Estrogen Receptor Α-positive Breast Cancer Research Articles

Oncomirnas and Tumor Suppressors In Microvesicles From Four Types Of Cancer

ObjectivesMicrovesicles (MVs) are small vesicles that are shed from almost all cell types including cancer cells into their surroundings. These secreted MVs bear surface receptors/ligands and miRNA of the original cells and play a role in cancer. Our aim in this study was to identify MVs miRNAs that might play important roles both in solid and non-solid tumors. MethodsThe MVs miRNA expression from 4 kinds of cancer cell lines (SMMC-7721, K562, Nalm6 and Jurkat) and healthy peripheral blood cells were analyzed by miRNA microarray. Quantitative real-time polymerase chain reaction (qRT-PCR) was further performed to verify the expression of 4 dysregulated miRNAs. ResultsThe miRNA microarray revealed aberrantly expressed miRNAs of MVs between the four types of cancer cells and normal cells. In the MVs from cancer groups, 73 miRNAs were up-regulated and 148 were down-regulated in all the four types of cancer cell lines, indicating that solid and non-solid tumors shared some dysregulated MVs miRNAs. qRT-PCR verified statistically consistent expression of four selected miRNAs with microarray analysis. The top six shared upregulated MVs miRNAs in the four cancer cells lines were miR-1290, miR-1268, miR-1246, miR-125a-3p, miR-1305, miR-1226*. The top seven shared downregulated MVs miRNAs in the four cancer cells lines were miR-335, miR-1, miR-363, miR-122, miR-223, miR-338-3p and miR-340.The general roles of the upregulated MVs miRNAs were oncomiRNAs in multicancer. MiR-1290 and its potential targets are associated with characteristics of estrogen receptor α-positive breast cancer. Up-regulations of miR-1290 impaired cytokinesis and affected the reprogramming of colon cancer cells. miR-1246 might serve as a likely link of the p53 family with Down syndrome and proposed a new p53-miR-1246-DYRK1A-NFAT pathway in cancer.The functional roles of the downregulated MVs miRNAs were tumor suppressors in many kinds of cancer. MiR-335 acted as a candidate tumor suppressor and inhibited tumor reinitiation and represented an invasion suppressor gene by targeting Bcl-w in many kinds of cancer. HBx regulated miR-338-3p in HCC and miR-338-3p inhibited proliferation by regulating CyclinD1, and miR-338-3p suppressed the invasion of liver cancer cell by targeting smoothened gene in liver cancer. miR-340 inhibited of breast cancer cell migrations and invasions through targeting of oncoprotein c-Met and may play an important role in breast cancer progression. ConclusionOur study identifies oncomiRNAs and tumor suppressors in MVs from four types of cancer. These MVs miRNAs may contribute to the developments of solid and non-solid tumors and should be further evaluated as a novel biomarker for cancers and may provide potential targets for novel therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Autocrine IGF-I/insulin receptor axis compensates for inhibition of AKT in ER-positive breast cancer cells with resistance to estrogen deprivation

IntroductionEstrogen receptor α-positive (ER+) breast cancers adapt to hormone deprivation and acquire resistance to antiestrogen therapies. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT pathway. We examined the effects of AKT inhibition and its compensatory upregulation of insulin-like growth factor (IGF)-I/InsR signaling in ER+ breast cancer cells with acquired resistance to estrogen deprivation.MethodsInhibition of AKT using the catalytic inhibitor AZD5363 was examined in four ER+ breast cancer cell lines resistant to long-term estrogen deprivation (LTED) by western blotting and proliferation assays. Feedback upregulation and activation of receptor tyrosine kinases (RTKs) was examined by western blotting, real-time qPCR, ELISAs, membrane localization of AKT PH-GFP by immunofluorescence and phospho-RTK arrays. For studies in vivo, athymic mice with MCF-7 xenografts were treated with AZD5363 and fulvestrant with either the ATP-competitive IGF-IR/InsR inhibitor AZD9362 or the fibroblast growth factor receptor (FGFR) inhibitor AZD4547.ResultsTreatment with AZD5363 reduced phosphorylation of the AKT/mTOR substrates PRAS40, GSK3α/β and S6K while inducing hyperphosphorylation of AKT at T308 and S473. Inhibition of AKT with AZD5363 suppressed growth of three of four ER+ LTED lines and prevented emergence of hormone-independent MCF-7, ZR75-1 and MDA-361 cells. AZD5363 suppressed growth of MCF-7 xenografts in ovariectomized mice and a patient-derived luminal B xenograft unresponsive to tamoxifen or fulvestrant. Combined treatment with AZD5363 and fulvestrant suppressed MCF-7 xenograft growth better than either drug alone. Inhibition of AKT with AZD5363 resulted in upregulation and activation of RTKs, including IGF-IR and InsR, upregulation of FoxO3a and ERα mRNAs as well as FoxO- and ER-dependent transcription of IGF-I and IGF-II ligands. Inhibition of IGF-IR/InsR or PI3K abrogated AKT PH-GFP membrane localization and T308 P-AKT following treatment with AZD5363. Treatment with IGFBP-3 blocked AZD5363-induced P-IGF-IR/InsR and T308 P-AKT, suggesting that receptor phosphorylation was dependent on increased autocrine ligands. Finally, treatment with the dual IGF-IR/InsR inhibitor AZD9362 enhanced the anti-tumor effect of AZD5363 in MCF-7/LTED cells and MCF-7 xenografts in ovariectomized mice devoid of estrogen supplementation.ConclusionsThese data suggest combinations of AKT and IGF-IR/InsR inhibitors would be an effective treatment strategy against hormone-independent ER+ breast cancer.

Association of MiR-1290 and its potential targets with characteristics of estrogen receptor α-positive breast cancer.

614 Background: Recent analyses have identified heterogeneity in estrogen receptor (ER) α-positive breast cancer. Subtypes called luminal A and luminal B have been identified, and the tumor characteristics, such as response to endocrine therapy and prognosis are different in these subtypes. However, little is known about how the biological characteristics of ER-positive breast cancer are determined. Methods: In this study, expression profiles of microRNAs (miRNAs) and mRNAs in ER-positive breast cancer tissue were compared between ERhigh Ki67low tumors and ERlow Ki67hightumors by miRNA and mRNA microarrays. Results: Unsupervised hierarchical clustering analyses revealed distinct expression patterns of miRNAs and mRNAs. We identified a down-regulation of miR-1290 and up-regulations of 6 miRNAs in ERhigh Ki67lowtumors. We picked up 11 genes that were potential target genes of the selected miRNAs. Protein expression patterns of the selected target genes were analyzed in ER-positive breast cancer samples by immunohistochemistry: miR-1290 and its 4 putative targets, BCL2, forkhead box A1 (FOXA1), microtubule associated protein tau (MAPT) and N-acetyltransferase-1 (NAT1) were identified. Transfection experiments revealed that introduction of miR-1290 into ER-positive breast cancer cells decreased mRNA and protein expression of NAT1 and FOXA1. Conclusions: Our results suggest that miR-1290 and its potential targets, NAT1 and FOXA1, might be associated with characteristics of ER-positive breast cancer.

Vacuolar H+ ATPase expression and activity is required for Rab27B‐dependent invasive growth and metastasis of breast cancer

The secretory Rab27B small GTPase promotes invasive growth and metastasis in estrogen receptor (ER) α-positive breast cancer cells by orchestrating the peripheral targeting of vesicles secreting proinvasive growth regulators. Increased Rab27B expression is associated with poor prognosis in breast cancer patients. The molecular mechanisms of peripheral Rab27B secretory vesicle distribution are poorly understood. Mass spectrometry analysis on green fluorescent protein (GFP)-Rab27B vesicles prepared from GFP-Rab27B transfected MCF-7 human breast cancer cells detected eight subunits of the vacuolar H(+)-ATPase (V-ATPase) and the presence of V0a1 and V0d1 subunits was confirmed by Western blot analysis. Reversible inhibition of V-ATPase activity by bafilomycin A1 or transient silencing of V0a1 or V0d1 subunits demonstrated that V-ATPase controls peripheral localization and size of Rab27B vesicles. V-ATPase expression and activity further controls Rab27B-induced collagen type I invasion, cell-cycle progression and invasive growth in the chorioallantoic membrane assay. In agreement, Rab27B-dependent extracellular heat shock protein90α release and matrix metalloprotease-2 activation is markedly reduced by bafilomycin A1 and transient silencing of V0a1 and V0d1 subunits. Poor prognosis ERα-positive primary breast tumors expressing high levels of Rab27B also expressed multiple V-ATPase subunits and showed a strong cytoplasmic and peripheral V-ATPase V1E expression. In conclusion, inhibiting V-ATPase activity by interfering agents and drugs might be an effective strategy for blocking Rab27B-dependent proinvasive secretory vesicle trafficking in ERα-positive breast cancer patients.

MiR-1290 and its potential targets are associated with characteristics of estrogen receptor α-positive breast cancer

Recent analyses have identified heterogeneity in estrogen receptor α (ERα)-positive breast cancer. Subtypes called luminal A and luminal B have been identified, and the tumor characteristics, such as response to endocrine therapy and prognosis, are different in these subtypes. However, little is known about how the biological characteristics of ER-positive breast cancer are determined. In this study, expression profiles of microRNAs (miRNAs) and mRNAs in ER-positive breast cancer tissue were compared between ER(high) Ki67(low) tumors and ER(low) Ki67(high) tumors by miRNA and mRNA microarrays. Unsupervised hierarchical clustering analyses revealed distinct expression patterns of miRNAs and mRNAs in these groups. We identified a downregulation of miR-1290 in ER(high) Ki67(low) tumors. Among 11 miRNAs that were upregulated in ER(high) Ki67(low) tumors, quantitative RT-PCR detection analysis using 64 samples of frozen breast cancer tissue identified six miRNAs (let-7a, miR-15a, miR-26a, miR-34a, miR-193b, and miR-342-3p). We picked up 11 genes that were potential target genes of the selected miRNAs and that were differentially expressed in ER(high) Ki67(low) tumors and ER(low) Ki67(high) tumors. Protein expression patterns of the selected target genes were analyzed in 256 ER-positive breast cancer samples by immunohistochemistry: miR-1290 and its putative targets, BCL2, FOXA1, MAPT, and NAT1, were identified. Transfection experiments revealed that introduction of miR-1290 into ER-positive breast cancer cells decreased expression of NAT1 and FOXA1. Our results suggest that miR-1290 and its potential targets might be associated with characteristics of ER-positive breast cancer.

Insulin-like growth factor-I regulates GPER expression and function in cancer cells

Functional cross talk between insulin-like growth factor-I (IGF-I) system and estrogen signaling has been largely reported, although the underlying molecular mechanisms remain to be fully elucidated. As GPR30/GPER mediates rapid cell responses to estrogens, we evaluated the potential of IGF-I to regulate GPER expression and function in estrogen receptor (ER)α-positive breast (MCF-7) and endometrial (Ishikawa) cancer cells. We found that IGF-I transactivates the GPER promoter sequence and upregulates GPER mRNA and protein levels in both cells types. Similar data were found, at least in part, in carcinoma-associated fibroblasts. The upregulation of GPER expression by IGF-I involved the IGF-IR/PKCδ/ERK/c-fos/AP1 transduction pathway and required ERα, as ascertained by specific pharmacological inhibitors and gene-silencing. In both MCF-7 and Ishikawa cancer cells, the IGF-I-dependent cell migration required GPER and its main target gene CTGF, whereas the IGF-I-induced proliferation required both GPER and cyclin D1. Our data demonstrate that the IGF-I system regulates GPER expression and function, triggering the activation of a signaling network that leads to the migration and proliferation of cancer cells.

Resistance to Antiestrogen Arzoxifene Is Mediated by Overexpression of Cyclin D1

ABSTRACT Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)α-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ERα-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ERα transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to a new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ERα in the presence of arzoxifene. In this altered conformation, ERα still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ERα-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ERα antagonist into an agonist. This can be explained by a stabilization of the ERα/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ERα upon binding to antiestrogen are at the basis of resistance to antiestrogens.

Effect of estrogen receptor-positive progenitor cells on a tamoxifen resistance phenotype in breast cancer cells

1042 Background: The antiestrogen tamoxifen and aromatase inhibitors are the most frequently prescribed hormonal agents for the treatment of estrogen receptor (ER) α-positive breast cancer. An important question is whether there is a group of hormone resistant, ERα-positive patients who may derive additional benefit from the addition of chemotherapy to endocrine therapy, or who may be candidates for “targeted” biologics. Dicer1 is an RNase III-containing enzyme that processes microRNA precursors into mature microRNA, which have been implicated in breast tumor invasion and metastasis. BCRP1 is a transmembrane transport protein known to efflux a number of chemotherapeutic agents, but also steroid hormones. In the present study, we investigated whether Dicer might affect response to tamoxifen in breast cancer cells, and generated estrogen receptor-positive MCF-7 human breast cancer cells stably overexpressing Dicer1, and they exhibited elevated BCRP1 protein. Methods: We utilized preclinical approaches to study the function of BCRP1 in Dicer-overexpressing breast cancer cells using in vitro growth assays in soft agar, mammosphere formation assays, and in vivo tumor initiation. Results: Microarray analyses of human breast tumors, suggested that Dicer overexpression was associated with tamoxifen resistance. Dicer-overexpressing MCF-7 cells express elevated levels of BCRP1, ALDH, and cErbB2/HER-2 evident by immunoblot analysis. The Dicer1-overexpressing cells formed soft agar colonies in the presence of tamoxifen, however Fumitremorgin C (FTC) or MBLI-97, both BCRP inhibitors, reversed resistance, and sensitized cells to tamoxifen therapy. Preclinical in vivo tumor xenograft experiments confirmed the tamoxifen-resistant phenotype. Mammosphere potential was enhanced in Dicer-overexpressing cells suggesting an enrichment of stem-like breast cancer cells. Conclusions: Our results suggest that Dicer-overexpressing breast cancer cells are a novel preclinical model for an estrogen receptor-positive breast cancer progenitor phenotype and tamoxifen resistance. Based on our data Dicer1 is a potential predictive biomarker in breast cancer, and predicts that clinical BCRP1 inhibition may facilitate tumor sensitization to hormonal therapy. No significant financial relationships to disclose.

MTA2 is a metastasis promoter in breast cancer cells.

Abstract Abstract #2047 Background: Breast cancer mortality is often the result of distant metastasis. It is believed that epigenetic regulation of gene expression plays an important roles in cancer biology. We have previously shown that metastasis tumor associated (MTA)2 is a repressor of estrogen signaling in estrogen receptor (ER) α-positive breast cancer cells and that overexpression of MTA2 rendered ERα-positive cells resistant to the effects of antiestrogens, such as tamoxifen. Knockdown of Rho GDP dissociation inhibitor (Rho GDIα), an inhibitor of the Rho family, conferred resistance to tamoxifen concomitant with MTA2 upregulation in ERα-positive cells. Furthermore, we found, using a retrospective cohort, that patients with high MTA2 and low Rho GDIα expression had poorer recurrence-free survival after tamoxifen treatment. Thus, MTA2 overexpression is associated with an aggressive, hormone-independent phenotype. We therefore hypothesize that MTA2 might similarly enhance the aggressiveness of ERα-negative breast cancer cells, and explored molecular mechanisms of metastasis.
 Materials and Methods: We compared the invasive and migratory phenotype of MDA-MB-231 cells stably overexpressing either MTA2 or empty vector using modified Boyden chamber invasion, wound healing, and soft agar colony formation assays. Immunoblot assay was used to assess changes in protein expression. MDA-MB-231 cells overexpressing MTA2 or vector were injected into the number four mammary gland as tumor xenografts in female athymic nude mice to test for changes in metastatic potential in vivo. Differential gene expression between the MTA2-overexpressing and vector control MDA-MB-231 cells was determined using Affymetrix microarray technology.
 Results: Stable MDA-MB-231 transfectants overexpressing MTA2 were generated. MTA2-overexpressing cells exhibited more cell to cell contacts, and fewer spindle-shaped cells than vector control. MTA2-overexpressing cells were approximately three times more invasive than vector control, and exhibited increased ability to form colonies in soft agar. However, they were less motile in wound healing assays. As xenografts, MTA2 stably transfected cells rapidly metastasized to the lung, lymph nodes, and skin. Metastases were not observed with vector control cells during the course of the experiment. Using microarray analysis, we identified enriched ontologies for a number of relevant pathways, including Rho signaling and cell adhesion. We have validated that MTA2-overexpressing cells have reduced expression of Rho GDIα, and we are exploring the role of Rho signaling as an effector of MTA2's impact on metastasis.
 Conclusion: MTA2 overexpression significantly alters the morphology and metastatic behavior of ERα-negative breast cancer cells. We hypothesize that MTA2 regulates pathways regulating cell adhesion, migration, and the Rho signaling pathway, thus increasing the metastatic ability of MTA2-overexpressing cells. In other models, MTA2's activity relies on its associated histone deacetylase activity, thus HDAC inhibitors may be able to block the effects of MTA2 overexpression, making MTA2 a potential therapeutic target. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2047.

Estrogen receptor-β: why may it influence clinical outcome in estrogen receptor-α positive breast cancer?

In the previous issue of the journal, Lin and coworkers present data demonstrate that increased expression of estrogen receptor (ER)-β in ER-α-positive breast cancer cells antagonizes a defined group of ER-α/estrogen stimulated genes that are involved in cell cycle regulation and DNA replication. Similar expression patterns for these genes were found human ER-α positive breast tumors expressing higher levels or ER-β, and this correlated with better clinical outcome. The implications for these data, which suggest that ER-β is a positive actor and diagnostic marker for therapeutic outcome, are discussed.

Control of estrogen receptor by tumor suppressor protein PTEN in breast cancer cells

Excessive exposure to estrogen has long been associated with an increased risk for developing breast cancer and anti-estrogen therapy is the gold standard of care in the treatment of estrogen receptor (ER) α-positive breast cancers. However, there are several mysteries concerning both anti-estrogen, tamoxifen, and estrogen. The most important of these are: (1) some ERα-positive breast cancers do not respond to tamoxifen; (2) some ERα-negative breast cancers do respond to tamoxifen; (3) initial or acquired resistance to tamoxifen occurs with recurrent tumors; (4) estrogen can cause marked tumor regression in long-term tamoxifen-resistant ERα-positive breast cancer. These mysteries indicate that we do not know enough about estrogen signaling to understand the effects of targeting these receptors in cancer. The discovery of ERβ, the second estrogen receptor, has added another level of complexity to estrogen signaling. This review summarizes recent publications and makes an updated portrait of ERα and ERβ in breast carcinogenesis and endocrine cancer therapy.

The Expression of ERβcx in Human Breast Cancer and the Relationship to Endocrine Therapy and Survival

Estrogen receptor (ER) alpha-positive breast cancer is often treated with endocrine therapy using either antiestrogens or aromatase inhibitors. However, 30% of patients who receive endocrine therapy will derive no benefit from such treatments and may indeed suffer adverse effects. Currently, there are no ways to predict response to such treatments. ER beta cx, a variant of ER beta, has a dominant-negative effect over ER alpha, and its expression thought to modulate response to endocrine treatment may represent a predictor of response to endocrine therapy. We investigated the expression of the ER beta cx in 82 frozen breast samples (8 benign, 1 ductal carcinoma in situ, and 73 malignant) by Western blot analysis. The relationship between the expression of ER beta cx variants with prognosis and outcome of endocrine therapy was examined. There was a statistically significant association between the presence of ER beta cx and the response to endocrine therapy (Fisher's exact test, P = 0.04). We also examined the influence of the ER beta cx status of a tumor on time to progression and death. There was a relationship between the presence of ER beta cx and survival, with patients whose tumors express ER beta cx having a longer survival rate (P = 0.05). Cell-type specificity of expression was assessed by immunohistochemistry on a selection of histological samples. On the basis of this small group of patients, we conclude that the expression of ER beta cx correlated with favorable response to endocrine therapy. A larger study involving the staining of archival material is currently underway to confirm these preliminary results.