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.

Activation of STAT3 by the c-Fes protein-tyrosine kinase.

STATs (signal transducers and activators of transcription) are transcription factors that contain SH2 domains and are activated by tyrosine phosphorylation, often in response to cytokine stimulation. Recent evidence indicates that the transforming tyrosine kinases encoded by the v-Src, v-Abl, and v-Fps oncogenes can induce STAT activation, suggesting that their normal cellular homologs may contribute to STAT activation under physiological conditions. In this report, we provide direct evidence that c-Fes, the normal human homolog of v-Fps, potently activates STAT3. Transient transfection of human 293T cells with STAT3 and Fes resulted in strong stimulation of STAT3 DNA binding activity. In contrast, only modest activation of STAT5 by Fes was observed in this system, indicative of possible selectivity. To determine whether Fes-induced STAT3 activation is dependent upon endogenous mammalian kinases, co-expression studies were also performed in Sf-9 insect cells. Fes also induced a dramatic increase in STAT3 DNA binding activity in this system, whereas no activation of STAT5 was observed. As a positive control, both STAT3 and STAT5 were shown to be activated by the Bcr-Abl tyrosine kinase in Sf-9 cells. Fes induced strong tyrosine phosphorylation of STAT3 in both expression systems, consistent with the gel-shift results. Fes and STAT3 have been independently linked to myeloid differentiation. Results presented here suggest that these proteins may cooperate to promote differentiation signaling in response to hematopoietic cytokines.

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.

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.

The coding sequences of mouse H2A and H3 histone genes contains a conserved seven nucleotide element that interacts with nuclear factors and is necessary for normal expression.

Expression of replication-dependent histone genes of all classes is up-regulated coordinately at the onset of DNA synthesis. The cellular signals involved in coordinate regulation of these genes are not known. Here we report identification of an alpha element, present within the mouse histone coding region activating sequence (CRAS). We show evidence that this element is present in histone genes from two classes, H2a and H3, in the mouse. This element has two biological functions in histone gene expression, i.e. the element interacts with nuclear proteins in regulation of gene expression, as well as encoding the amino acids of the histone proteins. We present both in vivo and in vitro evidence that interaction of nuclear proteins with this element is required for normal expression. The binding site for nuclear protein(s) has been precisely defined by means of synthetic oligonucleotides, as well as DNase I protection and methylation interference. It is interesting to note that the histone CRAS alpha element is mutated in a replication-independent H3.3 gene; 5 of 7 nt in the CRAS alpha box are changed in this gene.

A common transcriptional activator is located in the coding region of two replication-dependent mouse histone genes.

There is a region in the mouse histone H3 gene protein-encoding sequence required for high expression. The 110-nucleotide coding region activating sequence (CRAS) from codons 58 to 93 of the H3.2 gene restored expression when placed 520 nucleotides 5' of the start of transcription in the correct orientation. Since identical mRNA molecules are produced by transcription of the original deletion gene and the deletion gene with the CRAS at -520, effects of the deletions on mRNA stability or other posttranscriptional events are completely ruled out. Inversion of the CRAS sequence in its proper position in the H3 gene resulted in only a threefold increase in expression, and placing the CRAS sequence 5' of the deleted gene in the wrong orientation had no effect on expression. In-frame deletions in the coding region of an H2a.2 gene led to identification of a 105-nucleotide sequence in the coding region between amino acids 50 and 85 necessary for high expression of the gene. Additionally, insertion of the H3 CRAS into the deleted region of the H2a.2 gene restored expression of the H2a gene. Thus, the CRAS element has an orientation-dependent, position-independent effect. Gel mobility shift competition studies indicate that the same proteins interact with both the H3 and H2a CRAS elements, suggesting that a common factor is involved in expression of histone genes.