealed a decrease in MMP-2, MMP-9, and cmet expression in the S3DN cells. These results are consistent with those from the in vitro experiments. Finally, we show that S3DN may interfere with Stat3/cmet interaction, as evidenced by co-immunoprecipitation experiments, preventing the activation of cmet at the cell membrane. Ongoing studies will further address the effect of S3DN expression on cmet signaling and the effect this has on the HGF/c-met/Stat3 signaling loop and tumor cell invasion. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B40.