Identification of drugs including a dopamine receptor antagonist that selectively target cancer stem cells.

Selective targeting of cancer stem cells (CSCs) offers promise for a new generation of therapeutics. However, assays for both human CSCs and normal stem cells that are amenable to robust biological screens are limited. Using a discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells (hPSC), we identify small molecules from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal. Surprisingly, thioridazine, an antipsychotic drug, selectively targets the neoplastic cells, and impairs human somatic CSCs capable of in vivo leukemic disease initiation while having no effect on normal blood SCs. The drug antagonizes dopamine receptors that are expressed on CSCs and on breast cancer cells as well. These results suggest that dopamine receptors may serve as a biomarker for diverse malignancies, demonstrate the utility of using neoplastic hPSCs for identifying CSC-targeting drugs, and provide support for the use of differentiation as a therapeutic strategy.

The Role of Serotonin in Breast Cancer Stem Cells.

Breast tumors were the first tumors of epithelial origin shown to follow the cancer stem cell model. The model proposes that cancer stem cells are uniquely endowed with tumorigenic capacity and that their aberrant differentiation yields non-tumorigenic progeny, which constitute the bulk of the tumor cell population. Breast cancer stem cells resist therapies and seed metastases; thus, they account for breast cancer recurrence. Hence, targeting these cells is essential to achieve durable breast cancer remissions. We identified compounds including selective antagonists of multiple serotonergic system pathway components required for serotonin biosynthesis, transport, activity via multiple 5-HT receptors (5-HTRs), and catabolism that reduce the viability of breast cancer stem cells of both mouse and human origin using multiple orthologous assays. The molecular targets of the selective antagonists are expressed in breast tumors and breast cancer cell lines, which also produce serotonin, implying that it plays a required functional role in these cells. The selective antagonists act synergistically with chemotherapy to shrink mouse mammary tumors and human breast tumor xenografts primarily by inducing programmed tumor cell death. We hypothesize those serotonergic proteins of diverse activity function by common signaling pathways to maintain cancer stem cell viability. Here, we summarize our recent findings and the relevant literature regarding the role of serotonin in breast cancer.

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells.

BACKGROUND: Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). METHODS: We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. RESULTS: We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. CONCLUSIONS: Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.

Monoamine oxidase-A activity is required for clonal tumorsphere formation by human breast tumor cells.

BACKGROUND: Breast tumor growth and recurrence are driven by an infrequent population of breast tumor-initiating cells (BTIC). We and others have reported that the frequency of BTIC is orders of magnitude higher when breast tumor cells are propagated in vitro as clonal spheres, termed tumorspheres, by comparison to adherent cells. We exploited the latter to screen > 35,000 small molecules to identify agents capable of targeting BTIC. We unexpectedly discovered that selective antagonists of serotonin signaling were among the hit compounds. To better understand the relationship between serotonin and BTIC we expanded our analysis to include monoamine oxidase-A (MAO-A), an enzyme that metabolizes serotonin. METHODS: We used the Nanostring technology and Western blotting to determine whether MAO-A is expressed in human breast tumor cell lines cultured as tumorspheres by comparison to those grown as adherent cells. We then determined whether MAO-A activity is required for tumorsphere formation, a surrogate in vitro assay for BTIC, by assessing whether selective MAO-A inhibitors affect the frequency of tumorsphere-forming cells. To learn whether MAO-A expression in breast tumor cells is associated with other reported properties of BTIC such as anticancer drug resistance or breast tumor recurrence, we performed differential gene expression analyses using publicly available transcriptomic datasets. RESULTS: Tumorspheres derived from human breast tumor cell lines representative of every breast cancer clinical subtype displayed increased expression of MAO-A transcripts and protein by comparison to adherent cells. Surprisingly, inhibition of MAO-A activity with selective inhibitors reduced the frequency of tumorsphere-forming cells. We also found that increased MAO-A expression is a common feature of human breast tumor cell lines that have acquired anticancer drug resistance and is associated with poor recurrence-free survival (RFS) in patients that experienced high-grade, ER-negative (ER-) breast tumors. CONCLUSIONS: Our data suggests that MAO-A activity is required for tumorsphere formation and that its expression in breast tumor cells is associated with BTIC-related properties. The discovery that a selective MAO-A inhibitor targets tumorsphere-forming cells with potencies in the nanomolar range provides the first evidence of this agent's anticancer property. These data warrant further investigation of the link between MAO-A and BTIC.

Identification of Drug Candidates to Suppress Cigarette Smoke-induced Inflammation via Connectivity Map Analyses.

Cigarette smoking is the main risk factor for chronic obstructive pulmonary disease, and to date, existing pharmacologic interventions have been ineffective at controlling inflammatory processes associated with the disease. To address this issue, we used the Connectivity Map (cMap) database to identify drug candidates with the potential to attenuate cigarette smoke-induced inflammation. We queried cMap using three independent in-house cohorts of healthy nonsmokers and smokers. Potential drug candidates were validated against four publicly available human datasets, as well as six independent datasets from cigarette smoke-exposed mice. Overall, these analyses yielded two potential drug candidates: kaempferol and bethanechol. Subsequently, the efficacy of each drug was validated in vivo in a model of cigarette smoke-induced inflammation. BALB/c mice were exposed to room air or cigarette smoke and treated with each of the two candidate drugs either prophylactically or therapeutically. We found that kaempferol, but not bethanechol, was able to reduce cigarette smoke-induced neutrophilia, both when administered prophylactically and when administered therapeutically. Mechanistically, kaempferol decreased expression of IL-1α and CXCL5 concentrations in the lung. Our data suggest that cMap analyses may serve as a useful tool to identify novel drug candidates against cigarette smoke-induced inflammation.

RNAi screen identifies essential regulators of human brain metastasis-initiating cells.

Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-ß ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury.

Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Met(fl/fl); Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1-1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Met(fl/fl); Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Met(fl/fl); Wnt1Cre+ mice. Finally, Met(fl/fl); Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT: The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well understood. We show that a subset of adult calcitonin gene-related peptide (CGRP)-expressing myenteric neurons produce MET, the receptor for hepatocyte growth factor, and that loss of MET activity affects peristalsis in response to mucosal stroking, reduces MET-immunoreactive neurites, and increases susceptibility to dextran sodium sulfate-induced bowel injury. These observations may be relevant for understanding and treating intestinal motility disorders and also suggest that enhancing the activity of MET-expressing CGRP neurons might be a useful strategy to reduce bowel inflammation.

Methylation-dependent regulation of hypoxia inducible factor-1 alpha gene expression by the transcription factor Kaiso.

Low oxygen tension (hypoxia) is a common characteristic of solid tumors and strongly correlates with poor prognosis and resistance to treatment. In response to hypoxia, cells initiate a cascade of transcriptional events regulated by the hypoxia inducible factor-1 (HIF-1) heterodimer. Since the oxygen-sensitive HIF-1α subunit is stabilized during hypoxia, it functions as the regulatory subunit of the protein. To date, while the mechanisms governing HIF-1α protein stabilization and function have been well studied, those governing HIF1A gene expression are not fully understood. However, recent studies have suggested that methylation of a HIF-1 binding site in the HIF1A promoter prevents its autoregulation. Here we report that the POZ-ZF transcription factor Kaiso modulates HIF1A gene expression by binding to the methylated HIF1A promoter in a region proximal to the autoregulatory HIF-1 binding site. Interestingly, Kaiso's regulation of HIF1A occurs primarily during hypoxia, which is consistent with the finding that Kaiso protein levels peak after 4 h of hypoxic incubation and return to normoxic levels after 24 h. Our data thus support a role for Kaiso in fine-tuning HIF1A gene expression after extended periods of hypoxia.

The effects of lymph node status on predicting outcome in ER+ /HER2- tamoxifen treated breast cancer patients using gene signatures.

BACKGROUND: Lymph node (LN) status is the most important prognostic variable used to guide ER positive (+) breast cancer treatment. While a positive nodal status is traditionally associated with a poor prognosis, a subset of these patients respond well to treatment and achieve long-term survival. Several gene signatures have been established as a means of predicting outcome of breast cancer patients, but the development and indication for use of these assays varies. Here we compare the capacity of two approved gene signatures and a third novel signature to predict outcome in distinct LN negative (-) and LN+ populations. We also examine biological differences between tumours associated with LN- and LN+ disease. METHODS: Gene expression data from publically available data sets was used to compare the ability of Oncotype DX and Prosigna to predict Distant Metastasis Free Survival (DMFS) using an in silico platform. A novel gene signature (Ellen) was developed by including patients with both LN- and LN+ disease and using Prediction Analysis of Microarrays (PAM) software. Gene Set Enrichment Analysis (GSEA) was used to determine biological pathways associated with patient outcome in both LN- and LN+ tumors. RESULTS: The Oncotype DX gene signature, which only used LN- patients during development, significantly predicted outcome in LN- patients, but not LN+ patients. The Prosigna gene signature, which included both LN- and LN+ patients during development, predicted outcome in both LN- and LN+ patient groups. Ellen was also able to predict outcome in both LN- and LN+ patient groups. GSEA suggested that epigenetic modification may be related to poor outcome in LN- disease, whereas immune response may be related to good outcome in LN+ disease. CONCLUSIONS: We demonstrate the importance of incorporating lymph node status during the development of prognostic gene signatures. Ellen may be a useful tool to predict outcome of patients regardless of lymph node status, or for those with unknown lymph node status. Finally we present candidate biological processes, unique to LN- and LN+ disease, that may indicate risk of relapse.

A novel stem cell culture model of recurrent glioblastoma.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with average disease relapse at 9 months and median survival rarely extending beyond 15 months. Brain tumor stem cells (BTSCs) have been implicated in not only initiating GBM but also conferring resistance to therapy. However, it is not clear whether the BTSC population that initiates tumor growth is also responsible for GBM recurrence. In this study, we have developed a novel in vitro treatment model to profile the evolution of primary treatment-naïve GBM BTSCs through chemoradiotherapy. We report that our in vitro model enriched for a CD15+/CD133- BTSC population, mirroring the phenotype of BTSCs in recurrent GBM. We also show that in vitro treatment increased stem cell gene expression as well as self-renewal capacity of primary GBMs. In addition, the chemoradiotherapy-refractory gene signature obtained from gene expression profiling identified a hyper-aggressive subtype of glioma. The delivery of in vitro chemoradiotherapy to primary GBM BTSCs models several aspects of recurrent GBM biology, and could be used as a discovery and drug-screening platform to uncover new biological drivers and therapeutic targets in GBM.

Immunotherapy-induced CD8+ T cells instigate immune suppression in the tumor.

Despite clear evidence of immunogenicity, cancer vaccines only provide a modest clinical benefit. To evaluate the mechanisms that limit tumor regression following vaccination, we have investigated the weak efficacy of a highly immunogenic experimental vaccine using a murine melanoma model. We discovered that the tumor adapts rapidly to the immune attack instigated by tumor-specific CD8+ T cells in the first few days following vaccination, resulting in the upregulation of a complex set of biological networks, including multiple immunosuppressive processes. This rapid adaptation acts to prevent sustained local immune attack, despite continued infiltration by increasing numbers of tumor-specific T cells. Combining vaccination with adoptive transfer of tumor-specific T cells produced complete regression of the treated tumors but did not prevent the adaptive immunosuppression. In fact, the adaptive immunosuppressive pathways were more highly induced in regressing tumors, commensurate with the enhanced level of immune attack. Examination of tumor infiltrating T-cell functionality revealed that the adaptive immunosuppression leads to a progressive loss in T-cell function, even in tumors that are regressing. These novel observations that T cells produced by therapeutic intervention can instigate a rapid adaptive immunosuppressive response within the tumor have important implications for clinical implementation of immunotherapies.

FoxG1 interacts with Bmi1 to regulate self-renewal and tumorigenicity of medulloblastoma stem cells.

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Groups 1 and 2 are characterized by relatively good clinical outcomes and activation of the Wnt and Shh pathways, respectively. In contrast, groups 3 and 4 ("non-Shh/Wnt MBs") are distinguished by metastatic disease, poor patient outcome, and lack a molecular pathway phenotype. Current gene expression platforms have not detected brain tumor-initiating cell (BTIC) self-renewal genes in groups 3 and 4 MBs as BTICs typically comprise a minority of tumor cells and may therefore go undetected on bulk tumor analyses. Since increasing BTIC frequency has been associated with increasing tumor aggressiveness and poor patient outcome, we investigated the subgroup-specific gene expression profile of candidate stem cell genes within 251 primary human MBs from four nonoverlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston) and 74 NanoString-subgrouped MBs (Vancouver). We assessed the functional relevance of two genes, FoxG1 and Bmi1, which were significantly enriched in non-Shh/Wnt MBs and showed these genes to mediate MB stem cell self-renewal and tumor initiation in mice. We also identified their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional BTIC assays, which may then be used to develop novel BTIC self-renewal mechanisms amenable to therapeutic targeting.

Estrogen independent gene expression defines clinically relevant subgroups of estrogen receptor positive breast cancer.

BACKGROUND: Human breast cancer represents a significantly heterogeneous disease. Global gene expression profiling measurements have been used to classify tumors into multiple molecular subtypes. The capacity to define subtypes of breast tumors provides a framework to enable improved understanding of the mechanisms of breast oncogenesis, as well as to provide opportunities for improved therapeutic intervention in patients. METHODS: We used publicly available gene expression profiling data to identify 'estrogen independent' genes in estrogen receptor alpha (ER+) breast tumors, and subsequently identified 6 subgroups of ER+breast tumors. RESULTS: Each of the 6 identified subgroups exhibited distinct clinical behaviors and biology. Patients whose tumors comprised subgroups 2,5&6 experienced excellent long-term survival, whereas those patients whose tumors belonged to subgroups 1&4 experienced much poorer survival. Breast tumor cell lines representative of the different subgroups responded to therapeutic compounds in accordance with their subgroup classification. CONCLUSIONS: These data support the existence of 6 distinct subgroups of ER+breast cancer and suggest that knowledge of the ER+subgroup status of patient samples have the potential to guide therapy choice.

Impaired c-src activation and motility defects in PEA3-null fibroblasts.

Null mutations in the pea3 allele compromise the capacity of mammary tumors to metastasize in MMTV-Neu/ErbB2/HER2 transgenic mice, indicating a motility defect in PEA3-null cells. Cellular and biochemical analyses of established PEA3-null fibroblasts show impaired motility and aberrant localization of adhesion proteins in spreading cells. Our results show that PEA3-/- cells express normal levels of key adhesion components, but that spreading PEA3-null cells fail to activate c-src and to downregulate phospho-FAK(Y397), suggesting that focal adhesion signaling is impaired. Supporting this, biochemical analysis revealed that adhesion complex-associated proteins such as p130Cas failed to undergo tyrosine phosphorylation and dissociated from the adhesion complex with delayed kinetics. Overall our data show that the motility defects observed in PEA3-null cells are due to altered adhesion signaling.

Targeted disruption of beta1-integrin in a transgenic mouse model of human breast cancer reveals an essential role in mammary tumor induction.

Despite evidence demonstrating the role of beta1-integrin in the regulation of cancer cell proliferation in vitro, the importance of this cell adhesion receptor during the initiation and progression of epithelial tumors in vivo remains unclear. Here we have used the Cre/LoxP1 recombination system to disrupt beta1-integrin function in the mammary epithelium of a transgenic mouse model of human breast cancer. Using this approach, we show that beta1-integrin expression is critical for the initiation of mammary tumorigenesis in vivo, and for maintaining the proliferative capacity of late-stage tumor cells. These observations provide a direct demonstration that beta1-integrin plays a critical role in both the initiation and maintenance of mammary tumor growth in vivo.

ERM is required for transcriptional control of the spermatogonial stem cell niche.

Division of spermatogonial stem cells produces daughter cells that either maintain their stem cell identity or undergo differentiation to form mature sperm. The Sertoli cell, the only somatic cell within seminiferous tubules, provides the stem cell niche through physical support and expression of surface proteins and soluble factors. Here we show that the Ets related molecule (ERM) is expressed exclusively within Sertoli cells in the testis and is required for spermatogonial stem cell self-renewal. Mice with targeted disruption of ERM have a loss of maintenance of spermatogonial stem cell self-renewal without a block in normal spermatogenic differentiation and thus have progressive germ-cell depletion and a Sertoli-cell-only syndrome. Microarray analysis of primary Sertoli cells from ERM-deficient mice showed alterations in secreted factors known to regulate the haematopoietic stem cell niche. These results identify a new function for the Ets family transcription factors in spermatogenesis and provide an example of transcriptional control of a vertebrate stem cell niche.

The Pea3 Ets transcription factor regulates differentiation of multipotent progenitor cells during mammary gland development.

The Pea3 Ets transcription factor is overexpressed in breast tumors suggesting that it plays a role in mammary oncogenesis. However, the normal biological function of Pea3 in the mammary gland is not known. Here we report that Pea3 was expressed in the epithelium of the mouse mammary anlagen commensurate with their genesis, and at later times in the nipple and mammary ducts of female embryos. In adult mice Pea3 transcripts peaked at the onset of puberty and early pregnancy, times of active epithelial cell proliferation and differentiation. Pea3 was expressed in all progenitor cap cells and rare body cells of terminal end buds, and in the myoepithelial cells of ducts and alveoli. Analyses of the mammary glands of Pea3-null mice during puberty revealed an increased number of terminal end buds and an increased fraction of proliferating progenitor cells within these structures compared to their wild type littermates. Tissue transplant experiments demonstrated that these phenotypes were intrinsic to the Pea3-null mammary epithelium. During pregnancy, mammary glands isolated from Pea3-null females had impaired alveolar development as revealed by a decreased fraction of alveolar structures. We performed in vitro colony forming assays of mammary epithelial cells and discovered that loss of Pea3 altered the distribution of specific multipotent progenitor cells. Double-immunofluorescence confirmed that multipotential progenitors co-expressing markers of the myoepithelial and luminal epithelial lineage were amplified in the mammary glands of Pea3-null mice by comparison to their wild type counterparts. We propose that Pea3 functions in multipotential progenitors to regulate their lineage-specific differentiation potential.

The Antiarrhythmic Drug, Dronedarone, Demonstrates Cytotoxic Effects in Breast Cancer Independent of Thyroid Hormone Receptor Alpha 1 (THRα1) Antagonism.

Previous research has suggested that thyroid hormone receptor alpha 1 (THRα1), a hormone responsive splice variant, may play a role in breast cancer progression. Whether THRα1 can be exploited for anti-cancer therapy is unknown. The antiproliferative and antitumor effects of dronedarone, an FDA-approved anti-arrhythmic drug which has been shown to antagonize THRα1, was evaluated in breast cancer cell lines in vitro and in vivo. The THRα1 splice variant and the entire receptor, THRα, were also independently targeted using siRNA to determine the effect of target knockdown in vitro. In our study, dronedarone demonstrates cytotoxic effects in vitro and in vivo in breast cancer cell lines at doses and concentrations that may be clinically relevant. However, knockdown of either THRα1 or THRα did not cause substantial anti-proliferative or cytotoxic effects in vitro, nor did it alter the sensitivity to dronedarone. Thus, we conclude that dronedarone's cytotoxic effect in breast cancer cell lines are independent of THRα or THRα1 antagonism. Further, the depletion of THRα or THRα1 does not affect cell viability or proliferation. Characterizing the mechanism of dronedarone's anti-tumor action may facilitate drug repurposing or the development of new anti-cancer agents.

Kaiso depletion attenuates the growth and survival of triple negative breast cancer cells.

Triple negative breast cancers (TNBC) are highly aggressive and lack specific targeted therapies. Recent studies have reported high expression of the transcription factor Kaiso in triple negative tumors, and this correlates with their increased aggressiveness. However, little is known about the clinical relevance of Kaiso in the growth and survival of TNBCs. Herein, we report that Kaiso depletion attenuates TNBC cell proliferation, and delays tumor onset in mice xenografted with the aggressive MDA-231 breast tumor cells. We further demonstrate that Kaiso depletion attenuates the survival of TNBC cells and increases their propensity for apoptotic-mediated cell death. Notably, Kaiso depletion downregulates BRCA1 expression in TNBC cells expressing mutant-p53 and we found that high Kaiso and BRCA1 expression correlates with a poor overall survival in breast cancer patients. Collectively, our findings reveal a role for Kaiso in the proliferation and survival of TNBC cells, and suggest a relevant role for Kaiso in the prognosis and treatment of TNBCs.

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts.

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.