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.     

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.     

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.     

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.     

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.     

Nicastrin and Notch4 drive endocrine therapy resistance and epithelial to mesenchymal transition in MCF7 breast cancer cells

Introduction

Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ERα)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.

Methods

We used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen.

Results

We found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation.

Conclusions

ETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.     

ERβ1 represses basal-like breast cancer epithelial to mesenchymal transition by destabilizing EGFR

Introduction

Epithelial to mesenchymal transition (EMT) is associated with the basal-like breast cancer phenotypes. Sixty percent of basal-like cancers have been shown to express wild-type estrogen receptor beta (ERβ1). However, it is still unclear whether the ERβ expression is related to EMT, invasion and metastasis in breast cancer. In the present study, we examined whether ERβ1 through regulating EMT can influence invasion and metastasis in basal-like cancers.

Methods

Basal-like breast cancer cells (MDA-MB-231 and Hs578T), in which ERβ1 was either overexpressed or down-regulated were analyzed for their ability to migrate and invade (wound-healing assay, matrigel-coated Transwell assay) as well as for the expression of EMT markers and components of the EGFR pathway (immunoblotting, RT-PCR). Co-immunoprecipitation and ubiquitylation assays were employed to examine whether ERβ1 alters epidermal growth factor receptor (EGFR) protein degradation and the interaction between EGFR and the ubiquitin ligase c-Cbl. The metastatic potential of the ERβ1-expressing MDA-MB-231 cells was evaluated in vivo in a zebrafish xenotransplantation model and the correlation between ERβ1 and E-cadherin expression was examined in 208 clinical breast cancer specimens by immunohistochemistry.

Results

Here we show that ERβ1 inhibits EMT and invasion in basal-like breast cancer cells when they grow either in vitro or in vivo in zebrafish. The inhibition of EMT correlates with an ERβ1-mediated up-regulation of miR-200a/b/429 and the subsequent repression of ZEB1 and SIP1, which results in increased expression of E-cadherin. The positive correlation of ERβ1 and E-cadherin expression was additionally observed in breast tumor samples. Down-regulation of the basal marker EGFR through stabilization of the ubiquitin ligase c-Cbl complexes and subsequent ubiquitylation and degradation of the activated receptor is involved in the ERβ1-mediated repression of EMT and induction of EGFR signaling abolished the ability of ERβ1 to sustain the epithelial phenotype.

Conclusions

Taken together, the results of our study strengthen the association of ERβ1 with the regulation of EMT and propose the receptor as a potential crucial marker in predicting metastasis in breast cancer.     

The mechanistic target for rapamycin pathway is related to the phosphorylation score for estrogen receptor-α in human breast tumors in vivo

Introduction

A phosphorylation score for estrogen receptor-alpha (ERα), called P7 score, was shown previously to be an independent prognostic factor in breast cancer patients treated with tamoxifen. Since mechanistic target of rapamycin (mTOR) activation is implicated in resistance to endocrine therapy in breast cancer we determined whether mechanistic target of rapamycin complex 1 (mTORC1) activation, measured by phosphorylation on S2448 (p-mTOR), was associated with the P7-score and/or clinical outcome in the same cohort.

Methods

mTOR phosphorylation status was determined at S2448 residue in vivo by immunohistochemistry in a cohort of more than 400 well-characterized ERα positive breast tumors. MCF7 cells were treated with estrogen and activation of mTOR pathway was determined by Western blotting.

Results

Contrary to earlier reports, p-mTOR expression, measured by immunohistochemistry, was negatively associated with size and nodal status. Additionally, p-S2448 mTOR expression was positively correlated with p-S118- ERα, p-S167-ERα and p-S282-ERα but negatively correlated with p-T311- ERα. Consistent with these, p-S2448 mTOR was negatively associated with P7-score and was significantly associated with overall survival (OS) (hazard ratio (HR) = 0.61, P = 0.028, 95% confidence interval (CI) 0.39 to 0.95, n = 337) and relapse-free survival (HR = 0.58, P = 0.0032, 95% CI 0.41 to 0.83, n = 337) following univariate but not multivariate analysis. Furthermore, we show that estrogen can regulate phosphorylation of mTOR and its down stream target p70S6 kinase. Additionally, recombinant mTOR can phosphorylate ERα in vitro.

Conclusions

These data suggest that in breast tumors where there is intact estrogen regulated signaling, mTOR is regulated by estrogen and therefore associated with an increased likelihood of responsiveness to endocrine therapy.     

G-protein Coupled Estrogen Receptor 1 Expression in Primary Breast Cancers and Its Correlation with Clinicopathological Variables

Purpose

G-protein coupled estrogen receptor 1 (GPER) probably play important roles in the progression of breast cancer including endocrine therapeutic resistance. We evaluated GPER in primary breast cancers.

Methods

Immunohistochemistry was used to detect GPER in paraffin-embedded tissues of primary breast cancers from 423 patients and GPER expression was correlated with clinicopathological factors.

Results

GPER was expressed in 63.8% of specimens, coexpressed with estrogen receptor alpha (ERα) in 36.6% of tumors and was positive in 62.5% of the ERα-negative tumors. The expression of GPER had no relationship with the status of ERα, progesterone receptor and HER2. Although the expression of GPER was significantly inversely related with nodal status (p=0.045), no correlation between GPER expression and other clinicopathological variables (age, menstruation status, tumor size, stage, histologic grade, Nottingham Prognostic Index or pathological type) was found.

Conclusion

GPER and ERα exhibited independent expression pattern of distribution in primary breast cancers. A long-term follow-up and a more definite molecular phenotype for ER are necessary in confirming studies.     

Combination therapy with anti-DR5 antibody and tamoxifen for triple negative breast cancer

TRA-8, a monoclonal antibody targeting death receptor, has demonstrated high therapeutic effect for triple negative breast cancer (TNBC) in preclinical models. Tamoxifen, the standard of care for ERα-positive breast cancer, induces apoptosis via ERβ, which commonly presents in TNBC cells. The current study investigates the combination effects of TRA-8 and tamoxifen for TNBC. In vitro assays were implemented with two ERβ-positive TNBC cell lines, SUM159 and 2LMP, and in vivo therapy studies were followed using orthotopic breast tumor mouse models. IC₅₀ of tamoxifen for SUM159 and 2LMP were 29 μM and 38 μM, respectively. Synergy between TRA-8 (0–1000 ng/mL) and tamoxifen (20 μM) was observed for both the cell lines. Tamoxifen (400 mg/kg diet) markedly suppressed the growth of SUM159 tumors for 6 weeks after therapy initiation, but it did not induce antitumor effect for 2LMP tumors. TRA-8 (0.1 mg, weekly, i.p.) successfully arrested the growth of both SUM159 and 2LMP tumors during therapy, but an antagonistic effect was observed when tamoxifen was combined. TRA-8 uptake into tumors was not changed by tamoxifen treatment. Histological analysis confirmed that caspase-3 activation induced by TRA-8 was significantly decreased when tamoxifen was used in combination. In conclusion, our findings suggest that the combined use of TRA-8 and tamoxifen may cause antagonistic effects for TNBC patients.