The transcriptional modulator BCL6 as a molecular target for breast cancer therapy.

Inappropriate expression or activation of transcription factors can drive patterns of gene expression, leading to the malignant behavior of breast cancer cells. We have found that the transcriptional repressor BCL6 is highly expressed in breast cancer cell lines, and its locus is amplified in about half of primary breast cancers. To understand how BCL6 regulates gene expression in breast cancer cells, we used chromatin immunoprecipitation followed by deep sequencing to identify the BCL6 binding sites on a genomic scale. This revealed that BCL6 regulates a unique cohort of genes in breast cancer cell lines compared with B-cell lymphomas. Furthermore, BCL6 expression promotes the survival of breast cancer cells, and targeting BCL6 with a peptidomimetic inhibitor leads to apoptosis of these cells. Finally, combining a BCL6 inhibitor and a signal transducer and activator of transcription3 inhibitor provided enhanced cell killing in triple-negative breast cancer cell lines, suggesting that combination therapy may be particularly useful. Thus, targeting BCL6 alone or in conjunction with other signaling pathways may be a useful therapeutic strategy for treating breast cancer.

STAT5 represses BCL6 expression by binding to a regulatory region frequently mutated in lymphomas.

Deregulated expression of BCL6 is a pathogenic event in many lymphomas. BCL6 blocks cellular differentiation by repressing transcription of its target genes, and this may promote tumorigenesis. Conversely, the transcription factor signal transducers and activators of transcription (STAT)5 promotes differentiation in many systems. STAT5 upregulates a number of genes repressed by BCL6, raising the possibility that STAT5 and BCL6 have opposing roles in transcriptional regulation. Therefore, we sought to determine the effects of STAT5 activation on BCL6 expression and function. We found that activation of STAT5 downregulates BCL6 expression in B-lymphoma cells and other hematopoietic cell lines. We identified two potential STAT-binding regions in the first exon and first intron of BCL6 that fell within regions of high inter-species homology, suggesting conservation of regulatory function. STAT5 can bind inducibly and regulate transcription at one of these regions, identifying BCL6 as a STAT5 target gene. Additionally, STAT5-mediated downregulation of BCL6 results in loss of BCL6 repression of its target genes, confirming that STAT5 is a negative regulator of BCL6 function. The STAT5 responsive region of the BCL6 gene is mutated frequently in B-cell lymphomas, suggesting that loss of the repressive effects of STAT5 on BCL6 might contribute to the pathogenesis of these cancers.

The role of STATs in apoptosis.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate cytokine and growth factor induced signals that culminate in various biological responses, including proliferation and differentiation. Recent studies indicate a role for STATs in apoptosis as well. Depending upon the particular stimulus or cell type, STATs can mediate either pro-apoptotic signals or anti-apoptotic signals. STAT1 and, under some circums-tances. STAT3 are important for transducing pro-apoptotic signals whereas STAT3 and STAT5 have been implicated in promoting cell survival. Recent studies demonstrate that regulation of apoptotic pathways by STATs is largely due to transcriptional activation of genes that encode proteins that mediate or trigger the cell death process, such as Bcl-xL, caspases, Fas and TRAIL as well as those that regulate cell cycle progression, such as p21waf1. Interestingly, STAT proteins may also regulate apoptosis through a non-transcriptional mechanism by inhibiting the anti-apoptotic protein NF-kappaB. Considering that dysregulation of the STAT signaling pathway is commonly found in clinical tumor samples, understanding the mechanisms underlying STAT regulation of cell survival may lead to successful strategies for targeting STATs in cancer therapy.

The ribosomal S6 kinases, cAMP-responsive element-binding, and STAT3 proteins are regulated by different leukemia inhibitory factor signaling pathways in mouse embryonic stem cells.

Mouse embryonic stem (ES) cells remain "pluripotent" in vitro in the continuous presence of leukemia inhibitory factor (LIF). In the absence of LIF, ES cells are irreversibly committed to differentiate into various lineages. In this study we have set up an in vitro assay based on the anti-apoptotic activity of LIF to distinguish pluripotent from "differentiation-committed" ES cells. We have examined the phosphorylation profiles of known (STAT3 and ERKs) and identified new (ribosomal S6 kinases (RSKs) and cAMP-responsive element-binding protein (CREB)) LIF-regulated targets in ES and in ES-derived neuronal cells. We have demonstrated that although STAT3, a crucial player in the maintenance of ES cell pluripotency, is induced by LIF in all cell types tested, the LIF-dependent activation of RSKs is restricted to ES cells. We have shown that LIF-induced phosphorylation of RSKs in ES cells is dependent on ERKs, whereas STAT3 phosphorylation is not mediated by any known MAPK activities. Our results also demonstrate that the LIF-dependent phosphorylation of CREB is partially under the control of the RSK2 kinase.

CD40 activation of carcinoma cells increases expression of adhesion and major histocompatibility molecules but fails to induce either CD80/CD86 expression or T cell alloreactivity.

A major obstacle for the development of cancer immunotherapy is the poor capacity of most tumor cells to present antigen. It has previously been shown that ligation of CD40 on the surface of malignant B cells results in the induction of efficient antigen presentation primarily because of upregulated expression of MHC, costimulatory, and adhesion molecules. Ongoing clinical trials are testing the impact of CD40 ligation as immunotherapy for B cell malignancies. Because CD40 is also widely expressed in carcinomas, we studied whether CD40 activation of these cells using soluble recombinant trimeric human CD40 ligand (srhCD40L) can also induce T cell responses. Here, we show that carcinoma cells upregulate expression of CD54 and MHC molecules following in vitro exposure to srhCD40L but do not upregulate CD80 or CD86. CD40-activated carcinoma cells failed to trigger mixed lymphocyte reactions, in sharp contrast to CD40-activated lymphoma cells for which CD40 activation, as expected, resulted in increased expression of MHC, adhesion, and costimulatory molecules, and generated brisk allogeneic lymphocyte reactions. Retroviral-mediated expression of CD80 in carcinoma cells, with or without CD40 activation, triggered mixed lymphocyte reactions, provided cells were treated with IFN-gamma. Thus, the cell surface phenotype induced on carcinoma cells following CD40 activation is not fully capable of inducing T cell proliferation; however, these results support ongoing efforts to exploit costimulation in clinical efforts aimed at increasing carcinoma immunogenicity.

Growth, development, and behavior in early childhood following prenatal cocaine exposure: a systematic review.

CONTEXT: Despite recent studies that failed to show catastrophic effects of prenatal cocaine exposure, popular attitudes and public policies still reflect the belief that cocaine is a uniquely dangerous teratogen. OBJECTIVE: To critically review outcomes in early childhood after prenatal cocaine exposure in 5 domains: physical growth; cognition; language skills; motor skills; and behavior, attention, affect, and neurophysiology. DATA SOURCES: Search of MEDLINE and Psychological Abstracts from 1984 to October 2000. STUDY SELECTION: Studies selected for detailed review (1) were published in a peer-reviewed English-language journal; (2) included a comparison group; (3) recruited samples prospectively in the perinatal period; (4) used masked assessment; and (5) did not include a substantial proportion of subjects exposed in utero to opiates, amphetamines, phencyclidine, or maternal human immunodeficiency virus infection. DATA EXTRACTION: Thirty-six of 74 articles met criteria and were reviewed by 3 authors. Disagreements were resolved by consensus. DATA SYNTHESIS: After controlling for confounders, there was no consistent negative association between prenatal cocaine exposure and physical growth, developmental test scores, or receptive or expressive language. Less optimal motor scores have been found up to age 7 months but not thereafter, and may reflect heavy tobacco exposure. No independent cocaine effects have been shown on standardized parent and teacher reports of child behavior scored by accepted criteria. Experimental paradigms and novel statistical manipulations of standard instruments suggest an association between prenatal cocaine exposure and decreased attentiveness and emotional expressivity, as well as differences on neurophysiologic and attentional/affective findings. CONCLUSIONS: Among children aged 6 years or younger, there is no convincing evidence that prenatal cocaine exposure is associated with developmental toxic effects that are different in severity, scope, or kind from the sequelae of multiple other risk factors. Many findings once thought to be specific effects of in utero cocaine exposure are correlated with other factors, including prenatal exposure to tobacco, marijuana, or alcohol, and the quality of the child's environment. Further replication is required of preliminary neurologic findings.

A common epitope is shared by activated signal transducer and activator of transcription-5 (STAT5) and the phosphorylated erythropoietin receptor: implications for the docking model of STAT activation.

Erythropoietin (EPO) specifically activates the Janus kinase JAK2 and the transcription factor signal transducer and activator of transcription-5 (STAT5). All members of the STAT family are tyrosine phosphorylated in response to cytokine stimulation at a conserved carboxy-terminal tyrosine, Y694, in the case of STAT5. To determine structural features important for STAT signaling, we generated an activation-specific STAT5 antibody using a phosphopeptide containing amino acids 687 to 698 of STAT5 as antigen. This antibody specifically recognizes tyrosine- phosphorylated STAT5 but not nonphosphorylated STAT5. In immunoprecipitation reactions from cell lines and primary erythroblasts, 2 distinct polyclonal activation-specific STAT5 antibodies selectively immunoprecipitate the tyrosine phosphorylated EPO receptor (EPO-R) in addition to STAT5 under native and denaturing conditions. We propose that the activation-specific STAT5 antibody recognizes the 2 substrates to which the STAT5 SH2 domain interacts, namely, the tyrosine- phosphorylated EPO-R and STAT5 itself. Several studies have implicated EPO-R Y343, Y401, Y431, and Y479 in the recruitment of STAT5. Using a series of EPO-R tyrosine mutants expressed in Ba/F3 cells, we have shown that the activation-specific STAT5 antibody immunoprecipitates an EPO-R containing only 2 tyrosines at positions 343 and 401, confirming the importance of these tyrosines in STAT5 recruitment. These data uncover a novel aspect of STAT SH2 domain recognition and demonstrate the utility of activation-specific antibodies for examining the specificity of STAT-cytokine receptor interactions.

CD2 stimulation leads to the delayed and prolonged activation of STAT1 in T cells but not NK cells.

OBJECTIVE: T lymphocytes can be activated by soluble factors such as cytokines or through direct cell-cell interactions. Although cytokine receptors are known to signal through STAT family transcription factors, the mechanisms by which other cell-surface molecules, such as CD2, transduce signals is unclear. The goal of this study was to determine whether stimulation of T cells through CD2 recapitulates aspects of cytokine-induced T-cell activation by use of STAT transcription factors. MATERIALS AND METHODS: T cells were treated with anti-CD2 antibodies or cells bearing the natural CD2 ligand CD58, after which signaling through STAT transcription factors was assessed. RESULTS: Stimulation of CD2 on primary T lymphocytes leads to the tyrosine phosphorylation, nuclear translocation, and DNA binding of STAT1. In contrast to stimulation by cytokines, the activation of STAT1 in response to CD2 ligation is delayed and does not involve Jak kinases. Furthermore, while STAT phosphorylation induced by cytokines is generally transient, STAT1 phosphorylation following CD2 stimulation persists for a period of days. Transcription of key target genes such as IRF1 and c-fos proceeds with delayed kinetics following CD2 stimulation, suggesting that this unique pattern of STAT activation may lead to a distinct cellular response following CD2 ligation. This pathway appears to be restricted to T cells, as stimulation of CD2 on NK cells does not lead to STAT1 activation. CONCLUSION: Stimulation of T cells through cell-surface molecules such as CD2 involves activation of STAT transcription factors, thus recapitulating elements of cytokine signaling.

Inhibition of MAP kinase kinase causes morphological reversion and dissociation between soft agar growth and in vivo tumorigenesis in angiosarcoma cells.

Activated ras causes increased activity of several signal transduction systems, including the mitogen-activated protein kinase kinase (MAPKK) pathway and the phosphoinositol-3-kinase (PI-3-K) pathway. We have previously shown that the PI-3-K pathway plays a major role in regulation of ras-mediated tumor angiogenesis in angiosarcoma cells. However, the contribution of the MAPKK pathway to tumorigenesis and angiogenesis is not fully understood. Overexpression of constitutively active forms of MAPKK has previously been shown to transform nonmalignant NIH3T3 fibroblasts, but the effect of down-regulation of MAPKK on tumorigenesis and angiogenesis in a well established tumor has not been fully explored. We introduced a dominant negative MAPKK gene into SVR murine angiosarcoma cells. Introduction of a dominant negative MAPKK causes a significant decrease in proliferation rate in vitro and morphological reversion. Cells expressing the dominant negative MAPKK have a greatly decreased ability to form colonies in soft agar compared with wild-type cells. Despite the decreased cell growth in vitro and inability to grow in soft agar, the cells were equally tumorigenic in nude mice. Our results suggest that the MAPKK pathway is required for soft agar growth of angiosarcoma cells, and separates the phenotypes of soft agar growth versus in vivo tumorigenicity. These findings have implications in the development of signal transduction modulators as potential antineoplastic agents.

The treatment and outcome of cancer patients with thromboses on central venous catheters.

Thromboses are a common complication of central venous catheters in cancer patients. This study was performed to analyze retrospectively the treatment and outcome of all patients with venous thromboses related to central venous catheters at a major cancer center. From 1992 through 1995, 319 oncology patients with central venous catheters underwent radionuclide venography (RNV) at the Dana-Farber Cancer Institute for suspected catheter or venous thrombosis. The treatment and outcome of patients found to have venous thromboses were evaluated. Of the 319 patients, 112 were found to have evidence of venous thrombosis. The median age and platelet counts were not significantly different between the patients with and without thromboses. The most common indication for obtaining RNV was difficulty in aspirating or infusing material through the catheter. Patients who had pain or edema, or both, of the neck or upper extremity were more likely to have a venous thrombosis. Regardless of therapeutic intervention, including anticoagulation with heparin or coumadin, or both; line removal or replacement; or a combination thereof, no patients had a major adverse outcome such as pulmonary embolism, compromise of limb, or death. Only 4 patients did not have resolution of their presenting symptoms, all of whom were treated with line replacement. The overall survival of patients with and without thromboses was not significantly different. Either anticoagulation or removal of the central venous catheter (or both) appears to be adequate treatment for catheter-related thrombosis. A prospective trial to evaluate these approaches may be worthwhile so that the use of unnecessary procedures may be avoided in this patient population.

SCF and G-CSF lead to the synergistic induction of proliferation and gene expression through complementary signaling pathways.

Stem cell factor (SCF) is a potent costimulatory molecule for many cytokines. Its synergy with granulocyte colony-stimulating factor (G-CSF) results in important biologic and clinical effects, although the mechanism by which this occurs remains poorly understood. To investigate this interaction, this study used a retroviral vector to transduce the G-CSF receptor into MO7e cells, which are known to express the SCF receptor. The transduced G-CSF receptor is functionally active, and the resultant MO7e-G cells recapitulate the proliferative synergy between SCF and G-CSF. When treated with both cytokines, a marked shortening of the G(0)/G(1) phase of the cell cycle occurs, associated with a suppression of the cyclin-dependent kinase inhibitor p27(kip-1). In addition, SCF and G-CSF induce the synergistic activation of c-fos, a proto-oncogene involved in propagation of mitogenic signals in hematopoietic cells. G-CSF, but not SCF, induces the tyrosine phosphorylation of STAT1 and STAT3, transcription factors that can mediate the induction of c-fos. However, SCF induces phosphorylation of STAT3 on serine727 (ser727), which is necessary for maximal STAT transcriptional activity, and the combination of SCF and G-CSF leads to complete STAT3 phosphorylation on ser727. The pathways by which SCF and G-CSF lead to serine phosphorylation of STAT3 are distinct and are partially dependent on phosphatidylinositol-3 kinase and ERKs, pathways that are also necessary for the synergistic effects of SCF and G-CSF on proliferation and c-fos induction. Thus, MO7e-G cells provide a powerful system in which the molecular basis of the synergy between SCF and G-CSF can be dissected.

Impairment of STAT activation by IL-12 in a patient with atypical mycobacterial and staphylococcal infections.

IL-12 plays a pivotal role in the stimulation of immune responses against intracellular infections. This role is manifested in the increased susceptibility to atypical mycobacterial and salmonella infections among individuals whose lymphocytes lack expression of IL-12Rbeta1. Here, we report on a patient with Mycobacterium avium infection, recurrent Staphylococcus aureus sinusitis, and multiple adverse drug reactions whose T cells were unable to produce IFN-gamma or proliferate in response to IL-12 despite the expression of wild-type IL-12Rbeta1 and IL-12Rbeta2. The defect in these functional responses to IL-12 was selective, as cytolytic activity induced by IL-12 was intact, and lymphocytes were responsive to stimulation by IL-2. An examination of cytokine signaling revealed that STAT4 and extracellular regulated kinase 1 (ERK1) activation by IL-12 was intact, whereas the activation of STAT1, -3, and -5 by IL-12 was lost. This impairment of STAT activation was specific for IL-12, as STAT activation by IL-2, IL-15, and IFN-gamma was unaffected. These findings demonstrate that the activation of STAT4 alone is not sufficient for IL-12-induced IFN-gamma production and proliferation and suggest that other STATs play a role in these responses to IL-12. While the etiology of the impaired IL-12 signaling in this patient has not yet been elucidated, the absence of mutations in IL-12Rbeta1 or IL-12Rbeta2 and the preservation of STAT4 activation raise the possibility that there may be a mutation in an as yet undiscovered component of the IL-12 signaling complex that is normally required for the recruitment and activation of STAT1, -3, and -5.

STAT3 regulates the growth and immunoglobulin production of BCL(1) B cell lymphoma through control of cell cycle progression.

STAT3 is constitutively phosphorylated on tyrosine(705) in self-renewing, CD5(+) murine B-1 lymphocytes. Nuclear extracts from untreated primary B-1 or CD5(+) BCL(1) B lymphoma cells were found to contain immunoreactive STAT3 protein that binds to a sis-inducible element present in the promoter of the p21(waf1/cip1) tumor suppressor gene and is constitutively phosphorylated on serine(727). To determine the functional significance of constitutive STAT3 activation in B lymphoma cells, a specific STAT3 antisense oligonucleotide was developed and used to examine basal BCL(1) cell growth and IgM production. Abrogating STAT3 expression in BCL(1) cells inhibited their proliferative capacity and induced a corresponding decrease in secretion of IgM. Cell cycle analysis showed a block in progression through G1 in BCL(1) cells treated with the STAT3 antisense oligonucleotide. These results indicate that STAT3 controls cell growth and immunoglobulin secretion by enhancing progression through the G1 phase of the cell cycle in BCL(1) B cell lymphoma.

STAT signaling in the pathogenesis and treatment of leukemias.

Leukemias continue to cause significant mortality in adults and children, and the use of standard cytotoxic chemotherapy has reached a therapeutic plateau. Thus, there is great interest in treatments directed against inappropriately activated cell signaling pathways which stimulate the uncontrolled growth of neoplastic cells. Increasing evidence suggests that the STAT signaling cascade may be one target of these therapies. Signal transducer and activator of transcription (STAT) proteins are critical in mediating the response of hematopoietic cells to a diverse spectrum of cytokines. Constitutive STAT activation is present in many malignancies and has been especially well characterized in acute and chronic leukemias. While STAT activation is a common characteristic of leukemias, the specific pattern of activated STATs and the manner by which STAT activation occurs vary with each disease. STAT tyrosine phosphorylation can occur through inappropriate Jak activation or by direct activation of an oncoprotein such as Bcr/Abl, and STAT serine phosphorylation may play an important role in leukemias as well. Thus, the STAT signaling pathway is an attractive target for therapeutic intervention, and strategies designed to inhibit STAT activation and STAT mediated gene transcription may play an important role in the next generation of anti-leukemia therapies. Oncogene (2000).

TEL/PDGFbetaR fusion protein activates STAT1 and STAT5: a common mechanism for transformation by tyrosine kinase fusion proteins.

OBJECTIVE: TEL/PDGFbetaR is a tyrosine kinase fusion protein associated with the pathogenesis of chronic myelomonocytic leukemia. The following experiments were undertaken to understand the mechanisms whereby TEL/PDGFbetaR transforms cells. MATERIALS AND METHODS: Activation of JAK and STAT proteins was studied in an interleukin 3 (IL-3)-dependent cell line, Ba/F3, transformed to IL-3 independence by TEL/PDGFbetaR. RESULTS: TEL/PDGFbetaR activates STAT1 and STAT5 in transformed Ba/F3 cells through a JAK-independent pathway. Activation of STAT proteins requires the kinase activity of TEL/PDGFbetaR. JAK1, JAK2, JAK3, and TYK2 are not phosphorylated by TEL/PDGFbetaR. However, TEL/PDGFbetaR can phosphorylate STAT5 in transiently transfected COS cells, suggesting that TEL/PDGFbetaR may itself be the kinase involved in tyrosine phosphorylation of STAT proteins. In contrast, native PDGFbetaR stimulated by PDGF ligand does not activate STAT proteins to a significant degree in this hematopoietic context. STAT1 and STAT5 also are activated by TEL/ABL and TEL/JAK2 fusion proteins associated with human leukemia. CONCLUSIONS: STAT activation may be a common mechanism of transformation by leukemogenic tyrosine kinase fusion proteins.

Interleukin-2 enhances the response of natural killer cells to interleukin-12 through up-regulation of the interleukin-12 receptor and STAT4.

Interleukin (IL)-12 plays a critical role in modulating the activities of natural killer (NK) cells and T lymphocytes. In animal models, IL-12 has potent antitumor effects that are likely mediated by its ability to enhance the cytotoxic activity of NK cells and cytotoxic T lymphocytes, and to induce the production of interferon (IFN)-gamma by NK and T cells. In addition to IL-12, NK cells are responsive to IL-2, and may mediate some of the antitumor effects of IL-2. In this study, we examine the interaction between IL-2 and the signaling events induced by IL-12 in NK cells. We find that IL-2 not only up-regulates the expression of IL-12Rbeta1 and IL-12Rbeta2, it also plays an important role in up-regulating and maintaining the expression of STAT4, a critical STAT protein involved in IL-12 signaling in NK cells. In contrast to the effects of IL-2 alone, expression of IL-12 receptors and STAT4 are unaffected or decreased by IL-12 or the combination of IL-2 and IL-12. Through expression of high levels of IL-12 receptors and STAT4, IL-2-primed NK cells show enhanced functional responses to IL-12 as measured by IFN-gamma production and the killing of target cells. NK cells from cancer patients who received low-dose IL-2 treatment also exhibited increased expression of IL-12 receptor chains, suggesting that IL-2 may enhance the response to IL-12 in vivo. These findings provide a molecular framework to understand the interaction between IL-2 and IL-12 in NK cells, and suggest strategies for improving the effectiveness of these cytokines in the immunotherapy of cancer.