Person:

Walker, Sarah

STAT transcription factors are regulators of critical cellular processes such as proliferation, survival, and self-renewal. While the activity of these proteins is tightly regulated under physiological conditions, they can become constitutively activated in a broad range of human cancers. This inappropriate STAT activation leads to enhanced transcription of genes that can directly lead to the malignant phenotype. Since STATs are largely dispensable for normal cell function, this has raised the possibility that STATs might be key targets for cancer therapy. Although a number of structure-based strategies have been used to develop STAT inhibitors, an alternate approach is to use cell-based assays that make use of the transcriptional function of STATs. Employing these systems, one can screen large chemical libraries to identify compounds that specifically block the function of a given STAT. This approach can lead to the identification of compounds that inhibit STATs by a variety of mechanisms, and can suggest novel targets for therapy. This type of functional screening strategy has already identified a drug that potently inhibits STAT3, and which is now being evaluated in a clinical trial for patients with chronic lymphocytic leukemia.

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 utilized ChIP-seq 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 to 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 STAT3 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.

The transcription factor STAT3 regulates genes that control critical cellular processes such as proliferation, survival, pluripotency, and motility. Thus, under physiological conditions, the transcriptional function of STAT3 is tightly regulated as one part of a complex signaling matrix. When these processes are subverted through mutation or epigenetic events, STAT3 becomes highly active and drives elevated expression of genes underlying these phenotypes, leading to malignant cellular behavior. However, even in the presence of activated STAT3, other cellular modulators can have a major impact on the biological properties of a cancer cell, which is reflected in the clinical behavior of a tumor. Recent evidence has suggested that two such key modulators are the activation status of other STAT family members, particularly STAT5, and the expression of STAT3-regulated genes that are part of negative feedback circuits, including microRNAs such as miR-146b. With attention to these newly emerging areas, we will gain greater insight into the consequence of STAT3 activation in the biology of human cancers. In addition, understanding these subtleties of STAT3 signaling in cancer pathogenesis will allow the development of more rational molecular approaches to cancer therapy.

Interleukin-6 (IL-6)–mediated activation of signal transducer and activator of transcription 3 (STAT3) is a mechanism by which chronic inflammation can contribute to cancer and is a common oncogenic event. We discovered a pathway, the loss of which is associated with persistent STAT3 activation in human cancer. We found that the gene encoding the tumor suppressor microRNA miR-146b is a direct STAT3 target gene, and its expression was increased in normal breast epithelial cells but decreased in tumor cells. Methylation of the miR-146b promoter, which inhibited STAT3-mediated induction of expression, was increased in primary breast cancers. Moreover, we found that miR-146b inhibited nuclear factor κB (NF-κB)–dependent production of IL-6, subsequent STAT3 activation, and IL-6/STAT3–driven migration and invasion in breast cancer cells, thereby establishing a negative feedback loop. In addition, higher expression of miR-146b was positively correlated with patient survival in breast cancer subtypes with increased IL6 expression and STAT3 phosphorylation. Our results identify an epigenetic mechanism of crosstalk between STAT3 and NF-κB relevant to constitutive STAT3 activation in malignancy and the role of inflammation in oncogenesis.

The transcription factor signal STAT5 is constitutively activated in a wide range of leukemias and lymphomas, and drives the expression of genes necessary for proliferation, survival, and self-renewal. Thus, targeting STAT5 is an appealing therapeutic strategy for hematologic malignancies. Given the importance of bromodomain-containing proteins in transcriptional regulation, we considered the hypothesis that a pharmacologic bromodomain inhibitor could inhibit STAT5-dependent gene expression. We found that the small-molecule bromodomain and extra-terminal (BET) bromodomain inhibitor JQ1 decreases STAT5-dependent (but not STAT3-dependent) transcription of both heterologous reporter genes and endogenous STAT5 target genes. JQ1 reduces STAT5 function in leukemia and lymphoma cells with constitutive STAT5 activation, or inducibly activated by cytokine stimulation. Among the BET bromodomain subfamily of proteins, it seems that BRD2 is the critical mediator for STAT5 activity. In experimental models of acute T-cell lymphoblastic leukemias, where activated STAT5 contributes to leukemia cell survival, Brd2 knockdown or JQ1 treatment shows strong synergy with tyrosine kinase inhibitors (TKI) in inducing apoptosis in leukemia cells. In contrast, mononuclear cells isolated form umbilical cord blood, which is enriched in normal hematopoietic precursor cells, were unaffected by these combinations. These findings indicate a unique functional association between BRD2 and STAT5, and suggest that combinations of JQ1 and TKIs may be an important rational strategy for treating leukemias and lymphomas driven by constitutive STAT5 activation.

Inappropriate activation of the transcription factors STAT3 and STAT5 has been shown to drive cancer pathogenesis through dysregulation of genes involved in cell survival, growth, and differentiation. Although STAT3 and STAT5 are structurally related, they can have opposite effects on key genes, including BCL6. BCL6, a transcriptional repressor, has been shown to be oncogenic in diffuse large B cell lymphoma. BCL6 also plays an important role in breast cancer pathogenesis, a disease in which STAT3 and STAT5 can be activated individually or concomitantly. To determine the mechanism by which these oncogenic transcription factors regulate BCL6 transcription, we analyzed their effects at the levels of chromatin and gene expression. We found that STAT3 increases expression of BCL6 and enhances recruitment of RNA polymerase II phosphorylated at a site associated with transcriptional initiation. STAT5, in contrast, represses BCL6 expression below basal levels and decreases the association of RNA polymerase II at the gene. Furthermore, the repression mediated by STAT5 is dominant over STAT3-mediated induction. STAT5 exerts this effect by displacing STAT3 from one of the two regulatory regions to which it binds. These findings may underlie the divergent biology of breast cancers containing activated STAT3 alone or in conjunction with activated STAT5.

Loss of PTEN, a negative regulator of the phosphoinositide 3-kinase signaling pathway, is a frequent event in T-cell acute lymphoblastic leukemia, suggesting the importance of phosphoinositide 3-kinase activity in this disease. Indeed, hyperactivation of the phosphoinositide 3-kinase pathway is associated with the disease aggressiveness, poor prognosis and resistance to current therapies. To identify a molecular pathway capable of cooperating with PTEN deficiency to drive oncogenic transformation of leukocytes, we performed an unbiased transformation screen with a library of tyrosine kinases. We found that activation of NTRK2 is able to confer a full growth phenotype of Ba/F3 cells in an IL3-independent manner in the PTEN-null setting. NTRK2 activation cooperates with PTEN deficiency through engaging both phosphoinositide3-kinase/AKT and JAK/STAT3 pathway activation in leukocytes. Notably, pharmacological inhibition demonstrated that p110α and p110δ are the major isoforms mediating the phosphoinositide 3-kinase/AKT signaling driven by NTRK2 activation in PTEN-deficient leukemia cells. Furthermore, combined inhibition of phosphoinositide 3-kinase and STAT3 significantly suppressed proliferation of PTEN-mutant T-cell acute lymphoblastic leukemia both in culture and in mouse xenografts. Together, our data suggest that a unique conjunction of PTEN deficiency and NTRK2 activation in T-cell acute lymphoblastic leukemia, and combined pharmacologic inhibition of phosphoinositide 3-kinase and STAT3 signaling may serve as an effective and durable therapeutic strategy for T-cell acute lymphoblastic leukemia.

Since the neoplastic phenotype of a cell is largely driven by aberrant gene expression patterns, increasing attention has been focused on transcription factors that regulate critical mediators of tumorigenesis such as signal transducer and activator of transcription 3 (STAT3). As proteins that interact with STAT3 may be key in addressing how STAT3 contributes to cancer pathogenesis, we took a proteomics approach to identify novel STAT3-interacting proteins. We performed mass spectrometry-based profiling of STAT3-containing complexes from breast cancer cells that have constitutively active STAT3 and are dependent on STAT3 function for survival. We identified granulin (GRN) as a novel STAT3-interacting protein that was necessary for both constitutive and maximal leukemia inhibitory factor (LIF)induced STAT3 transcriptional activity. GRN enhanced STAT3 DNA binding and also increased the time-integrated amount of LIF-induced STAT3 activation in breast cancer cells. Furthermore, silencing GRN neutralized STAT3-mediated tumorigenic phenotypes including viability, clonogenesis, and migratory capacity. In primary breast cancer samples, GRN mRNA levels were positively correlated with STAT3 gene expression signatures and with reduced patient survival. These studies identify GRN as a functionally important STAT3-interacting protein that may serve as an important prognostic biomarker and potential therapeutic target in breast cancer.