Since their discovery as key mediators of cytokine signaling, considerable progress has been made in de®ning the structure-function relationships of Signal Transducers and Activators of Transcription (STATs). In addition to their central roles in normal cell signaling, recent studies have demonstrated that diverse oncoproteins can activate speci®c STATs (particularly Stat3 and Stat5) and that constitutively-activated STAT signaling directly contributes to oncogenesis. Furthermore, extensive surveys of primary tumors and cell lines derived from tumors indicate that inappropriate activation of speci®c STATs occurs with surprisingly high frequency in a wide variety of human cancers. Together, these ®ndings provide compelling evidence that aberrant STAT activation associated with oncogenesis is not merely adventitious but instead contributes to the process of malignant transformation. These studies are beginning to reveal the molecular mechanisms leading to STAT activation in the context of oncogenesis, and candidate genes regulated by STATs that may contribute to oncogenesis are being identi®ed. Recent studies suggest that activated STAT signaling participates in oncogenesis by stimulating cell proliferation and preventing apoptosis. This review presents the evidence for critical roles of STATs in oncogenesis and discusses the potential for development of novel cancer therapies based on mechanistic understanding of STAT signaling.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that were first identified as mediators of cellular responses to interferons (reviewed in references 12, 16 and 35). Signaling induced by the interaction of interferons and other cytokines with their cognate receptors is initiated by a cascade of events, including receptor aggregation and activation of Janus protein tyrosine kinases (JAKs) associated with the receptors. Subsequently, STAT proteins are recruited to the receptor-JAK complexes and activated by tyrosine phosphorylation, which promotes the formation of homodimers or heterodimers of STAT family members. Activated STATs, in turn, translocate to the nucleus and bind to specific DNA response elements that regulate gene expression. There are at least seven genes in the mammalian genome known to encode different STAT family members, which are activated in various combinations in response to stimulation by numerous cytokines (12,16,35).It has become evident that, in addition to cytokines, mitogenic growth factors, such as platelet-derived growth factor and epidermal growth factor, also induce STAT signaling, particularly Stat1, Stat3, and Stat5 (21,35). An emerging concept is that normal signaling by STAT proteins is involved in control of diverse biological processes regulated by cytokines and growth factors, including cell differentiation, proliferation, development, and apoptosis (2,

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological eects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our ®ndings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Purpose: Constitutive activation of signal transducer and activator of transcription 3 (Stat3) protein has been observed in a wide variety of tumors, including breast cancer, and contributes to oncogenesis at least in part by prevention of apoptosis. In a study of 45 patients with highrisk breast cancer enrolled in a phase II neoadjuvant chemotherapy trial with docetaxel and doxorubicin, we evaluated the levels of Stat3 activation and potentially associated molecular biomarkers in invasive breast carcinoma compared with matched nonneoplastic tissues. Experimental Design: Using immunohistochemistry and image analysis, we quantified the levels of phospho-Stat3 (pY-Stat3), phospho-Src (pY-Src), epidermal growth factor receptor, HER2/neu, Ki-67, estrogen receptor, Bcl-2, Bcl-x L , Survivin, and apoptosis in formalin-fixed, paraffin-embedded sections from invasive carcinomas and their paired nonneoplastic parenchyma. The levels of molecular biomarkers in nonneoplastic and tumor tissues were analyzed as continuous variables for statistically significant correlations. Results: Levels of activated pY-Stat3 and pY-Src measured by immunohistochemistry were significantly higher in invasive carcinoma than in nonneoplastic tissue (P < 0.001). In tumors, elevated levels of pY-Stat3 correlated with those of pY-Src and Survivin. Levels of pY-Stat3 were higher in partial pathologic responders than in complete pathologic responders. In partial pathologic responders, pY-Stat3 levels correlated with Survivin expression. Conclusions: Our findings suggest important roles for elevated activities of Stat3 and Src, as well as Survivin expression, in malignant progression of breast cancer. Furthermore, elevated Stat3 activity correlates inversely with complete pathologic response. These findings suggest that specific Stat3 or Src inhibitors could offer clinical benefits to patients with breast cancer.

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.

Recent data suggest that signal transduction may have a critical role in the development and regulation of the metastatic phenotype. Here, we investigated the role of c-Src activation in the process of human colon cancer metastasis to the liver. Our data, derived from two di erent sets of human colon cancer cell line metastatic variants, suggest that not only do highly-metastatic cells display constitutively elevated c-Src protein kinase activity when compared to poorly metastatic cells, but also that receptor tyrosine kinases participate in the ligand-activation of c-Src above basal levels. Speci®cally, the epidermal growth factor receptor (EGFR), p185HER2/Neu and the hepatocyte growth factor receptor (c-Met) appear to be linked to the process because they preferentially activate c-Src in highlymetastatic cells. EGFR was found to associate with c-Src in colon cancer cells and speci®c inhibitors of the EGFR resulted in a reduction of c-Src activity to basal levels. In addition, c-Src transfectants displayed partially-activated EGFRs, suggesting a feedback role for c-Src in the regulation of the EGFR. p185HER2/Neu was also identi®ed in immunocomplexes of c-Src following ligand activation of the EGFR, but only in highly-metastatic cells. Collectively, these observations suggest a paradigm whereby c-Src interacts with multiple cell-surface growth factors in a catalytic fashion for the development of tumor cells with metastatic potential.

Members of the STAT family of transcriptional regulators modulate the expression of a variety of gene products that promote cell proliferation, survival and transformation. Although initially identi®ed as mediators of cytokine signaling, the STAT proteins are also activated by, and thus may contribute to the actions of, polypeptide growth factors. To de®ne the mechanism by which these factors activate STATs, we examined the process of Stat3 activation in Balb/c-3T3 ®broblasts treated with platelet-derived growth factor (PDGF). As STATs are activated by tyrosine phosphorylation, and as PDGF receptors are ligand-activated tyrosine kinases, we considered the possibility that Stat3 interacts with and is phosphorylated by PDGF receptors. We ®nd that Stat3 associates with PDGF b receptors in both the presence and, surprisingly, the absence of PDGF. Moreover, Stat3 was phosphorylated on tyrosine in PDGF b receptor immunoprecipitates of PDGF-treated but not untreated cells. Although required, receptor activation was insucient for Stat3 activation. When added to cells in combination with a pharmacologic agent (PD180970) that speci®cally inhibits the activity of Src family tyrosine kinases, PDGF did not activate Stat3 as monitored by electrophoretic mobility shift assay. PD180970 did not aect MAPK activation by PDGF or the JAK-dependent activation of Stat3 by interleukin-6. The necessity of Src activity for Stat3 activation by PDGF was further evidenced by data showing the presence of Src in complexes containing both Stat3 and PDGF b receptors in PDGF-treated cells. These results suggest a novel mechanism of STAT activation in which inactive Stat3 pre-assembles with inactive PDGF receptors, and in response to ligand binding and in a manner dependent on Src kinase activity, is rapidly phosphorylated and activated. Additional data demonstrate that Src kinase activity is also required for PDGF stimulation of DNA synthesis in density-arrested cells.

Signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression in response to cytokine and growth factor stimulation. Emerging evidence indicates that STAT signaling is also frequently activated by oncogenes and in tumor cells. Constitutive activation of STAT proteins has been reported in cell lines stably transformed by diverse oncoproteins that directly or indirectly activate specific tyrosine kinase signaling pathways. In addition, STAT activation has been detected in a variety of human tumors and tumor cell lines, many of which are known to harbor activated tyrosine kinases. Recent findings support a model in which activation of STAT signaling in the context of oncogenesis induces gene expression that participates in malignant transformation.