The antimicrobial drug pyrimethamine inhibits STAT3 transcriptional activity by targeting the enzyme dihydrofolate reductase.

Cancer is often characterized by aberrant gene expression patterns caused by the inappropriate activation of transcription factors. Signal transducer and activator of transcription 3 (STAT3) is a key transcriptional regulator of many protumorigenic processes and is persistently activated in many types of human cancer. However, like many transcription factors, STAT3 has proven difficult to target clinically. To address this unmet clinical need, we previously developed a cell-based assay of STAT3 transcriptional activity and performed an unbiased and high-throughput screen of small molecules known to be biologically active in humans. We identified the antimicrobial drug pyrimethamine as a novel and specific inhibitor of STAT3 transcriptional activity. Here, we show that pyrimethamine does not significantly affect STAT3 phosphorylation, nuclear translocation, or DNA binding at concentrations sufficient to inhibit STAT3 transcriptional activity, suggesting a potentially novel mechanism of inhibition. To identify the direct molecular target of pyrimethamine and further elucidate the mechanism of action, we used a new quantitative proteome profiling approach called proteome integral solubility alteration coupled with a metabolomic analysis. We identified human dihydrofolate reductase as a target of pyrimethamine and demonstrated that the STAT3-inhibitory effects of pyrimethamine are the result of a deficiency in reduced folate downstream of dihydrofolate reductase inhibition, implicating folate metabolism in the regulation of STAT3 transcriptional activity. This study reveals a previously unknown regulatory node of the STAT3 pathway that may be important for the development of novel strategies to treat STAT3-driven cancers.

Essential role of the histone lysine demethylase KDM4A in the biology of malignant pleural mesothelioma (MPM).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a dismal prognosis. There is increasing interest in targeting chromatin regulatory pathways in difficult-to-treat cancers. In preliminary studies, we found that KDM4A (lysine-specific histone demethylase 4) was overexpressed in MPM. METHODS: KDM4A protein expression was determined by immunohistochemistry or immunoblotting. Functional inhibition of KDM4A by targeted knockdown and small molecule drugs was correlated to cell growth using cell lines and a xenograft mouse model. Gene expression profiling was performed to identify KDM4A-dependent signature pathways. RESULTS: Levels of KDM4A were found to be significantly elevated in MPM patients compared to normal mesothelial tissue. Inhibiting the enzyme activity efficiently reduced cell growth in vitro and reduced tumour growth in vivo. KDM4A inhibitor-induced apoptosis was further enhanced by the BH3 mimetic navitoclax. KDM4A expression was associated with pathways involved in cell growth and DNA repair. Interestingly, inhibitors of the DNA damage and replication checkpoint regulators CHK1 (prexasertib) and WEE1 (adavosertib) within the DNA double-strand break repair pathway, cooperated in the inhibition of cell growth. CONCLUSIONS: The results establish a novel and essential role for KDM4A in growth in preclinical models of MPM and identify potential therapeutic approaches to target KDM4A-dependent vulnerabilities.

Epigenetic targeting of Waldenström macroglobulinemia cells with BET inhibitors synergizes with BCL2 or histone deacetylase inhibition.

Aim: Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by overproduction of monoclonal IgM. To date, there are no therapies that provide a cure for WM patients, and therefore, it is important to explore new therapies. Little is known about the efficiency of epigenetic targeting in WM. Materials & methods: WM cells were treated with BET inhibitors (JQ1 and I-BET-762) and venetoclax, panobinostat or ibrutinib. Results: BET inhibition reduces growth of WM cells, with little effect on survival. This finding was enhanced by combination therapy, with panobinostat (LBH589) showing the highest synergy. Conclusion: Our studies identify BET inhibitors as effective therapy for WM, and these inhibitors can be enhanced in combination with BCL2 or histone deacetylase inhibition.

A single-cell landscape of high-grade serous ovarian cancer.

Malignant abdominal fluid (ascites) frequently develops in women with advanced high-grade serous ovarian cancer (HGSOC) and is associated with drug resistance and a poor prognosis1. To comprehensively characterize the HGSOC ascites ecosystem, we used single-cell RNA sequencing to profile ~11,000 cells from 22 ascites specimens from 11 patients with HGSOC. We found significant inter-patient variability in the composition and functional programs of ascites cells, including immunomodulatory fibroblast sub-populations and dichotomous macrophage populations. We found that the previously described immunoreactive and mesenchymal subtypes of HGSOC, which have prognostic implications, reflect the abundance of immune infiltrates and fibroblasts rather than distinct subsets of malignant cells2. Malignant cell variability was partly explained by heterogeneous copy number alteration patterns or expression of a stemness program. Malignant cells shared expression of inflammatory programs that were largely recapitulated in single-cell RNA sequencing of ~35,000 cells from additionally collected samples, including three ascites, two primary HGSOC tumors and three patient ascites-derived xenograft models. Inhibition of the JAK/STAT pathway, which was expressed in both malignant cells and cancer-associated fibroblasts, had potent anti-tumor activity in primary short-term cultures and patient-derived xenograft models. Our work contributes to resolving the HSGOC landscape3-5 and provides a resource for the development of novel therapeutic approaches.

Retraction Note: Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Endometriosis: A Malignant Fingerprint.

BACKGROUND: Endometriosis is complex, but identifying the novel biomarkers, inflammatory molecules, and genetic links holds the key to the enhanced detection, prediction and treatment of both endometriosis and endometriosis related malignant neoplasia. Here we review the literature relating to the specific molecular mechanism(s) mediating tumorigenesis arising within endometriosis. METHODS: Guidance (e.g. Cochrane) and published studies were identified. The Published studies were identified through PubMed using the systematic review methods filter, and the authors' topic knowledge. These data were reviewed to identify key and relevant articles to create a comprehensive review article to explore the molecular fingerprint associated with in endometriosis-driven tumorigenesis. RESULTS: An important focus is the link between C3aR1, PGR, ER1, SOX-17 and other relevant gene expression profiles and endometriosis-driven tumorigenesis. Further studies should also focus on the combined use of CA-125 with HE-4, and the role for OVA1/MIA as clinically relevant diagnostic biomarkers in the prediction of endometriosis-driven tumorigenesis. CONCLUSIONS: Elucidating endometriosis' molecular fingerprint is to understand the molecular mechanisms that drive the endometriosis-associated malignant phenotype. A better understanding of the predictive roles of these genes and the value of the biomarker proteins will allow for the derivation of unique molecular treatment algorithms to better serve our patients.

CDK5RAP3 is a co-factor for the oncogenic transcription factor STAT3.

The transcription factor STAT3 regulates genes governing critical cellular processes such as proliferation, survival, and self-renewal. While STAT3 transcriptional function is activated rapidly and transiently in response to physiologic signals, through a variety of mechanisms it can become constitutively activated in the pathogenesis of cancer. This leads to chronic expression of genes that underlie malignant cellular behavior. However, STAT3 is known to interact with other proteins, which may modulate its function. Understanding these interactions can provide insights into novel aspects of STAT3 function and may also suggest strategies to therapeutically target the large number of cancers driven by constitutively activated STAT3. To identify critical modulators of STAT3 transcriptional function, we performed an RNA-interference based screen in a cell-based system that allows quantitative measurement of STAT3 activity. From this approach, we identified CDK5 kinase regulatory-subunit associated protein 3 (CDK5RAP3) as an enhancer of STAT3-dependent gene expression. We found that STAT3 transcriptional function is modulated by CDK5RAP3 in cancer cells, and silencing CDK5RAP3 reduces STAT3-mediated tumorigenic phenotypes including clonogenesis and migration. Mechanistically, CDK5RAP3 binds to STAT3-regulated genomic loci, in a STAT3-dependent manner. In primary human breast cancers, the expression of CDK5RAP3 expression was associated with STAT3 gene expression signatures as well as the expression of individual STAT3 target genes. These findings reveal a novel aspect of STAT3 transcriptional function and potentially provide both a biomarker of enhanced STAT3-dependent gene expression as well as a unique mechanism to therapeutically target STAT3.

Atovaquone is active against AML by upregulating the integrated stress pathway and suppressing oxidative phosphorylation.

Atovaquone, a US Food and Drug Administration-approved antiparasitic drug previously shown to reduce interleukin-6/STAT3 signaling in myeloma cells, is well tolerated, and plasma concentrations of 40 to 80 µM have been achieved with pediatric and adult dosing. We conducted preclinical testing of atovaquone with acute myeloid leukemia (AML) cell lines and pediatric patient samples. Atovaquone induced apoptosis with an EC50 <30 µM for most AML lines and primary pediatric AML specimens. In NSG mice xenografted with luciferase-expressing THP-1 cells and in those receiving a patient-derived xenograft, atovaquone-treated mice demonstrated decreased disease burden and prolonged survival. To gain a better understanding of the mechanism of atovaquone, we performed an integrated analysis of gene expression changes occurring in cancer cell lines after atovaquone exposure. Atovaquone promoted phosphorylation of eIF2α, a key component of the integrated stress response and master regulator of protein translation. Increased levels of phosphorylated eIF2α led to greater abundance of the transcription factor ATF4 and its target genes, including proapoptotic CHOP and CHAC1. Furthermore, atovaquone upregulated REDD1, an ATF4 target gene and negative regulator of the mechanistic target of rapamycin (mTOR), and caused REDD1-mediated inhibition of mTOR activity with similar efficacy as rapamycin. Additionally, atovaquone suppressed the oxygen consumption rate of AML cells, which has specific implications for chemotherapy-resistant AML blasts that rely on oxidative phosphorylation for survival. Our results provide insight into the complex biological effects of atovaquone, highlighting its potential as an anticancer therapy with novel and diverse mechanisms of action, and support further clinical evaluation of atovaquone for pediatric and adult AML.

Fatty acids and cancer-amplified ZDHHC19 promote STAT3 activation through S-palmitoylation.

Signal transducer and activator of transcription 3 (STAT3) has a critical role in regulating cell fate, inflammation and immunity1,2. Cytokines and growth factors activate STAT3 through kinase-mediated tyrosine phosphorylation and dimerization3,4. It remains unknown whether other factors promote STAT3 activation through different mechanisms. Here we show that STAT3 is post-translationally S-palmitoylated at the SRC homology 2 (SH2) domain, which promotes the dimerization and transcriptional activation of STAT3. Fatty acids can directly activate STAT3 by enhancing its palmitoylation, in synergy with cytokine stimulation. We further identified ZDHHC19 as a palmitoyl acyltransferase that regulates STAT3. Cytokine stimulation increases STAT3 palmitoylation by promoting the association between ZDHHC19 and STAT3, which is mediated by the SH3 domain of GRB2. Silencing ZDHHC19 blocks STAT3 palmitoylation and dimerization, and impairs the cytokine- and fatty-acid-induced activation of STAT3. ZDHHC19 is frequently amplified in multiple human cancers, including in 39% of lung squamous cell carcinomas. High levels of ZDHHC19 correlate with high levels of nuclear STAT3 in patient samples. In addition, knockout of ZDHHC19 in lung squamous cell carcinoma cells significantly blocks STAT3 activity, and inhibits the fatty-acid-induced formation of tumour spheres as well as tumorigenesis induced by high-fat diets in an in vivo mouse model. Our studies reveal that fatty-acid- and ZDHHC19-mediated palmitoylation are signals that regulate STAT3, which provides evidence linking the deregulation of palmitoylation to inflammation and cancer.

Study of Cathepsin B inhibition in VEGFR TKI treated human renal cell carcinoma xenografts.

Several therapeutic options are available for metastatic RCC, but responses are almost never complete, and resistance to therapy develops in the vast majority of patients. Consequently, novel treatments are needed to combat resistance to current therapies and to improve patient outcomes. We have applied integrated transcriptome and proteome analyses to identify cathepsin B (CTSB), a cysteine proteinase of the papain family, as one of the most highly upregulated gene products in established human RCC xenograft models of resistance to vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors (TKI). We used established RCC models to test the significance of CTSB in the progression of renal cancer. Our evaluation of CTSB showed that stable CTSB knockdown suppressed RCC growth in vitro and in vivo. Stable over-overexpression of wild-type CTSB (CTSBwt/hi), but not of an CTSB active site mutant (CTSBN298A), rescued cell growth in CTSB knockdown cells and abolished the efficacy of VEGFR TKI treatment. Genome-wide transcriptome profiling of CTSB knockdown cells demonstrated significant effects on multiple metabolic and stem cell-related pathways, with ALDHA1A (ALDH1) as one of the most significantly downregulated genes. Importantly, survival analysis across 16 major TCGA cancers revealed that CTSB overexpression is associated with low rates of three and five year patient survival rates (P = 2.5e-08, HR = 1.4). These data strongly support a contribution of CTSB activity to RCC cell growth and tumorigenicity. They further highlight the promise of CTSB inhibition in development of novel combination therapies designed to improve efficacy of current TKI treatments of metastatic RCC.

The kinases IKBKE and TBK1 regulate MYC-dependent survival pathways through YB-1 in AML and are targets for therapy.

To identify novel therapeutic targets in acute myeloid leukemia (AML), we examined kinase expression patterns in primary AML samples. We found that the serine/threonine kinase IKBKE, a noncanonical IkB kinase, is expressed at higher levels in myeloid leukemia cells compared with normal hematopoietic cells. Inhibiting IKBKE, or its close homolog TANK-binding kinase 1 (TBK1), by either short hairpin RNA knockdown or pharmacological compounds, induces apoptosis and reduces the viability of AML cells. Using gene expression profiling and gene set enrichment analysis, we found that IKBKE/TBK1-sensitive AML cells typically possess an MYC oncogenic signature. Consistent with this finding, the MYC oncoprotein was significantly downregulated upon IKBKE/TBK1 inhibition. Using proteomic analysis, we found that the oncogenic gene regulator YB-1 was activated by IKBKE/TBK1 through phosphorylation, and that YB-1 binds to the MYC promoter to enhance MYC gene transcription. Momelotinib (CYT387), a pharmacological inhibitor of IKBKE/TBK1, inhibits MYC expression, reduces viability and clonogenicity of primary AML cells, and demonstrates efficacy in a murine model of AML. Together, these data identify IKBKE/TBK1 as a promising therapeutic target in AML.

IL-6 Mediates Cross-Talk between Tumor Cells and Activated Fibroblasts in the Tumor Microenvironment.

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers.Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR.

The STAT3 Target Gene TNFRSF1A Modulates the NF-κB Pathway in Breast Cancer Cells.

The transcription factor STAT3 is activated inappropriately in 70% of breast cancers, most commonly in triple negative breast cancer (TNBC). Although the transcriptional function of STAT3 is essential for tumorigenesis, the key target genes regulated by STAT3 in driving tumor pathogenesis have remained unclear. To identify critical STAT3 target genes, we treated TNBC cell lines with two different compounds that block STAT3 transcriptional function, pyrimethamine and PMPTP. We then performed gene expression analysis to identify genes whose expression is strongly down-regulated by both STAT3 inhibitors. Foremost among the down-regulated genes was TNFRSF1A, which encodes a transmembrane receptor for TNFα. We showed that STAT3 binds directly to a regulatory region within the TNFRSF1A gene, and that TNFRSF1A levels are dependent on STAT3 function in both constitutive and cytokine-induced models of STAT3 activation. Furthermore, TNFRSF1A is a major mediator of both basal and TNFα-induced NF-κB activity in breast cancer cells. We extended these findings to primary human breast cancers, in which we found that high TNFRSF1A transcript levels correlated with STAT3 activation. In addition, and consistent with a causal role, increased TNFRSF1A expression was associated with an NF-κB gene expression in signature in breast cancers. Thus, TNFRSF1A is a STAT3 target gene that regulates the NF-κB pathway. These findings reveal a novel functional crosstalk between STAT3 and NF-κB signaling in breast cancer. Furthermore, elevated TNFRSF1A levels may predict a subset of breast tumors that are sensitive to STAT3 transcriptional inhibitors, and may be a biomarker for response to inhibition of this pathway.

NTRK2 activation cooperates with PTEN deficiency in T-ALL through activation of both the PI3K-AKT and JAK-STAT3 pathways.

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.

Gene expression-based discovery of atovaquone as a STAT3 inhibitor and anticancer agent.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is frequently activated inappropriately in a wide range of hematological and solid cancers, but clinically available therapies targeting STAT3 are lacking. Using a computational strategy to identify compounds opposing the gene expression signature of STAT3, we discovered atovaquone (Mepron), an antimicrobial approved by the US Food and Drug Administration, to be a potent STAT3 inhibitor. We show that, at drug concentrations routinely achieved clinically in human plasma, atovaquone inhibits STAT3 phosphorylation, the expression of STAT3 target genes, and the viability of STAT3-dependent hematological cancer cells. These effects were also observed with atovaquone treatment of primary blasts isolated from patients with acute myelogenous leukemia or acute lymphocytic leukemia. Atovaquone is not a kinase inhibitor but instead rapidly and specifically downregulates cell-surface expression of glycoprotein 130, which is required for STAT3 activation in multiple contexts. The administration of oral atovaquone to mice inhibited tumor growth and prolonged survival in a murine model of multiple myeloma. Finally, in patients with acute myelogenous leukemia treated with hematopoietic stem cell transplantation, extended use of atovaquone for Pneumocystis prophylaxis was associated with improved relapse-free survival. These findings establish atovaquone as a novel, clinically accessible STAT3 inhibitor with evidence of anticancer efficacy in both animal models and humans.

Granulin, a novel STAT3-interacting protein, enhances STAT3 transcriptional function and correlates with poorer prognosis in breast cancer.

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.

Inhibiting STAT5 by the BET bromodomain inhibitor JQ1 disrupts human dendritic cell maturation.

Maturation of dendritic cells (DCs) is required to induce T cell immunity, whereas immature DCs can induce immune tolerance. Although the transcription factor STAT5 is suggested to participate in DC maturation, its role in this process remains unclear. In this study, we investigated the effect of STAT5 inhibition on LPS-induced maturation of human monocyte-derived DCs (Mo-DCs). We inhibited STAT5 by treating Mo-DCs with JQ1, a selective inhibitor of BET epigenetic readers, which can suppress STAT5 function. We found that JQ1 inhibits LPS-induced STAT5 phosphorylation and nuclear accumulation, thereby attenuating its transcriptional activity in Mo-DCs. The diminished STAT5 activity results in impaired maturation of Mo-DCs, as indicated by defective upregulation of costimulatory molecules and CD83, as well as reduced secretion of IL-12p70. Expression of constitutively activated STAT5 in JQ1-treated Mo-DCs overcomes the effects of JQ1 and enhances the expression of CD86, CD83, and IL-12. The activation of STAT5 in Mo-DCs is mediated by GM-CSF produced following LPS stimulation. Activated STAT5 then leads to increased expression of both GM-CSF and GM-CSFR, triggering an autocrine loop that further enhances STAT5 signaling and enabling Mo-DCs to acquire a more mature phenotype. JQ1 decreases the ability of Mo-DCs to induce allogeneic CD4(+) and CD8(+) T cell proliferation and production of proinflammatory cytokines. Furthermore, JQ1 leads to a reduced generation of inflammatory CD8(+) T cells and decreased Th1 differentiation. Thus, JQ1 impairs LPS-induced Mo-DC maturation by inhibiting STAT5 activity, thereby generating cells that can only weakly stimulate an adaptive-immune response. Therefore, JQ1 could have beneficial effects in treating T cell-mediated inflammatory diseases.

STAT3 Activity and Function in Cancer: Modulation by STAT5 and miR-146b.

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.