The role of Stat3 signaling in skin carcinogenesis

The primary goal of these studies is to gain a better understanding of the role of Stat3 signaling during skin cancer development. Stat3 has been shown to be important in the regulation of apoptosis, proliferation, motility, and angiogenesis, all hallmarks of malignancy. Our laboratory and others have shown that Stat3 activity is constitutive in several malignant cell types and is required for initiation, promotion and progression to a more malignant phenotype in skin SCC. We aim to address the following three problems: (1) How does all-trans retinoic acid (ATRA) administration affect Stat3 activity and tumor formation in 2-stage chemical carcinogenesis mouse models of skin cancer? We have previously used the well-established mouse skin 2-stage chemical carcinogenesis protocol to test the ability of ATRA to suppress skin tumor formation. Mice of the SENCAR strain develop benign papillomas which convert to malignant SCCs at a very low frequency after 30 weeks of TPA treatment. ATRA is a highly efficient suppressor of tumor formation in this model and we have shown that this effect correlates with the suppression of the B-Raf/Mek/ERK pathway. Interestingly, ATRA treatment also suppresses Stat3 activity during early stages of tumor promotion, but is unable to do so beyond 7 weeks. Unlike SENCAR mice, TPA-induced tumor formation in K5.Stat3C mice, which express a constitutively active form of Stat3, called Stat3C, under the control of the skin basal cell-specific cytokeratin 5 promoter, is not inhibited by ATRA. However, ATRA is still able to reduce the activity of Stat3 in the skin of these mice. These studies are the first description of the effect of ATRA treatment on Stat3 activity in the 2-stage skin chemical carcinogenesis model.;We determine that the HGF/cmet/Stat3 signaling cascade is critically involved in the motility and invasion of cancer cells, and importantly, disruption of this pathway by expression of S3DN diminishes cmet activity and inhibits cmet/Stat3 interaction. We speculate that Stat3 alone may not be sufficient for enhanced invasion, but a combination of high cmet and Stat3 activity, especially in an unregulated positive feedback loop, can drive cells toward invasive behavior. We successfully suppress this invasive potential with the expression of S3DN, which blocks cmet activity. Based on these results, we suggest that targeting the HGF/cmet/Stat3 pathway may be an especially effective target strategy for cancer therapy.;(3) What effect does overexpression of S3DN have on the survival of SCC cells and which genes can we identify as key regulators of Stat3 mediated survival? In the present study we demonstrated that S3DN cells had reduced expression of survivin, an inhibitor of apoptosis that is often up-regulated during tumorigenesis and is associated with chemotherapy resistance and poor patient survival. S3DN cells progressively lost expression of survivin during a time course of serum starvation, correlating with cell death. In vivo mouse experiments indicate that tumors generated from S3DN cells also have reduced expression of survivin. DNA microarray analysis reveals that several genes involved in the regulation of apoptosis, including survivin, as well as numerous cell cycle regulators, are differentially expressed in control and S3DN cells under conditions of serum deprivation. Further bioinformatics analysis of this microarray data has organized the differentially expressed genes into functional gene networks. Using arrays designed to detect secreted chemokines, we were able to analyze which chemokines differ between cells undergoing apoptosis (S3DN cells) and those able to survive (SRB12-p9 parental cells) in serum deprivation conditions. The chemokines identified in this study are promising candidates for future experiments, since they may be critical Stat3 regulated survival genes. (Abstract shortened by UMI.);(2) What role does Stat3 activation play in the motility and invasion of SCC cells and how is this activity related to the HGF/cmet signaling axis? In order to investigate the role of Stat3 in the induction of cell motility and invasion in greater detail, we have employed a cell culture model in which we have expressed a dominant-negative form of Stat3 in a tumorigenic squamous cell carcinoma cell line, SRB 12-p9. We showed that SRB12-p9 cell clones expressing FLAG-tagged Stat3beta-Y705F (S3DN cells) were significantly less invasive in both in vitro and in vivo assays, and this phenotype was accompanied by inhibition of HGF-induced motility. We also described a likely role for Stat3 at the cell membrane that contributes to the malignant phenotype of invading tumor cells.