An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1.

The goal of this study is to identify pharmacological inhibitors that target a recently identified novel mediator of breast cancer brain metastasis (BCBM), truncated glioma-associated oncogene homolog 1 (tGLI1). Inhibitors of tGLI1 are not yet available. To identify compounds that selectively kill tGLI1-expressing breast cancer, we screened 1527 compounds using two sets of isogenic breast cancer and brain-tropic breast cancer cell lines engineered to stably express the control, GLI1, or tGLI1 vector, and identified the FDA-approved antifungal ketoconazole (KCZ) to selectively target tGLI1-positive breast cancer cells and breast cancer stem cells, but not tGLI1-negative breast cancer and normal cells. KCZ's effects are dependent on tGLI1. Two experimental mouse metastasis studies have demonstrated that systemic KCZ administration prevented the preferential brain metastasis of tGLI1-positive breast cancer and suppressed the progression of established tGLI1-positive BCBM without liver toxicities. We further developed six KCZ derivatives, two of which (KCZ-5 and KCZ-7) retained tGLI1-selectivity in vitro. KCZ-7 exhibited higher blood-brain barrier penetration than KCZ/KCZ-5 and more effectively reduced the BCBM frequency. In contrast, itraconazole, another FDA-approved antifungal, failed to suppress BCBM. The mechanistic studies suggest that KCZ and KCZ-7 inhibit tGLI1's ability to bind to DNA, activate its target stemness genes Nanog and OCT4, and promote tumor proliferation and angiogenesis. Our study establishes the rationale for using KCZ and KCZ-7 for treating and preventing BCBM and identifies their mechanism of action.

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.

NEDD4 degrades TUSC2 to promote glioblastoma progression.

Whether tumor suppressor candidate 2 (TUSC2) plays an important role in glioblastoma (GBM) progression is largely unknown. Whether TUSC2 undergoes polyubiquitination is unknown. Herein, we report that TUSC2 protein expression is reduced/lost in GBM compared to normal brain due to protein destabilization; TUSC2 mRNA is equally expressed in both tissues. NEDD4 E3 ubiquitin ligase polyubiquitinates TUSC2 at residue K71, and the TUSC2-K71R mutant is resistant to NEDD4-mediated proteasomal degradation. Analysis of GBM specimens showed NEDD4 protein is highly expressed in GBM and the level is inversely correlated with TUSC2 protein levels. Furthermore, TUSC2 restoration induces apoptosis and inhibits patient-derived glioma stem cells (PD-GSCs) in vitro and in vivo. Conversely, TUSC2-knockout promotes PD-GSCs in vitro and in vivo. RNA-Seq analysis and subsequent validations showed GBM cells with TUSC2-knockout expressed increased Bcl-xL and were more resistant to apoptosis induced by a Bcl-xL-specific BH3 mimetic. A TUSC2-knockout gene signature created from the RNA-seq data predicts poor patient survival. Together, these findings establish that NEDD4-mediated polyubiquitination is a novel mechanism for TUSC2 degradation in GBM and that TUSC2 loss promotes GBM progression in part through Bcl-xL upregulation.

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.

Transgenic mouse models of breast cancer.

Transgenic breast cancer mouse models are critical tools for preclinical studies of human breast cancer. Genetic editing of the murine mammary gland allows for modeling of abnormal genetic events frequently found in human breast cancers. Genetically engineered mouse models (GEMMs) of breast cancer employ tissue-specific genetic manipulation for tumorigenic induction within the mammary tissue. Under the transcriptional control of mammary-specific promoters, transgenic mouse models can simulate spontaneous mammary tumorigenesis by expressing one or more putative oncogenes, such as MYC, HRAS, and PIK3CA. Alternatively, the Cre-Lox system allows for tissue-specific deletion of tumor suppressors, such as p53, Rb1, and Brca1, or specific knock-in of putative oncogenes. Thus, GEMMs can be designed to implement one or more genetic events to induce mammary tumorigenesis. Features of GEMMs, such as age of transgene expression, breeding quality, tumor latency, histopathological characteristics, and propensity for local and distant metastasis, are variable and strain-dependent. This review aims to summarize currently available transgenic breast cancer mouse models that undergo spontaneous mammary tumorigenesis upon genetic manipulation, their varying characteristics, and their individual genetic manipulations that model aberrant signaling events observed in human breast cancers.

Combined inhibition of JAK2-STAT3 and SMO-GLI1/tGLI1 pathways suppresses breast cancer stem cells, tumor growth, and metastasis.

Triple-negative breast cancer (TNBC) and HER2-positive breast cancer are particularly aggressive and associated with unfavorable prognosis. TNBC lacks effective treatments. HER2-positive tumors have treatment options but often acquire resistance to HER2-targeted therapy after initial response. To address these challenges, we determined whether novel combinations of JAK2-STAT3 and SMO-GLI1/tGLI1 inhibitors synergistically target TNBC and HER2 breast cancer since these two pathways are concurrently activated in both tumor types and enriched in metastatic tumors. Herein, we show that novel combinations of JAK2 inhibitors (ruxolitinib and pacritinib) with SMO inhibitors (vismodegib and sonidegib) synergistically inhibited in vitro growth of TNBC and HER2-positive trastuzumab-resistant BT474-TtzmR cells. Synergy was also observed against breast cancer stem cells. To determine if the combination is efficacious in inhibiting metastasis, we treated mice with intracardially inoculated TNBC cells and found the combination to inhibit lung and liver metastases, and prolong host survival without toxicity. The combination inhibited orthotopic growth, VEGF-A expression, and tumor vasculature of both TNBC and HER2-positive trastuzumab-refractory breast cancer. Lung metastasis of orthotopic BT474-TtzmR xenografts was suppressed by the combination. Together, our results indicated that dual targeting of JAK2 and SMO resulted in synergistic suppression of breast cancer growth and metastasis, thereby supporting future clinical testing.

Hedgehog Signaling and Truncated GLI1 in Cancer.

The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform.

TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment.

Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and metastasis-initiating cells, we examined TGLI1 for its involvement in breast CSCs and found TGLI1 to transcriptionally activate stemness genes CD44, Nanog, Sox2, and OCT4 leading to CSC renewal, and TGLI1 outcompetes with GLI1 for binding to target promoters. We next examined whether astrocyte-priming underlies TGLI1-mediated brain tropism and found that TGLI1-positive CSCs strongly activated and interacted with astrocytes in vitro and in vivo. These findings demonstrate, for the first time, that TGLI1 mediates breast cancer metastasis to the brain, in part, through promoting metastasis-initiating CSCs and activating astrocytes in BCBM microenvironment.

The role of fit-for-purpose assays within tiered testing approaches: A case study evaluating prioritized estrogen-active compounds in an in vitro human uterotrophic assay.

Chemical risk assessment relies on toxicity tests that require significant numbers of animals, time and costs. For the >30,000 chemicals in commerce, the current scale of animal testing is insufficient to address chemical safety concerns as regulatory and product stewardship considerations evolve to require more comprehensive understanding of potential biological effects, conditions of use, and associated exposures. We demonstrate the use of a multi-level new approach methodology (NAMs) strategy for hazard- and risk-based prioritization to reduce animal testing. A Level 1/2 chemical prioritization based on estrogen receptor (ER) activity and metabolic activation using ToxCast data was used to select 112 chemicals for testing in a Level 3 human uterine cell estrogen response assay (IKA assay). The Level 3 data were coupled with quantitative in vitro to in vivo extrapolation (Q-IVIVE) to support bioactivity determination (as a surrogate for hazard) in a tissue-specific context. Assay AC50s and Q-IVIVE were used to estimate human equivalent doses (HEDs), and HEDs were compared to rodent uterotrophic assay in vivo-derived points of departure (PODs). For substances active both in vitro and in vivo, IKA assay-derived HEDs were lower or equivalent to in vivo PODs for 19/23 compounds (83%). Activity exposure relationships were calculated, and the IKA assay was as or more protective of human health than the rodent uterotrophic assay for all IKA-positive compounds. This study demonstrates the utility of biologically relevant fit-for-purpose assays and supports the use of a multi-level strategy for chemical risk assessment.

Trk receptor tyrosine kinases in metastasis and cancer therapy.

Tropomyosin receptor kinases (TRKs) were first identified in 1986 when NTRK1 was discovered as part of an oncogenic fusion gene in colorectal cancer and the discovery of NTRK2 and NTRK3 followed shortly after. In the decades since their discovery, TRKs have been implicated in a number of cancer types due to their canonical roles in promoting cell proliferation and survival. Studies have shown that increased expression and/or activity of TRKs can be indicative of metastatic potential, suggesting that TRKs can be therapeutic targets in aggressive cancers. While predominantly known for forming oncogenic gene fusions, aberrant alternative splicing does not appear to be a prerequisite for TRK-mediated metastasis. However, expression and activity of each TRK can confer either a pro-apoptotic or pro-survival effect in different tissue types, predicting a complex treatment paradigm for patients exhibiting abnormalities in TRK expression or activity. While preclinical studies on TRK kinases continue, clinical advances in TRK inhibition were achieved upon Larotrectinib (Vitrakvi) becoming the first FDA-approved pan-TRK inhibitor. This review summarizes findings regarding TRK expression and activity in different tissue types, the biological impact of aberrant TRK signaling, and the potential for additional inhibitor design.

An in vitro approach for prioritization and evaluation of chemical effects on glucocorticoid receptor mediated adipogenesis.

Rising obesity rates worldwide have socio-economic ramifications. While genetics, diet, and lack of exercise are major contributors to obesity, environmental factors may enhance susceptibility through disruption of hormone homeostasis and metabolic processes. The obesogen hypothesis contends that chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes and predisposing for obesity and metabolic disease. We previously developed a primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis. Here, the assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes. To evaluate the role of environmental compounds on adipocyte differentiation, progenitor cells were treated with 19 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, and multiplexed assays were used to confirm a GR-dependent mode of action. Five chemicals were found to be strong agonists. The assay was also modified to evaluate GR-antagonists, and 8/10 of the hypothesized antagonists inhibited adipogenesis. The in vitro bioactivity data was put into context with extrapolated human steady state concentrations (Css) and clinical exposure data (Cmax). These data support using a human adipose-derived stem cell differentiation assay to test the potential of chemicals to alter human GR-dependent adipogenesis.

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.