Argonaute 2 Expression Correlates with a Luminal B Breast Cancer Subtype and Induces Estrogen Receptor Alpha Isoform Variation.
ABSTRACT: Estrogen receptor alpha (ER?) signaling pathways are frequently disrupted in breast cancer and contribute to disease progression. ER? signaling is multifaceted and many ER? regulators have been identified including transcription factors and growth factor pathways. More recently, microRNAs (miRNAs) are shown to deregulate ER? activity in breast carcinomas, with alterations in both ER? and miRNA expression correlating to cancer progression. In this study, we show that a high expression of Argonaute 2 (AGO2), a translation regulatory protein and mediator of miRNA function, correlates with the luminal B breast cancer subtype. We further demonstrate that a high expression of AGO2 in ER?+ tumors correlates with a poor clinical outcome. MCF-7 breast cancer cells overexpressing AGO2 (MCF7-AGO2) altered ER? downstream signaling and selective ER? variant expression. Enhanced ER?-36, a 36 kDa ER? isoform, protein and gene expression was observed in vitro. Through quantitative polymerase chain reaction (qPCR), we demonstrate decreased basal expression of the full-length ER? and progesterone receptor genes, in addition to loss of estrogen stimulated gene expression in vitro. Despite the loss, MCF-7-AGO2 cells demonstrated increased estrogen stimulated tumorigenesis in vivo. Together with our clinical findings on AGO2 expression and the luminal B subtype, we suggest that AGO2 is a regulator of altered ER? signaling in breast tumors.
Project description:BACKGROUND: Within estrogen receptor-positive breast cancer (ER+ BC), the expression levels of proliferation-related genes can define two clinically distinct molecular subtypes. When treated with adjuvant tamoxifen, those ER+ BCs that are lowly proliferative have a good prognosis (luminal-A subtype), however the clinical outcome of those that are highly proliferative is poor (luminal-B subtype). METHODS: To investigate the biological basis for these observations, gene set enrichment analysis (GSEA) was performed using microarray data from 246 ER+ BC samples from women treated with adjuvant tamoxifen monotherapy. To create an in vitro model of growth factor (GF) signaling activation, MCF-7 cells were treated with heregulin (HRG), an HER3 ligand. RESULTS: We found that a gene set linked to GF signaling was significantly enriched in the luminal-B tumors, despite only 10% of samples over-expressing HER2 by immunohistochemistry. To determine the biological significance of this observation, MCF-7 cells were treated with HRG. These cells displayed phosphorylation of HER2/3 and downstream ERK and S6. Treatment with HRG overcame tamoxifen-induced cell cycle arrest with higher S-phase fraction and increased anchorage independent colony formation. Gene expression profiles of MCF-7 cells treated with HRG confirmed enrichment of the GF signaling gene set and a similar proliferative signature observed in human ER+ BCs resistant to tamoxifen. CONCLUSION: These data demonstrate that activation of GF signaling pathways, independent of HER2 over-expression, could be contributing to the poor prognosis of the luminal-B ER+ BC subtype.
Project description:Aberrant Notch signaling is implicated in several cancers, including breast cancer. However, the mechanistic details of the specific receptors and function of ligand-mediated Notch signaling that promote breast cancer remains elusive. In our studies we show that DLL1, a Notch signaling ligand, is significantly overexpressed in ER?<sup>+</sup> luminal breast cancer. Intriguingly, DLL1 overexpression correlates with poor prognosis in ER?<sup>+</sup> luminal breast cancer, but not in other subtypes of breast cancer. In addition, this effect is specific to DLL1, as other Notch ligands (DLL3, JAGGED1, and JAGGED2) do not influence the clinical outcome of ER?<sup>+</sup> patients. Genetic studies show that DLL1-mediated Notch signaling in breast cancer is important for tumor cell proliferation, angiogenesis, and cancer stem cell function. Consistent with prognostic clinical data, we found the tumor-promoting function of DLL1 is exclusive to ER?<sup>+</sup> luminal breast cancer, as loss of DLL1 inhibits both tumor growth and lung metastasis of luminal breast cancer. Importantly, we find that estrogen signaling stabilizes DLL1 protein by preventing its proteasomal and lysososmal degradations. Moreover, estrogen inhibits ubiquitination of DLL1. Together, our results highlight an unexpected and novel subtype-specific function of DLL1 in promoting luminal breast cancer that is regulated by estrogen signaling. Our studies also emphasize the critical role of assessing subtype-specific mechanisms driving tumor growth and metastasis to generate effective subtype-specific therapeutics.
Project description:Argonaute (Ago) 2 is the catalytic engine of mammalian RNA interference, but little is known concerning the regulation of Ago2 by cell-signaling pathways. In this study we show that expression of Ago2, but not Ago1, Ago3, or Ago4, is elevated in estrogen receptor (ER) alpha-negative (ERalpha(-)) vs. ERalpha-positive (ERalpha+) breast cancer cell lines, and in ERalpha(-) breast tumors. In MCF-7 cells the low level of Ago2 was found to be dependent upon active ERalpha/estrogen signaling. Interestingly, the high expression of Ago2 in ERalpha(-) cells was severely blunted by inhibition of the epidermal growth factor (EGF) receptor/MAPK signaling pathway, using either a pharmacological MAPK kinase inhibitor, U0126, or a small interfering RNA directed against EGF receptor. Half-life studies using cycloheximide indicated that EGF enhanced, whereas U0126 decreased, Ago2 protein stability. Furthermore, a proteosome inhibitor, MG132, blocked Ago2 protein turnover. The functional consequences of elevated Ago2 levels were examined by stable transfection of ERalpha+ MCF-7 cells with full-length and truncated forms of Ago2. The full-length Ago2 transfectants displayed enhanced proliferation, reduced cell-cell adhesion, and increased migratory ability, as shown by proliferation, homotypic aggregation, and wound healing assays, respectively. Overexpression of full-length Ago2, but not truncated forms of Ago2 or an empty vector control, reduced the levels of E-cadherin, beta-catenin, and beta-actin, as well as enhanced endogenous miR-206 activity. These data indicate that Ago2 is regulated at both the transcriptional and posttranslational level, and also implicate Ago2 and enhanced micro-RNA activity in the tumorigenic progression of breast cancer cell lines.
Project description:The non-receptor tyrosine kinase, PTK6/BRK, is highly expressed in multiple tumor types, including prostate, ovarian, and breast cancers, and regulates oncogenic phenotypes such as proliferation, migration, and survival. PTK6 inhibition also overcomes targeted therapy resistance of HER2+ breast cancer. Although PTK6 is highly expressed in ER+ Luminal breast cancers, the role of PTK6 in this subtype has not been elucidated. In this study, we investigated the functions of PTK6 in ER+ Luminal breast cancer cells, including those that are relatively resistant to estrogen deprivation or targeted endocrine therapies used in the treatment of ER+ cancers. Enhanced expression of PTK6 in ER+ breast cancer cells enhances growth of ER+ breast cancer cells, including tamoxifen-treated cells. Downregulation of PTK6 in ER+ breast cancer cells, including those resistant to tamoxifen, fulvestrant, and estrogen deprivation, induces apoptosis, as evidenced by increased levels of cleaved PARP, and an increase in the AnnexinV+ population. PTK6 downregulation impairs growth of these cells in 3D MatrigelTM cultures, and virtually abrogates primary tumor growth of both tamoxifen-sensitive and resistant MCF-7 xenografts. Finally, we show that p38 MAPK activation is critical for PTK6 downregulation-induced apoptosis, a mechanism that we previously reported for survival of HER2+ breast cancer cells, highlighting conserved mechanisms of survival regulation by PTK6 across breast cancer subtypes. In conclusion, our studies elucidate critical functions of PTK6 in ER+ Luminal breast cancers and support PTK6 as an attractive therapeutic target for ER+ breast cancers.
Project description:We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance.
Project description:Tumors that express estrogen receptor alpha (ER?+) comprise 75% of breast cancers in women. While treatments directed against this receptor have successfully lowered mortality rates, many primary tumors initially or later exhibit resistance. The paucity of murine models of this "luminal" tumor subtype has hindered studies of factors that promote their pathogenesis and modulate responsiveness to estrogen-directed therapeutics. Since epidemiologic studies closely link prolactin and the development of ER?+ tumors in women, we examined characteristics of the aggressive ER?+ and ER?- carcinomas which develop in response to mammary prolactin in a murine transgenic model (neu-related lipocalin- prolactin (NRL-PRL)). To evaluate their relationship to clinical tumors, we determined phenotypic relationships among these carcinomas, other murine models of breast cancer, and features of luminal tumors in women.We examined a panel of prolactin-induced tumors for characteristics relevant to clinical tumors: histotype, ER?/progesterone receptor (PR) expression and estrogen responsiveness, Activating Protein 1 (AP-1) components, and phosphorylation of signal transducer and activator of transcription 5 (Stat5), extracellular signal regulated kinase (ERK) 1/2 and AKT. We compared levels of transcripts in the ER?-associated "luminal" signature that defines this subtype of tumors in women and transcripts enriched in various mammary epithelial lineages to other well-studied genetically modified murine models of breast cancer. Finally, we used microarray analyses to compare prolactin-induced ER?+ and ER?- tumors, and examined responsiveness to estrogen and the anti-estrogen, Faslodex, in vivo.Prolactin-induced carcinomas were markedly diverse with respect to histotype, ER?/PR expression, and activated signaling cascades. They constituted a heterogeneous, but distinct group of murine mammary tumors, with molecular features of the luminal subtype of human breast cancer. In contrast to morphologically normal and hyperplastic structures in NRL-PRL females, carcinomas were insensitive to ER?-mediated signals. These tumors were distinct from mouse mammary tumor virus (MMTV)-neu tumors, and contained elevated transcripts for factors associated with luminal/alveolar expansion and differentiation, suggesting that they arose from physiologic targets of prolactin. These features were shared by ER?+ and ER?- tumors, suggesting a common origin, although the former exhibited transcript profiles reflecting greater differentiation.Our studies demonstrate that prolactin can promote diverse carcinomas in mice, many of which resemble luminal breast cancers, providing a novel experimental model to examine the pathogenesis, progression and treatment responsiveness of this tumor subtype.
Project description:The luminal A phenotype is the most common breast cancer subtype and is characterized by estrogen receptor ? expression (ER?). Identification of the key regulator that governs the luminal phenotype of breast cancer will clarify the pathogenic mechanism and provide novel therapeutic strategies for this subtype of cancer. ER? signaling pathway sustains the epithelial phenotype and inhibits the epithelial-mesenchymal transition (EMT) of breast cancer. In this study, we demonstrate that Notch3 positively associates with ER? in both breast cancer cell lines and human breast cancer tissues. We found that overexpression of Notch3 intra-cellular domain, a Notch3 active form (N3ICD), in ER? negative breast cancer cells re-activated ER?, while knock-down of Notch3 reduced ER? transcript and proteins, with alteration of down-stream genes, suggesting its ability to regulate ER?. Mechanistically, our results show that Notch3 specifically binds to the CSL binding element of the ER? promoter and activates ER? expression. Moreover, Notch3 suppressed EMT, while suppression of Notch3 promoted EMT in cellular assay. Overexpressing N3ICD in triple-negative breast cancer suppressed tumorigenesis and metastasis in vivo. Conversely, depletion of Notch3 in luminal breast cancer promoted metastasis in vivo. Furthermore, Notch3 transcripts were significantly associated with prolonged relapse-free survival in breast cancer, in particular in ER? positive breast cancer patients. Our observations demonstrate that Notch3 governs the luminal phenotype via trans-activating ER? expression in breast cancer. These findings delineate the role of a Notch3/ER? axis in maintaining the luminal phenotype and inhibiting tumorigenesis and metastasis in breast cancer, providing a novel strategy to re-sensitize ER? negative or low-expressing breast cancers to hormone therapy.
Project description:Estrogen Receptor alpha (ER?) activation by estrogenic hormones induces luminal breast cancer cell proliferation. However, ER? plays also important hormone-independent functions to maintain breast tumor cells epithelial phenotype. We reported previously by RNA-Seq that in MCF-7 cells in absence of hormones ER? down-regulation changes the expression of several genes linked to cellular development, representing a specific subset of estrogen-induced genes. Here, we report regulation of long non-coding RNAs from the same experimental settings. A list of 133 Apo-ER?-Regulated lncRNAs (AER-lncRNAs) was identified and extensively characterized using published data from cancer cell lines and tumor tissues, or experiments on MCF-7 cells. For several features, we ran validation using cell cultures or fresh tumor biopsies. AER-lncRNAs represent a specific subset, only marginally overlapping estrogen-induced transcripts, whose expression is largely restricted to luminal cells and which is able to perfectly classify breast tumor subtypes. The most abundant AER-lncRNA, DSCAM-AS1, is expressed in ER?+ breast carcinoma, but not in pre-neoplastic lesions, and correlates inversely with EMT markers. Down-regulation of DSCAM-AS1 recapitulated, in part, the effect of silencing ER?, i.e. growth arrest and induction of EMT markers. In conclusion, we report an ER?-dependent lncRNA set representing a novel luminal signature in breast cancer cells.
Project description:Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ER?(+)) cells in vitro, the molecular mechanisms that regulate estrogen/ER? signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ER?(+) human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ER? attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ER? decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen-H19 signaling axis in breast tumors and found that ER?(+) tumors exhibited a higher expression of H19 as compared with ER?(-) tumors and that H19 expression showed a positive correlation with ER? expression in those tumors. Taken together, the present results indicate that the estrogen-ER?-H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER(+) breast cancer tumors.
Project description:Human primary breast cancers and breast cancer cell lines are classified by microarray-defined molecular subtypes, which reflect differentiation characteristics. Estrogen receptor (ER) expression is indicative of the luminal molecular subtype. We have previously established IPH-926, the first well-characterized cell line from infiltrating lobular breast cancer. IPH-926 displays an ER/PR/ErbB2 triple-negative immunophenotype, which is due to a loss of ER expression in its in vivo clonal ancestry. Loss of ER might indicate a fundamental change of cellular differentiation and it is unclear whether a luminal subtype is preserved beyond ER conversion. Using Affymetrix microarray analysis, seven different classifier gene lists (PAM305, DISC256, TN1288, PAM50, UNC1300, LAB704, INT500) and a background population of 50 common mammary carcinoma cell lines, we have now determined the molecular subtype of IPH-926. Strikingly, the IPH-926 expression profile is highly consistent with a luminal subtype. It is nearest to luminal/ER-positive breast cancer cell lines and far apart from basal breast cancer cell lines. Quantitative real-time RT-PCR confirmed enhanced expression of luminal marker genes (AGR2, CLU, CA12, EMP2, CLDN3) and low or absent expression of basal marker genes (KRT5, CD44, CAV1, VIM). Moreover, IPH-926 lacked androgen receptor (AR) expression, a transcription factor previously associated with luminal-like gene expression in a subset of triple-negative or molecular apocrine breast cancers. In conclusion, IPH-926 is triple-negative but belongs to the luminal subtype. Luminal differentiation characteristics can be preserved beyond ER conversion and might not require a compensatory expression of AR.