Tammy Bowman

STATS IN ONCOGENESIS

Since their discovery as key mediators of cytokine signaling, considerable progress has been made in defining 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 specific 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 specific STATs occurs with surprisingly high frequency in a wide variety of human cancers. Together, these findings 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 identified. 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.

Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

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 effects 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 findings, 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.

Persistent Activation of Stat3 Signaling Induces Survivin Gene Expression and Confers Resistance to Apoptosis in Human Breast Cancer Cells

Purpose: Signal transducer and activator of transcription 3 (Stat3) protein is persistently activated in breast cancer and promotes tumor cell survival. To gain a better understanding of the role of constitutive Stat3 signaling in breast cancer progression, we evaluated the expression profile of potential Stat3-regulated genes that may confer resistance to apoptosis. Experimental Design: Stat3 signaling was blocked with antisense oligonucleotides in human MDA-MB-435s breast cancer cells and Affymetrix GeneChip microarray analysis was done. The candidate Stat3 target gene Survivin was further evaluated in molecular assays using cultured breast cancer cells and immunohistochemistry of breast tumor specimens. Results: Survivin, a member of the inhibitor of apoptosis protein family, was identified as a potential Stat3-regulated gene by microarray analysis. This was confirmed in Survivin gene promoter studies and chromatin immunoprecipitation assays showing that Stat3 directly binds to and regulates the Survivin promoter. Furthermore, direct inhibition of Stat3 signaling blocked the expression of Survivin protein and induced apoptosis in breast cancer cells. Direct inhibition of Survivin expression also induced apoptosis. Increased Survivin protein expression correlates significantly (P = 0.001) with elevated Stat3 activity in primary breast tumor specimens from high-risk patients who were resistant to chemotherapy treatment. Conclusions: We identify Survivin as a direct downstream target gene of Stat3 in human breast cancer cells that is critical for their survival in culture. Our findings suggest that activated Stat3 signaling contributes to breast cancer progression and resistance to chemotherapy by, at least in part, inducing expression of the antiapoptotic protein, Survivin.

Induction of p21WAF1/CIP1 and cyclin D1 expression by the Src oncoprotein in mouse fibroblasts: role of activated STAT3 signaling.

While the activated viral Src oncoprotein, v-Src, induces uncontrolled cell growth, the mechanisms underlying cell cycle deregulation by v-Src have not been fully defined. Previous studies demonstrated that v-Src induces constitutively active STAT3 signaling that is required for cell transformation and recent data have implicated STAT3 in the transcriptional control of critical cell cycle regulators. Here we show in mouse fibroblasts stably transformed by v-Src that mRNA and protein levels of p21 (WAF1/CIP1), cyclin D1, and cyclin E are elevated. Using reporter constructs in transient-transfection assays, the cyclin D1 and p21 promoters were both found to be transcriptionaly induced by v-Src in a STAT3-dependent manner. The kinase activities of cyclin D/CDK4, 6 and cyclin E/CDK2 complexes were only slightly elevated, consistent with the findings that coordinate increases in p21, cyclin D1 and cyclin E resulted in an increase in cyclin/CDK/p21 complexes. Similar results were obtained in NIH3T3 and BALB/c 3T3 cells stably transformed by v-Src, indicating that these regulatory events associated with STAT3 signaling represent common mechanisms independent of cell line or clonal variation. These findings suggest that STAT3 has an essential role in the regulation of critical cell cycle components in v-Src transformed mouse fibroblasts.

Requirement for Ras/Rac1-Mediated p38 and c-Jun N-Terminal Kinase Signaling in Stat3 Transcriptional Activity Induced by the Src Oncoprotein

ABSTRACT Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

Activation of stat3 in primary tumors from high-risk breast cancer patients is associated with elevated levels of activated SRC and survivin expression.

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 high-risk 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-xL, 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.

An H3 coding region regulatory element is common to all four nucleosomal classes of mouse histone-encoding genes

We have previously identified the alpha element within the mouse H2A and H3 histone gene coding region activating sequences (CRAS). This common element is required for normal in vivo expression of these two replication-dependent genes and interacts with nuclear factor(s). Here we report that the CRAS alpha element is present in the coding region sequences of two other replication-dependent mouse H genes, H2B and H4. The DNA-protein interactions were examined by DNase I footprinting and methylation-interference assays, and are very similar, if not identical, for these replication-dependent genes, confirming that the alpha element is the binding site for common nuclear protein(s) in H genes of all four nucleosomal classes. Moreover, we show that the same nuclear factor is involved in these DNA-protein interactions. Our findings, together with the fact that a replication-independent H gene, H3.3, has a mutated alpha element that fails to interact with nuclear proteins, suggest that this regulatory element is involved in the coordinate expression of the replication-dependent core H genes in the eukaryotic cell cycle.