STAT family of transcription factors in breast cancer: Pathogenesis and therapeutic opportunities and challenges.

Breast cancer is the most commonly diagnosed cancer and second-leading cause of cancer deaths in women. Breast cancer stem cells (BCSCs) promote metastasis and therapeutic resistance contributing to tumor relapse. Through activating genes important for BCSCs, transcription factors contribute to breast cancer metastasis and therapeutic resistance, including the signal transducer and activator of transcription (STAT) family of transcription factors. The STAT family consists of six major isoforms, STAT1, STAT2, STAT3, STAT4, STAT5, and STAT6. Canonical STAT signaling is activated by the binding of an extracellular ligand to a cell-surface receptor followed by STAT phosphorylation, leading to STAT nuclear translocation and transactivation of target genes. It is important to note that STAT transcription factors exhibit diverse effects in breast cancer; some are either pro- or anti-tumorigenic while others maintain dual, context-dependent roles. Among the STAT transcription factors, STAT3 is the most widely studied STAT protein in breast cancer for its critical roles in promoting BCSCs, breast cancer cell proliferation, invasion, angiogenesis, metastasis, and immune evasion. Consequently, there have been substantial efforts in developing cancer therapeutics to target breast cancer with dysregulated STAT3 signaling. In this comprehensive review, we will summarize the diverse roles that each STAT family member plays in breast cancer pathobiology, as well as, the opportunities and challenges in pharmacologically targeting STAT proteins and their upstream activators in the context of breast cancer treatment.

Breast cancer extracellular vesicles-derived miR-1290 activates astrocytes in the brain metastatic microenvironment via the FOXA2→CNTF axis to promote progression of brain metastases.

Mechanisms underlying breast cancer brain metastasis (BCBM) are still unclear. In this study, we observed that extracellular vesicles (EVs) secreted from breast cancer cells with increased expression of tGLI1, a BCBM-promoting transcription factor, strongly activated astrocytes. EV-derived microRNA/miRNA microarray revealed tGLI1-positive breast cancer cells highly secreted miR-1290 and miR-1246 encapsulated in EVs. Genetic knockin/knockout studies established a direct link between tGLI1 and both miRNAs. Datamining and analysis of patient samples revealed that BCBM patients had more circulating EV-miRs-1290/1246 than those without metastasis. Ectopic expression of miR-1290 or miR-1246 strongly activated astrocytes whereas their inhibitors abrogated the effect. Conditioned media from miR-1290- or miR-1246-overexpressing astrocytes promoted mammospheres. Furthermore, miRs-1290/1246 suppressed expression of FOXA2 transcription repressor, leading to CNTF cytokine secretion and subsequent activation of astrocytes. Finally, we conducted a mouse study to demonstrate that astrocytes overexpressing miR-1290, but not miR-1246, enhanced intracranial colonization and growth of breast cancer cells. Collectively, our findings demonstrate, for the first time, that breast cancer EV-derived miR-1290 and miR-1246 activate astrocytes in the brain metastatic microenvironment and that EV-derived miR-1290 promotes progression of brain metastases through the novel EV-miR-1290→FOXA2→CNTF signaling axis.

IL-6/JAK/STAT3 Signaling in Breast Cancer Metastasis: Biology and Treatment.

Breast cancer is the most commonly diagnosed cancer in women. Metastasis is the primary cause of mortality for breast cancer patients. Multiple mechanisms underlie breast cancer metastatic dissemination, including the interleukin-6 (IL-6)-mediated signaling pathway. IL-6 is a pleiotropic cytokine that plays an important role in multiple physiological processes including cell proliferation, immune surveillance, acute inflammation, metabolism, and bone remodeling. IL-6 binds to the IL-6 receptor (IL-6Rα) which subsequently binds to the glycoprotein 130 (gp130) receptor creating a signal transducing hexameric receptor complex. Janus kinases (JAKs) are recruited and activated; activated JAKs, in turn, phosphorylate signal transducer and activator of transcription 3 (STAT3) for activation, leading to gene regulation. Constitutively active IL-6/JAK/STAT3 signaling drives cancer cell proliferation and invasiveness while suppressing apoptosis, and STAT3 enhances IL-6 signaling to promote a vicious inflammatory loop. Aberrant expression of IL-6 occurs in multiple cancer types and is associated with poor clinical prognosis and metastasis. In breast cancer, the IL-6 pathway is frequently activated, which can promote breast cancer metastasis while simultaneously suppressing the anti-tumor immune response. Given these important roles in human cancers, multiple components of the IL-6 pathway are promising targets for cancer therapeutics and are currently being evaluated preclinically and clinically for breast cancer. This review covers the current biological understanding of the IL-6 signaling pathway and its impact on breast cancer metastasis, as well as, therapeutic interventions that target components of the IL-6 pathway including: IL-6, IL-6Rα, gp130 receptor, JAKs, and STAT3.

TrkA Interacts with and Phosphorylates STAT3 to Enhance Gene Transcription and Promote Breast Cancer Stem Cells in Triple-Negative and HER2-Enriched Breast Cancers.

JAK2-STAT3 and TrkA signaling pathways have been separately implicated in aggressive breast cancers; however, whether they are co-activated or undergo functional interaction has not been thoroughly investigated. Herein we report, for the first time that STAT3 and TrkA are significantly co-overexpressed and co-activated in triple-negative breast cancer (TNBC) and HER2-enriched breast cancer, as shown by immunohistochemical staining and data mining. Through immunofluorescence staining-confocal microscopy and immunoprecipitation-Western blotting, we found that TrkA and STAT3 co-localize and physically interact in the cytoplasm, and the interaction is dependent on STAT3-Y705 phosphorylation. TrkA-STAT3 interaction leads to STAT3 phosphorylation at Y705 by TrkA in breast cancer cells and cell-free kinase assays, indicating that STAT3 is a novel substrate of TrkA. ß-NGF-mediated TrkA activation induces TrkA-STAT3 interaction, STAT3 nuclear transport and transcriptional activity, and the expression of STAT3 target genes, SOX2 and MYC. The co-activation of both pathways promotes breast cancer stem cells. Finally, we found that TNBC and HER2-enriched breast cancer with JAK2-STAT3 and TrkA co-activation are positively associated with poor overall metastasis-free and organ-specific metastasis-free survival. Collectively, our study uncovered that TrkA is a novel activating kinase of STAT3, and their co-activation enhances gene transcription and promotes breast cancer stem cells in TNBC and HER2-enriched breast cancer.

Editor's Highlight: Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis.

The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue.

Editor's Highlight: Development of an In vitro Assay Measuring Uterine-Specific Estrogenic Responses for Use in Chemical Safety Assessment.

A toxicity pathway approach was taken to develop an in vitro assay using human uterine epithelial adenocarcinoma (Ishikawa) cells as a replacement for measuring an in vivo uterotrophic response to estrogens. The Ishikawa cell was determined to be fit for the purpose of recapitulating in vivo uterine response by verifying fidelity of the biological pathway components and the dose-response predictions to women of child-bearing age. Expression of the suite of estrogen receptors that control uterine proliferation (ERα66, ERα46, ERα36, ERß, G-protein coupled estrogen receptor (GPER)) were confirmed across passages and treatment conditions. Phenotypic responses to ethinyl estradiol (EE) from transcriptional activation of ER-mediated genes, to ALP enzyme induction and cellular proliferation occurred at concentrations consistent with estrogenic activity in adult women (low picomolar). To confirm utility of this model to predict concentration-response for uterine proliferation with xenobiotics, we tested the concentration-response for compounds with known uterine estrogenic activity in humans and compared the results to assays from the ToxCast and Tox21 suite of estrogen assays. The Ishikawa proliferation assay was consistent with in vivo responses and was a more sensitive measure of uterine response. Because this assay was constructed by first mapping the key molecular events for cellular response, and then ensuring that the assay incorporated these events, the resulting cellular assay should be a reliable tool for identifying estrogenic compounds and may provide improved quantitation of chemical concentration response for in vitro-based safety assessments.