Stat’s (signal transducers and activators of transcription) are a family of latent transcription factors that are activated in response to many cytokines and growth factors. Stat activation is dependent upon tyrosine phosphorylation, which induces dimerization via reciprocal phosphotyrosine–src homology domain 2 (phosphotyrosine–SH2) interaction between two Stat molecules. Activated Stat’s translocate to the nucleus where they bind to consensus promoter sequences of target genes and activate their transcription (1) (Figure ​(Figure1).1). Many tyrosine kinases, including JAKs (Janus kinases), RTKs (receptor tyrosine kinases), and non-RTKs can phosphorylate Stat proteins. In normal cells, Stat tyrosine phosphorylation is transient, lasting from 30 minutes to several hours. However, in numerous cancer-derived cell lines or in primary tumors, Stat proteins (in particular Stat3) are persistently tyrosine phosphorylated either as a consequence of deregulated positive effectors of Stat activation such as tyrosine kinases or negative regulators of Stat phosphorylation, e.g., phosphatases, suppressor of cytokine signaling, protein inhibitor of activated stats) (2). Inhibition of Stat3 activity in tumor-derived cell lines by the introduction of antisense, small interfering RNA, dominant-negative Stat3 constructs, and/or blockade of tyrosine kinases has been associated with growth arrest and apoptosis (2). Furthermore, the introduction of a constitutively activated Stat3 molecule (Stat3C) into immortalized cell lines leads to transformation, indicating an oncogenic role for activated Stat3 (3, 4). A possible mechanism for transformation by activated Stat3 is the transcriptional upregulation of genes known to be involved in proliferation and apoptosis, including Bcl-xL, c-Myc, cyclin D1, Vegf, and Survivin (3, 5–8). In addition to its role as a transcription factor, phosphorylated Stat3 has been described in a recent report as a component of focal adhesions (sites of cell contact with the extracellular matrix) that may contribute to the invasiveness of ovarian cancer cells (9). The in vivo role of Stat3 in tumorigenesis has not been addressed until now. In this issue of the JCI, Chan and colleagues demonstrate in two different murine models of skin tumor development that Stat3 is required for de novo tumorigenesis (10).

K5Cre.Stat3^fl/fl transgenic mice, whose epidermal and follicular keratinocytes lack Stat3, are viable and develop normally. However, they have defects in hair cycle processes as well as impaired wound healing, and they develop spontaneous ulcers with age. In vitro, keratinocytes derived from these mice had no defects in proliferation, but growth factor–dependent migration was markedly impaired in contrast to control keratinocytes (17). In the study described in this issue of the JCI, Chan et al. used K5Cre.Stat3^fl/fl transgenic mice to investigate the role of Stat3 in chemically induced carcinogenesis of the skin (10). The role of Stat3 in tumor initiation, the first step of chemically induced carcinogenesis, was addressed both in vitro, in DMBA-treated primary keratinocytes, and in vivo, by topical treatment with this mutagen. Stat3 mutant keratinocytes underwent enhanced apoptosis following DMBA treatment, compared to control keratinocytes. Expression of Ha-ras homolog (v-Ha-ras) into cultured primary keratinocytes in vitro was used to generate initiated keratinocytes. Upon the introduction of a Stat3 decoy molecule (a high-affinity double-stranded DNA binding site for Stat3), the v-Ha-ras–initiated keratinocytes underwent apoptosis with a concomitant decrease in Bcl-xL levels. In general, inhibiting Stat3 function in cancer-derived cell lines containing abundant phosphorylated Stat3 leads to apoptosis or growth arrest. In contrast, cell lines which contain low or no levels of detectable tyrosine-phosphorylated Stat3 are relatively unaffected by Stat3 inhibitory therapies. It therefore remains unclear how Stat3 protects keratinocytes against DMBA-induced apoptosis, since DMBA does not induce tyrosine phosphorylation of Stat3 in primary keratinocytes, nor is it likely that v-Ha-ras–containing keratinocytes contain abundant levels of phosphorylated Stat3 (18, 19). Perhaps the low amounts of phosphorylated Stat3 present in these cells are sufficient to drive transcription of antiapoptotic genes such as Bcl-xL. Alternatively, nonphosphorylated Stat3 may be playing a role as a transcription factor as has been demonstrated for Stat1 (20). There are a few notable examples where relatively low levels of phosphorylated Stat3 are sufficient to mediate protection from growth arrest or apoptosis (21, 22). Thus, determination of the relative levels of phosphorylated Stat3 required to impart a phenotype is likely to be cell-type specific and remains an important objective. It has been shown that phosphorylated Stat1 levels are markedly enhanced in Stat3 null hepatocytes (23). Given that Stat1 activation has been implicated in promoting growth arrest as well as apoptosis, it would be of interest to determine whether the enhanced apoptosis observed in the Stat3 null keratinocytes correlates with increased levels of activated Stat1.

Tumor promotion, the second stage of chemically induced skin carcinogenesis, occurs following the topical application of TPA, leading to the proliferation of epidermal cells and the subsequent formation of benign papillomas in wild-type animals. Perhaps the most important observation made by Chan et al. (10) is that mice deficient in Stat3 were completely resistant to skin tumor development. Given that DMBA treatment resulted in keratinocyte apoptosis in the K5Cre.Stat3^fl/fl animals, there was undoubtedly a decreased number of keratinocytes capable of proliferating in response to TPA. Nevertheless, the role of TPA alone (in the absence of the initiation stage) was assessed in vivo by applying TPA topically. Marked epidermal hyperproliferation and BrdU uptake was observed in wild-type animals. In contrast, significantly less proliferation was seen in the K5Cre.Stat3^fl/fl animals. TPA induced the tyrosine phosphorylation of Stat3, perhaps in part through increased production of EGF leading to activation of the EGFR (13). An increase in some of the known putative Stat3 targets, including cyclin D1 and c-Myc, was observed in the skin of wild-type animals while in the K5Cre.Stat3^fl/fl animals a delay in the increased level of these proteins was seen. These data demonstrate that upregulation of these genes is partly dependent upon Stat3. The requirement of Stat3 in tumor promotion was also examined in transgenic animals expressing v-Ha-ras targeted to keratinocytes. Topical TPA treatment of these animals led to benign papilloma formation, which was abrogated upon the introduction of a Stat3 decoy molecule. The use of a Stat3 decoy was first described as an inhibitor of tubular morphogenesis in cells grown in culture (27). It was subsequently shown to inhibit growth of squamous cell carcinoma–derived cell lines in vitro and more recently in vivo (28) (J. Grandis, personal communication).