Identification of New Mono/Dihydroxynaphthoquinone as Lead Agents That Inhibit the Growth of Refractive and Triple-Negative Breast Cancer Cell Lines.

Human epidermal growth factor receptor 2 (HER2) is overexpressed in nearly 20-30% of breast cancers and is associated with metastasis resulting in poor patient survival and high recurrence. The dual EGFR/HER2 kinase inhibitor lapatinib has shown promising clinical results, but its limitations have also led to the resistance and activation of tumor survival pathways. Following our previous investigation of quinones as HER2 kinase inhibitors, we synthesized several naphthoquinone derivatives that significantly inhibited breast tumor cells expressing HER2 and trastuzumab-resistant HER2 oncogenic isoform, HER2Δ16. Two of these compounds were shown to be more effective than lapatinib at the inhibition of HER2 autophosphorylation of Y1248. Compounds 7 (5,8-dihydroxy-2-methylnaphthalene-1,4-dione) and 9 (2-(bromomethyl)-5,8-dihydroxynaphthalene-1,4-dione) inhibited HER2-expressing MCF-7 cells (IC50 0.29 and 1.76 µM, respectively) and HER2Δ16-expressing MCF-7 cells (IC50 0.51 and 1.76 µM, respectively). Compound 7 was also shown to promote cell death in multiple refractory breast cancer cell lines with IC50 values ranging from 0.12 to 2.92 µM. These compounds can function as lead compounds for the design of a new series of nonquinonoid structural compounds that can maintain a similar inhibition profile.

Ablating ErbB4 in PV neurons attenuates synaptic and cognitive deficits in an animal model of Alzheimer's disease.

Accumulation of amyloid ß (Aß) induces neuronal, synaptic, and cognitive deficits in patients and animal models of Alzheimer's disease (AD). The underlying mechanisms, however, remain to be fully elucidated. In the present study, we found that Aß interacted with ErbB4, a member of the receptor tyrosine kinase family and mainly expressed in GABAergic interneurons. Deleting ErbB4 in parvalbumin-expressing neurons (PV neurons) significantly attenuated oligomeric Aß-induced suppression of long term potentiation (LTP). Furthermore, specific ablation of ErbB4 in PV neurons via Cre/loxP system greatly improved spatial memory and synaptic plasticity in the hippocampus of hAPP-J20 mice. The deposition of Aß detected by 3D6 and Thioflavin S staining and the proteolytic processing of hAPP analyzed by western blotting were not affected in the hippocampus of hAPP-J20 mice by deleting ErbB4 in PV neurons. Our data suggested that ErbB4 in PV neurons mediated Aß-induced synaptic and cognitive dysfunctions without affecting Aß levels.

Trastuzumab stimulation of ribosomal protein S6 kinase 1 (S6K1) predicts de novo trastuzumab resistance.

Breast cancer is a complex disease with at least five different molecular subtypes identified. The breast tumor molecular subtypes guide stratification of patients for specific targeted therapy regimens and each subtype is associated with significantly different patient outcomes. For example, patients with the HER2 positive molecular subtype benefit from the HER2 targeted therapy trastuzumab. Unfortunately, women with the HER2 positive molecular subtype have the worst overall prognosis and nearly 70% of women with HER2 positive breast cancer exhibit de novo or acquired resistance to trastuzumab. Identification of tumor markers predicting trastuzumab response can be used to further stratify patients for life-saving personalized therapeutic options. The aim of this study was to identify clinically useful tumor markers predicting de novo tumor cell resistance to trastuzumab treatment. To identify oncogenic signaling pathways activated in response to trastuzumab treatment, we performed a Human Phospho-Kinase Proteome Profiler Array analysis comparing trastuzumab sensitive MCF-7/HER2.2 and trastuzumab resistant MCF-7/HER2Δ16H cells following acute treatment with 20 µg/ml of trastuzumab for 2 h. We found that of the 43 phosphorylation activated human kinases represented on the array, S6K1 was the only kinase altered greater than 1.5-fold in response to trastuzumab treatment of the trastuzumab resistant MCF-7/HER2Δ16H cells. Trastuzumab activation of S6K1 was confirmed in the two trastuzumab resistant SUM190 and SUM225 cell lines. Significantly, trastuzumab failed to stimulate S6K1 activation in the trastuzumab sensitive MCF-7/HER2.2, BT474, and SKBR3 cell lines suggesting that trastuzumab activation of S6K1 is a tumor cell marker for trastuzumab resistance. Consistent with a role for mTORC1/S6K1 signaling promoting trastuzumab resistance, all cell lines were sensitive to S6K1 inactivation with significant growth inhibition following treatment with the mTORC1 inhibitor rapamycin. In conclusion, characterizing rapid trastuzumab induced molecular alterations resulted in the identification of activated S6K1 as an early breast tumor cell marker for trastuzumab resistance. Our results further suggest that trastuzumab resistant breast tumor cells are addicted to mTORC1/S6K1 oncogenic signaling and targeting mTORC1 with rapamycin reverses trastuzumab resistance.

Intrinsic HER4/4ICD transcriptional activation domains are required for STAT5A activated gene expression.

The epidermal growth factor receptor family member HER4 undergoes proteolytic processing at the cell surface to release the HER4 intracellular domain (4ICD) nuclear protein. Interestingly, 4ICD directly interacts with STAT5 and functions as an obligate STAT5 nuclear chaperone. Once in the nucleus 4ICD binds with STAT5 at STAT5 target genes, dramatically potentiating STAT5 transcriptional activation. These observations raise the possibility that 4ICD directly coactivates STAT5 gene expression. Using both yeast and mammalian transactivation reporter assays, we performed truncations of 4ICD fused to a GAL4 DNA binding domain and identified two independent 4ICD transactivation domains located between residues 1022 and 1090 (TAD1) and 1192 and 1225 (TAD2). The ability of the 4ICD DNA binding domain fusions to transactivate reporter gene expression required deletion of the intrinsic tyrosine kinase domain. In addition, we identified the 4ICD carboxyl terminal TVV residues, a PDZ domain binding motif (PDZ-DBM), as a potent transcriptional repressor. The transactivation activity of the HER4 carboxyl terminal domain lacking the tyrosine kinase (CTD) was significantly lower than similar EGFR or HER2 CTD. However, deletion of the HER4 CTD PDZ-DBM enhanced HER4 CTD transactivation to levels equivalent to the EGFR and HER2 CTDs. To determine if 4ICD TAD1 and TAD2 have a physiologically relevant role in STAT5 transactivation, we coexpressed 4ICD or 4ICD lacking TAD2 or both TAD1 and TAD2 with STAT5 in a luciferase reporter assay. Our results demonstrate that each 4ICD TAD contributes additively to STAT5A transactivation and the ability of STAT5A to transactivate the ß-casein promoter requires the 4ICD TADs. Taken together, published data and our current results demonstrate that both 4ICD nuclear chaperone and intrinsic coactivation activities are essential for STAT5 regulated gene expression.

The transcription factor NeuroD2 coordinates synaptic innervation and cell intrinsic properties to control excitability of cortical pyramidal neurons.

KEY POINTS: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks to allow proper brain function. Disrupting this balance may lead to autism spectral disorders and epilepsy. We show the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo. We identify two genes potentially downstream of NeuroD2-mediated transcription that regulate these parameters: gastrin-releasing peptide and the small conductance, calcium-activated potassium channel, SK2. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability. Our results offer insight into how synaptic innervation and intrinsic excitability are coordinated during cortical development. ABSTRACT: Synaptic excitation and inhibition must be properly balanced in individual neurons and neuronal networks for proper brain function. Disruption of this balance during development may lead to autism spectral disorders and epilepsy. Synaptic excitation is counterbalanced by synaptic inhibition but also by attenuation of cell-intrinsic neuronal excitability. To maintain proper excitation levels during development, neurons must sense activity over time and regulate the expression of genes that control these parameters. While this is a critical process, little is known about the transcription factors involved in coordinating gene expression to control excitatory/inhibitory synaptic balance. We show here that the basic helix-loop-helix transcription factor NeuroD2 promotes inhibitory synaptic drive but also decreases cell-intrinsic neuronal excitability of cortical pyramidal neurons both in vitro and in vivo as shown by ex vivo analysis of a NeuroD2 knockout mouse. Using microarray analysis and comparing wild-type and NeuroD2 knockout cortical networks, we identified two potential gene targets of NeuroD2 that contribute to these processes: gastrin-releasing peptide (GRP) and the small conductance, calcium-activated potassium channel, SK2. We found that the GRP receptor antagonist RC-3059 and the SK2 specific blocker apamin partially reversed the effects of increased NeuroD2 expression on inhibitory synaptic drive and action potential repolarization, respectively. Our results reveal an important function for NeuroD2 in balancing synaptic neurotransmission and intrinsic excitability and offer insight into how these processes are coordinated during cortical development.

Direct coupling of the HER4 intracellular domain (4ICD) and STAT5A signaling is required to induce mammary epithelial cell differentiation.

The HER4 receptor tyrosine kinase and STAT5A cooperate to promote mammary luminal progenitor cell maturation and mammary epithelial cell differentiation. Coupled HER4 and STAT5A signaling is mediated, in part, through association of the HER4 intracellular domain (4ICD) with STAT5A at STAT5A target gene promoters where 4ICD functions as a STAT5A transcriptional coactivator. Despite an essential role for coupled 4ICD and STAT5A signaling in mammary gland development, the mechanistic basis of 4ICD and STAT5A cooperative signaling remains unexplored. Here we show for the first time that 4ICD and STAT5A directly interact through STAT5A recruitment and binding to HER4/4ICD residue Y984. Accordingly, altering the 4ICD Y984 to phenylalanine results in a dramatic reduction of STAT5A and 4ICD-Y984F interacting complexes coimmunoprecipitated with HER4 or STAT5A specific antibodies. We further show that disrupting the 4ICD and STAT5A interaction has an important physiological impact on mammary epithelial cell differentiation. HC11 mammary epithelial cells with stable expression of 4ICD undergo differentiation with significantly increased expression of the STAT5A target genes and differentiation markers ß-casein and WAP. In contrast, HC11 cells stably expressing 4ICD-Y984F failed to undergo differentiation with basal expression levels of ß-casein and WAP. Differentiation in this cell system was induced in the absence of exogenous prolactin indicating that 4ICD activity is sufficient to induce mammary epithelial cell differentiation. Finally, we show that suppression of STAT5A expression abolishes the ability of 4ICD to induce HC11 differentiation and activate ß-casein or WAP expression. Taken together our results demonstrate for the first time that direct coupling of 4ICD and STAT5A is both necessary and sufficient to drive mammary epithelial differentiation. In conclusion, our findings that 4ICD and STAT5A directly interact to form a physiologically important transcriptional activation complex, provide a mechanistic basis for the in vivo observations that HER4/4ICD and STAT5A cooperate to promote mammary gland progenitor cell maturation and initiate lactation at parturition.

A class of genes in the HER2 regulon that is poised for transcription in breast cancer cell lines and expressed in human breast tumors.

HER2-positive breast cancer accounts for 25% of all cases and has a poor prognosis. Although progress has been made in understanding signal transduction, little is known of how HER2 achieves gene regulation. We performed whole genome expression analysis on a HER2⁺ and HER2⁻ breast cancer cell lines and compared these results to expression in 812 primary tumors stratified by their HER2 expression level. Chip-on-chip with anti-RNA polymerase II was compared among breast cancer cell lines to identify genes that are potentially activated by HER2. The expression levels of these HER2-dependent POL II binding genes were determined for the 812 HER2+/- breast cancer tissues. Genes differentially expressed between HER2+/- cell lines were generally regulated in the same direction as in breast cancer tissues. We identified genes that had POLII binding in HER2⁺ cell lines, but without significant gene expression. Of 737 such genes "poised" for expression in cell lines, 113 genes were significantly differentially expressed in breast tumors in a HER2-dependent manner. Pathway analysis of these 113 genes revealed that a large group of genes were associated with stem cell and progenitor cell control as indicated by networks centered on NANOG, SOX2, OCT3/4. HER2 directs POL II binding to a large number of genes in breast cancer cells. A "poised" class of genes in HER2⁺ cell lines with POLII binding and low RNA expression but is differentially expressed in primary tumors, strongly suggests a role of the microenvironment and further suggests a role for stem cells proliferation in HER2-regulated breast cancer tissue.

MicroRNA-7 inhibits multiple oncogenic pathways to suppress HER2Δ16 mediated breast tumorigenesis and reverse trastuzumab resistance.

The oncogenic isoform of HER2, HER2Δ16, is expressed with HER2 in nearly 50% of HER2 positive breast tumors where HER2Δ16 drives metastasis and resistance to multiple therapeutic interventions including tamoxifen and trastuzumab. In recent years microRNAs have been shown to influence multiple aspects of tumorigenesis and tumor cell response to therapy. Accordingly, the HER2Δ16 oncogene alters microRNA expression to promote endocrine resistance. With the goal of identifying microRNA suppressors of HER2Δ16 oncogenic activity we investigated the contribution of altered microRNA expression to HER2Δ16 mediated tumorigenesis and trastuzumab resistance. Using a gene array strategy comparing microRNA expression profiles of MCF-7 to MCF-7/HER2Δ16 cells, we found that expression of HER2Δ16 significantly altered expression of 16 microRNAs by 2-fold or more including a 4.8 fold suppression of the miR-7 tumor suppressor. Reestablished expression of miR-7 in the MCF-7/HER2Δ16 cell line caused a G1 cell cycle arrest and reduced both colony formation and cell migration activity to levels of parental MCF-7 cells. Suppression of miR-7 in the MCF-7 cell line resulted in enhanced colony formation activity but not cell migration, indicating that miR-7 suppression is sufficient to drive tumor cell proliferation but not migration. MiR-7 inhibited MCF-7/HER2Δ16 cell migration through a mechanism involving suppression of the miR-7 target gene EGFR. In contrast, miR-7 inhibition of MCF-7/HER2Δ16 cell proliferation involved a pathway where miR-7 expression resulted in the inactivation of Src kinase independent of suppressed EGFR expression. Also independent of EGFR suppression, reestablished miR-7 expression sensitized refractory MCF-7/HER2Δ16 cells to trastuzumab. Our results demonstrate that reestablished miR-7 expression abolishes HER2Δ16 induced cell proliferation and migration while sensitizing HER2Δ16 expressing cells to trastuzumab therapy. We propose that miR-7 regulated pathways, including EGFR and Src kinase, represent targets for the therapeutic intervention of refractory and metastatic HER2Δ16 driven breast cancer.

Hypoxia-inducible factor-1α induces ErbB4 signaling in the differentiating mammary gland.

Conditional knock-out of Hif1a in the mouse mammary gland impairs lobuloalveolar differentiation during lactation. Here, we demonstrate that expression of ErbB4 was reduced in the lobulalveoli of mice with mammary gland-specific deletion of Hif1a. Erbb4 was not, however, a direct target gene for transcriptional regulation by HIF-1α in vitro. HIF-1α overexpression or HIF accumulating prolyl hydroxylase inhibitors reduced ErbB4 endocytosis, promoted transcriptional co-regulatory activity of ErbB4, and stimulated ErbB4-induced differentiation of mammary carcinoma cells. Consistently, RNA interference-mediated down-regulation of HIF-1α resulted in reduced ErbB4 protein amount and reduced mammary carcinoma cell differentiation. These findings indicate that HIF-1α is a physiologically relevant regulator of ErbB4 and that ErbB4 is involved in HIF-regulated differentiation of the mammary gland.

Basal cell signaling by p63 controls luminal progenitor function and lactation via NRG1.

The mammary epithelium is organized as a bilayer of luminal and basal/myoepithelial cells. During pregnancy, the luminal compartment expands for milk production, while basal cells are thought to provide structural and contractile support. Here, we reveal a pregnancy-specific role of basal epithelia as a central coordinator of lactogenesis. We demonstrate that genetic deletion of the transcription factor p63 (Trp63) gene exclusively within basal cells of the adult gland during pregnancy leads to dramatic defects in luminal cell proliferation and differentiation, resulting in lactation failure. This phenotype is explained by direct transcriptional activation of the epidermal growth factor family ligand gene Nrg1 by p63 selectively in basal cells, which is required for luminal ERBB4/STAT5A activation and consequent luminal progenitor cell maturation. Thus, paracrine basal-to-luminal cell signaling, controlled by p63 via NRG1, orchestrates the entire lactation program. Collectively, these findings redefine the paradigm for cellular interactions specifying the functional maturation of the mammary gland.

Identification of quinones as HER2 inhibitors for the treatment of trastuzumab resistant breast cancer.

HER2 overexpression is associated with aggressive breast cancer with high recurrence rate and poor patient prognosis. Treatment of HER2 overexpressing patients with the HER2 targeting therapy trastuzumab results in acquired resistance within a year. The HER2/EGFR dual kinase inhibitor lapatinib was shown to inhibit some trastuzumab resistant breast cancer cell lines and is currently in clinical trials. Our group has found two new quinone compounds that show excellent inhibition of breast tumor cells expressing HER2 or the trastuzumab resistant HER2 oncogenic isoform, HER2Δ16. Compound 4 ((1R,2S,3S)-1,2,3,5,8-pentahydroxy-1,2,3,4-tetrahydroanthracene-9,10-dione) and compound 5 (5,8-dihydroxy-2,3-bis(hydroxymethyl)naphthalene-1,4-dione) showed sub-micromolar inhibition potency against these cell lines. These compounds also inhibit auto-phosphorylation of the Y1248 and Y1068 residues of HER2 and EGFR, respectively.

HER4 selectively coregulates estrogen stimulated genes associated with breast tumor cell proliferation.

The EGFR-family member HER4 undergoes regulated intramembrane proteolysis (RIP) to generate an intracellular domain (4ICD) that functions as a transcriptional coactivator. Accordingly, 4ICD coactivates the estrogen receptor (ER) and associates with ER at target gene promoters in breast tumor cells. However, the extent of 4ICD coactivation of ER and the functional significance of the 4ICD/ER transcriptional complex is unclear. To identify 4ICD coactivated genes we performed a microarray gene expression analysis of ß-estradiol treated cells comparing control MCF-7 breast cancer cells to MCF-7 cells where HER4 expression was stably suppressed using a shRNA. In the MCF-7 cell line, ß-estradiol significantly stimulated or repressed by 2-fold or more 726 or 53 genes, respectively. Significantly, HER4/4ICD was an obligate coactivator for 277 or 38% of the ß-estradiol stimulated genes. Ingenuity Pathway Analysis of ß-estradiol regulated genes identified significant associations with multiple cellular functions regulating cellular growth and proliferation, cell cycle progression, cancer metastasis, decreased hypoplasia, tumor cell migration, apoptotic resistance of tumor cells, and increased transcription. Genes coactivated by 4ICD displayed functional specificity by only significantly contributing to cellular growth and proliferation, cell cycle progression, and decreased hypoplasia. In direct concordance with these in situ results we show that HER4 knockdown in MCF-7 cells results in a loss of estrogen stimulated tumor cell proliferation and cell cycle progression, whereas, estrogen stimulated tumor cell migration was unaffected by loss of HER4 expression. In summary, we demonstrate for the first time that a cell surface receptor functions as an obligate ER coactivator with functional specificity associated with breast tumor cell proliferation and cell cycle progression. Nearly 90% of ER positive tumors coexpress HER4, therefore we predict that the majority of breast cancer patients would benefit from a strategy to therapeutic disengage ER/4ICD coregulated tumor cell proliferation.

Interaction with ErbB4 promotes hypoxia-inducible factor-1α signaling.

The receptor-tyrosine kinase ErbB4 was identified as a direct regulator of hypoxia-inducible factor-1α (HIF-1α) signaling. Cleaved intracellular domain of ErbB4 directly interacted with HIF-1α in the nucleus, and stabilized HIF-1α protein in both normoxic and hypoxic conditions by blocking its proteasomal degradation. The mechanism of HIF stabilization was independent of VHL and proline hydroxylation but dependent on RACK1. ErbB4 activity was necessary for efficient HRE-driven promoter activity, transcription of known HIF-1α target genes, and survival of mammary carcinoma cells in vitro. In addition, mammary epithelial specific targeting of Erbb4 in the mouse significantly reduced the amount of HIF-1α protein in vivo. ERBB4 expression also correlated with the expression of HIF-regulated genes in a series of 4552 human normal and cancer tissue samples. These data demonstrate that soluble ErbB4 intracellular domain promotes HIF-1α stability and signaling via a novel mechanism.

Jumonji/ARID1 B (JARID1B) protein promotes breast tumor cell cycle progression through epigenetic repression of microRNA let-7e.

MicroRNAs (miRs) function as tumor suppressors or oncogenes in multiple tumor types. Although miR expression is tightly regulated, the molecular basis of miR regulation is poorly understood. Here, we investigated the influence of the histone demethylase Jumonji/ARID1 B (JARID1B) on miR regulation in breast tumor cells. In MCF-7 cells with stable RNAi-mediated suppression of JARID1B expression we identified altered regulation of multiple miRs including let-7e, a member of the let-7 family of tumor suppressor miRs. Chromatin immunoprecipitation analysis demonstrated JARID1B binding to the let-7e promoter region as well as removal of the of H3K4me3 histone mark associated with active gene expression. These results suggest that JARID1B epigenetically represses let-7e expression. JARID1B stimulates tumor cell proliferation by promoting the G(1) to S transition. As predicted, suppression of JARID1B resulted in an accumulation of MCF-7 cells in G(1). We confirmed that cyclin D1, which also promotes G(1) progression, is a direct target of let-7e, and we show that cyclin D1 expression is suppressed in JARID1B knockdown cells. Cyclin D1 expression and cell cycle progression were restored following inhibition of let-7e, suggesting that JARID1B repression of let-7e contributes to cyclin D1 expression and JARID1B-mediated cell cycle progression. Our results indicate that the JARID1B demethylase contributes to tumor cell proliferation through the epigenetic repression of a tumor suppressor miR.

The ERBB4 intracellular domain (4ICD) regulates NRG1-induced gene expression in hippocampal neurons.

The NRG1 growth factor and ERBB4 receptor have been identified as leading schizophrenia risk genes. Although NRG1 and ERBB4 have been shown to modulate neuronal functions involved in schizophrenia, including both GABAergic and glutamatergic synapses, the exact molecular mechanisms remain poorly understood. Here we investigated ERBB4 intracellular domain, 4ICD, transactivator function in rat hippocampal cultures by inhibiting γ-secretase mediated ERBB4 regulated intramembrane proteolysis (RIP). NRG1 stimulation resulted in a dramatic increase in the number of hippocampal cells displaying nuclear 4ICD which was abolished in cultures pretreated with the γ-secretase inhibitor compound E (CE). To identify NRG1-4ICD transactivated genes we compared global gene expression profiles of hippocampal cultures stimulated with NRG1 in the absence or presence of CE. In concordance with the contribution of NRG1-ERBB4 signaling to dendritic spine maturation and schizophrenia, global gene expression analysis followed by Ingenuity Pathway Analysis of the dataset identified NRG1-4ICD regulated genes significantly represented in semaphorin signaling and actin cytoskeletal plasticity and multiple genes with confirmed roles in dendritic spine morphogenesis. Using the power of global gene expression analysis our data provides a proof-of-concept supporting a role for non-canonical NRG1-4ICD signaling in the regulation of gene expression contributing to normal and schizophrenic neuronal function.

Neuregulin receptor ErbB4 functions as a transcriptional cofactor for the expression of surfactant protein B in the fetal lung.

Sufficient pulmonary surfactant production is required for the fetal-neonatal transition, especially in preterm infants. Neuregulin (NRG) and its transmembrane receptor ErbB4 positively regulate the onset of fetal surfactant synthesis. Details of this signaling process remain to be elucidated. ErbB4 is known to regulate gene expression in the mammary gland, where the receptor associates with the signal transducer and activator of transcription Stat5a to transactivate the ß-casein gene promoter. We hypothesized that in the fetal lung, ErbB4 functions as a transcriptional regulator for surfactant protein B (Sftpb), the most critical surfactant protein gene. Re-expressing full-length ErbB4 in primary fetal ErbB4-depleted Type II epithelial cells led to an increased expression of Sftpb mRNA. This stimulatory effect required the nuclear translocation of ErbB4 and association with Stat5a, with the resultant binding to and activation of the Sftpb promoter. We conclude that ErbB4 directly regulates important aspects of fetal lung maturation that help prepare for the fetal-neonatal transition.

Downregulation of miR-342 is associated with tamoxifen resistant breast tumors.

BACKGROUND: Tumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive. RESULTS: Here we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators. CONCLUSIONS: Our findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.

Inhibition of breast cancer cell invasion by melatonin is mediated through regulation of the p38 mitogen-activated protein kinase signaling pathway.

INTRODUCTION: The pineal gland hormone, melatonin, has been shown by numerous studies to inhibit the proliferation of estrogen receptor α (ERα)-positive breast cancer cell lines. Here, we investigated the role of melatonin in the regulation of breast cancer cell invasion. METHODS: Three invasive MCF-7 breast cancer cell clones - MCF-7/6, MCF-7/Her2.1, and MCF-7/CXCR4 cells - were employed in these studies. All three cell lines exhibited elevated phosphorylation of the ERK1/2 and p38 mitogen-activated protein kinase (MAPK) as determined by Western blot analysis. The effect of melatonin on the invasive potential of these human breast cancer cells was examined by matrigel invasion chamber assays. The expression and proteinase activity of two matrix metalloproteinases (MMPs), MMP-2 and MMP-9, were analyzed by Western blot analysis and gelatin zymography, respectively. RESULTS: Melatonin (10-9 M) significantly suppressed the invasive potential of MCF-7/6 and MCF-7/Her2.1 cells as measured by matrigel invasion chamber assays, and significantly repressed the proteinase activity of MMP-2 and MMP-9. In MCF-7/CXCR4 cells, melatonin significantly inhibited stromal-derived factor-1 (SDF-1/CXCL12) induced cell invasion and activity of MMP-9. Elevated expression of the MT1 melatonin receptor further enhanced, while luzindole, an MT1/MT2 antagonist, abrogated melatonin's anti-invasive effect, suggesting that melatonin's effect on invasion is mediated, principally, through the MT1 receptor. Furthermore, melatonin repressed the phosphorylation of p38 MAPK in MCF-7/Her2.1 cells and blocked stromal-derived factor-1 (SDF-1) induced p38 phosphorylation in MCF-7/CXCR4 cells. SB230580, a p38 inhibitor, was able to mimic, while transfection of the cells with a constitutively-active MKK6b construct blocked melatonin's effect on cell invasion, suggesting that the anti-invasive action of melatonin is mediated through the p38 pathway. CONCLUSIONS: Melatonin exerts an inhibitory effect on breast cancer cell invasion through down-regulation of the p38 pathway, and inhibition of MMP-2 and MMP-9 expression and activity.

Oncogenic HER2{Delta}16 suppresses miR-15a/16 and deregulates BCL-2 to promote endocrine resistance of breast tumors.

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.