The role of estrogen receptor alpha in mediating chemoresistance in breast cancer cells

Introduction

Previous studies suggested that estrogen receptor alpha (ERα) plays an important role in the chemoresistance of breast cancers. However, large random trials failed to demonstrate any benefit of the concurrent estrogen antagonist tamoxifen on the chemotherapy efficacy. Thus, in the present study, the importance of the role of ERα in the chemoresistance of breast cancer cells was investigated.

Methods

The ERα-transfected Bcap37 cells and natural ERα-positive T47D breast cancer cells were treated using chemotherapeutic agents with or without 17-beta estradiol (E2) pretreatment. Their viabilities were assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays. The dead cell rates were determined using propidium iodide dye exclusion tests, and the expression levels of Bcl-2 and Bax were detected through Western blot analysis. The effects of E2 on the growth of breast cancer cells were also determined via cell growth curve and cell cycle analysis.

Results

ERα activation by E2 increased the sensitivity of natural ERα-positive T47D breast cancer cells to chemotherapeutic agents. However, the increase in ERα expression in ERα-negative Bcap37 breast cancer cells also significantly increased their resistance. These phenomena cannot be explained by asserting that ERα mediated the chemoresistance of breast cancer cells by regulating the expression of Bcl-2 and Bax. Our findings show that ERα activation upregulated the expression of Bcl-2 in natural ERα-positive T47D breast cancer cells, whereas ERα activation by E2 downregulated and upregulated the Bcl-2 and Bax expression levels, respectively, in ERα-transfected Bcap37 cells. This phenomenon was due to the influence of ERα on the growth of breast cancer cells. Specifically, ERα activation enhanced the growth of natural ERα-positive breast cancer cells and thus increased their sensitivity to chemotherapeutic agents. However, ERα activation also inhibited the growth of ERα-transfected Bcap37 cells and increased the resistance of cancer cells to chemotherapeutic agents. Chemoresistance of ERα-transfected Bcap37 cells was only due to the specific growth inhibition by E2, which is not applicable to common ERα-positive breast cancer cells.

Conclusions

Although ERα was associated with chemoresistance of breast cancers, ERα itself did not mediate this resistance process.     

Calcitriol induces estrogen receptor α expression through direct transcriptional regulation and epigenetic modifications in estrogen receptor-negative breast cancer cells

Patients with estrogen receptor (ER) α-negative breast tumors have a poor prognosis and are not suitable for hormone therapy. Previously, we demonstrated that calcitriol, the active metabolite of vitamin D, induces ERα expression and re-establishes the response to antiestrogens in ER-negative breast cancer cells. However, the mechanisms involved in this process have not been elucidated. Therefore, the present study was undertaken to investigate the mechanisms implicated in the calcitriol-induced ERα expression in ER-negative breast cancer cells. Using EMSA and ChIP assays, we found that the calcitriol/vitamin D receptor (VDR)/retinoic X receptor (RXR) complex binds to putative vitamin D response elements (VDREs) in the ERα gene promoter region. In addition, we established by a fluorometric assay that calcitriol decreased DNA-methyltransferase and histone deacetylase activities. Flow cytometry and qPCR analyses showed that co-treatment of calcitriol with inhibitors of the histone deacetylase and DNA methyltransferase, and genistein significantly increased ERα expression, compared to that observed with the compounds alone. In conclusion, the calcitriol-dependent ERα induction in ER-negative breast cancer cells results from binding of the VDR-RXR complex to VDREs in the ERα gene promoter region, including the downregulation of enzymes with chromatin-remodeling activities. These results may bring forth novel mechanistic knowledge into the actions of calcitriol in ERα-negative breast cancer.     

Estradiol–estrogen receptor: A key interplay of the expression of syndecan‐2 and metalloproteinase‐9 in breast cancer cells

Estrogens are related with the growth and development of target tissues and play a critical role in breast cancer progression. The effects of estrogens are mediated by the estrogen receptors ERα and ERβ, which are members of the nuclear steroid receptor superfamily. To date, it is not known how these hormones elicit many of their effects on extracellular matrix molecules and how these effects can be connected with ER expression. For this purpose, the effect of estradiol on ER expression as well as on proteoglycan and metalloproteinase expression was studied. The effect of E2 on extracellular matrix molecule expression has been studied using ERα suppression in breast cancer cells. Our studies using ERα‐positive MCF‐7 cells show that estradiol affects the expression of syndecan‐2, but not of syndecan‐4, through ERα. Furthermore, the ability of estradiol to affect MMP‐9 and TIMP‐1 expression is connected with ERα status. Together, these data demonstrate the significant role of ERα on mediating the effect of estradiol on extracellular matrix molecules.     

Cryptotanshinone inhibits breast cancer cell growth by suppressing estrogen receptor signaling

Estrogen receptor (ER) is a major therapeutic target for the treatment of breast cancer, because of the crucial role of estrogen signaling deregulation in the development and progression of breast cancer. In this study, we report the identification of a novel ERα binding compound, cryptotanshinone (CPT), by screening the CADD database. We also show that CPT effectively inhibits estrogen-induced ER transactivation and gene expression of ER target genes. Furthermore, we showed that CPT suppressed breast cancer cell growth mainly in an ERα dependent manner. Finally, we confirmed the potential therapeutic efficiency of CPT using xenograft experiments in vivo. Taken together, our results describe a novel mechanism for the anticancer activity of CPT and provide supporting evidence for its use as a potential therapeutic agent to treat patients with ERα positive breast cancer.     

Obesity enhances nongenomic estrogen receptor crosstalk with the PI3K/Akt and MAPK pathways to promote in vitro measures of breast cancer progression

Introduction

Epidemiological and clinical studies indicate that obesity is associated with a worse postmenopausal breast cancer prognosis and an increased risk of endocrine therapy resistance. However, the mechanisms mediating these effects remain poorly understood. Here we investigate the molecular pathways by which obesity-associated circulating factors in the blood enhance estrogen receptor alpha (ERα) positive breast cancer cell viability and growth.

Methods

Blood serum was collected from postmenopausal breast cancer patients and pooled by body mass index (BMI) category (Control: 18.5 to 24.9 kg/m²; Obese: ≥30.0 kg/m²). The effects of patient sera on MCF-7 and T47D breast cancer cell viability and growth were examined by MTT and colony formation assays, respectively. Insulin-like growth factor receptor 1(IGF-1R), Akt, and ERK1/2 activation and genomic ERα activity were assessed to determine their possible contribution to obese patient sera-induced cell viability and growth. To further define the relative contribution of these signaling pathways, cells grown in patient sera were treated with various combinations of ERα, PI3K/Akt and MAPK targeted therapies. Comparisons between cells exposed to different experimental conditions were made using one-way analysis of variance (ANOVA) and Student''s t test.

Results

Cells grown in media supplemented with obese patient sera displayed greater cell viability and growth as well as IGF-1R, Akt and ERK1/2 activation relative to control sera. Despite the lack of a significant difference in genomic ERα activity following growth in obese versus control patient sera, we observed a dramatic reduction in cell viability and growth after concurrent inhibition of the ERα and PI3K/Akt signaling pathways. Further, we demonstrated that ERα inhibition was sufficient to attenuate obese serum-induced Akt and ERK1/2 activation. Together, these data suggest that obesity promotes greater ERα positive breast cancer cell viability and growth through enhanced crosstalk between nongenomic ERα signaling and the PI3K/Akt and MAPK pathways.

Conclusions

Circulating factors in the serum of obese postmenopausal women stimulate ERα positive breast cancer cell viability and growth by facilitating non-genomic ERα crosstalk with the PI3K/Akt and MAPK signaling pathways. These findings provide valuable insight into one mechanism by which obesity may promote ERα positive postmenopausal breast cancer progression and endocrine therapy resistance.     

NOP14 suppresses breast cancer progression by inhibiting NRIP1/Wnt/β-catenin pathway

NOP14, which is functionally conserved among eukaryotes, has been implicated in cancer development. Here, we show that NOP14 is poorly expressed in breast cancer cells and invasive breast cancer tissues. In vivo and in vitro studies indicated that NOP14 suppressed the tumorigenesis and metastasis of breast cancer cells. Further investigations revealed that NOP14 enhanced ERα expression and inhibited the Wnt/β-catenin pathway by up-regulating NRIP1 expression. Survival analysis indicated that low NOP14 expression was significantly associated with poor overall survival (P = 0.0006) and disease-free survival (P = 0.0007), suggesting that NOP14 is a potential prognostic factor in breast cancer. Taken together, our findings reveal that NOP14 may suppress breast cancer progression and provide new insights into the development of targeted therapeutic agents for breast cancer.     

Estrogen Receptor β Expression and Its Clinical Implication in Breast Cancers: Favorable or Unfavorable?

There are two estrogen receptor (ER) genes (ESR1/ERα and ESR2/ERβ) in humans. Of those. ERβ, the second ER isotype identified in 1996, is differentially expressed in different phenotypes and molecular subtypes of breast cancer (BCa), and is highly expressed in ERα-negative BCa and triple-negative BCa (TNBC). This review summarizes the potential clinical relevance of ERβ in BCa and the challenges associated with studies on the role of ERβ in BCa. The experimental and clinical studies evaluating clinical outcomes and associations with clinical characteristics and responses to endocrine therapy on targeting ERβ reviewed herein indicate that ERβ is a clinically important biomarker in BCa. The reviewed studies also suggest that each ERβ isoform has a distinct role in BCa subtypes and the potential of novel- targeted therapies in BCa, especially ERα-negative BCa and TNBC. However, the findings of many studies on ERβ are inconsistent, and the exact role of ERβ in BCa remains elusive; this may potentially be attributed to the complexity of ERβ isoforms, but also to the lack of standardized testing protocol. Thus, successful clinical application of ERβ requires the development of standardized, reproducible, and objective measurement methods for ERβ that can be widely and routinely applied in clinical setting.     

RhoB modifies estrogen responses in breast cancer cells by influencing expression of the estrogen receptor

Introduction

RhoB has been reported to exert positive and negative effects on cancer pathophysiology but an understanding of its role in breast cancer remains incomplete. Analysis of data from the Oncomine database showed a positive correlation between RhoB expression and positivity for both estrogen receptor alpha (ERα) and progesterone receptor (PR).

Methods

This finding was validated by our analysis of a tissue microarray constructed from a cohort of 113 patients and then investigated in human cell models.

Results

We found that RhoB expression in tissue was strongly correlated with ERα and PR expression and inversely correlated with tumor grade, tumor size and count of mitosis. In human breast cancer cell lines, RhoB attenuation was associated with reduced expression of both ERα and PR, whereas elevation of RhoB was found to be associated with ERα overexpression. Mechanistic investigations suggested that RhoB modulates ERα expression, controlling both its protein and mRNA levels, and that RhoB modulates PR expression by accentuating the recruitment of ERα and other major co-regulators to the promoter of PR gene. A major consequence of RhoB modulation was that RhoB differentially regulated the proliferation of breast cancer cell lines. Interestingly, we documented crosstalk between RhoB and ERα, with estrogen treatment leading to RhoB activation.

Conclusion

Taken together, our findings offer evidence that in human breast cancer RhoB acts as a positive function to promote expression of ERα and PR in a manner correlated with cell proliferation.     

The evolving role of oestrogen receptor β in clinical breast cancer

Controversy surrounds the potential clinical importance of oestrogen receptor (ER)β in breast cancer, and three recent papers have sought to resolve this. In the present issue of Breast Cancer Research Novelli and colleagues explored the significance of ERβ1 expression in 936 breast cancer patients, and they showed diverse relationships according to lymph node status. A second paper examined 442 breast cancers in which ERβ1 was an independent predictor of recurrence, disease-free survival and overall survival. Finally a third paper showed that ERβ2 was a powerful prognostic indicator in 757 breast cancers but this was dependent on cellular location, with nuclear ERβ2 expression predicting good survival whilst cytoplasmic expression predicted worse outcome. These papers point to a clinical role for ERβ in breast cancer and shall be discussed.     

17β-estradiol regulates giant vesicle formation via estrogen receptor-alpha in human breast cancer cells

A significant proportion of the genes regulated by 17-beta-estradiol (E2) via estrogen receptor alpha (ERα) have roles in vesicle trafficking in breast cancer. Intracellular vesicle trafficking and extracellular vesicles have important roles in tumourigenesis. Here we report the discovery of giant (3-42μm) intracellular and extracellular vesicles (GVs) and the role of E2 on vesicle formation in breast cancer (BC) cell lines using three independent live cell imaging techniques. Large diameter vesicles, GVs were also identified in a patient-derived xenograft BC model, and in invasive breast carcinoma tissue. ERα-positive (MCF-7 and T47D) BC cell lines demonstrated a significant increase in GV formation after stimulation with E2 which was reversed by tamoxifen. ERα-negative (MDA-MB-231 and MDA-MB-468) BC cell lines produced GVs independently of E2 and tamoxifen. These results indicate the existence of both intracellular and extracellular vesicles with considerably larger dimensions than generally recognised with BC cells and suggest that the GVs are regulated by E2 via ERα in ERα-positive BC but by E2-independent mechanisms in ER-ve BC.     

Upregulation of CYP1B1 by hypoxia is mediated by ERα activation in breast cancer cells

Endocrine therapy for breast cancer often leads to drug resistance and tumor recurrence; tumor hypoxia is also associated with mortality and tumor relapse. Cytochrome P450 1B1 (CYP1B1) regulates estrogen metabolism in breast cells and is known to be overexpressed in breast cancer tissue. Although the individual association of hypoxia-induced hypoxia-inducible factor-1-alpha (HIF-1α) and CYP1B1 with tumorigenesis is well known, the association between HIF-1α and CYP1B1 leading to tumorigenesis has not been investigated. Here, we investigated the correlation between hypoxia and CYP1B1 expression in breast cancer cells for tumorigenesis-related mechanisms. Hypoxia was induced in the human breast cancer cell lines MCF-7 (Er-positive) and MDA-MB-231 (triple-negative) and the normal breast epithelial cell line MCF10A; these cell lines were then subjected to immunoblotting, transient transfection, luciferase assays, gene silencing using small interfering RNA, polymerase chain reaction analysis, chromatin immunoprecipitation, co-immunoprecipitation, and mammalian two-hybrid assays. Furthermore, immunofluorescence analysis of the tumor microarrays was performed, and the pub2015 and the Cancer Genome Atlas patient datasets were analyzed. HIF-1α expression in response to hypoxia occurred in both normal and breast cancer cells, whereas CYP1B1 was induced only in estrogen receptor α (ERα)-positive breast cancer cells under hypoxia. HIF-1α activated ERα through direct binding and in a ligand-independent manner to promote CYP1B1 expression. Therefore, we suggested the mechanism by which hypoxia and ER-positivity orchestrate breast cancer relapse.     

Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer

The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERβ) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.