ERα/LKB1 complex upregulates E-cadherin expression and stimulates breast cancer growth and progression upon adiponectin exposure

Adiponectin is the major adipocytes-secreted protein involved in obesity-related breast cancer growth and progression. We proved that adiponectin promotes proliferation in ERα-positive breast cancer cells, through ERα transactivation and the recruitment of LKB1 as ERα-coactivator. Here, we showed that adiponectin-mediated ERα transactivation enhances E-cadherin expression. Thus, we investigated the molecular mechanism through which ERα/LKB1 complex may modulate the expression of E-cadherin, influencing tumor growth, progression and distant metastasis. We demonstrated that adiponectin increases E-cadherin expression in ERα-positive 2D and higher extent in 3D cultures. This occurs through a direct activation of E-cadherin gene promoter by ERα/LKB1-complex. The impact of E-cadherin on ERα-positive breast cancer cell proliferation comes from the evidence that in the presence of E-cadherin siRNA the proliferative effects of adiponectin is no longer noticeable. Since E-cadherin connects cell polarity and growth, we investigated if the adiponectin-enhanced E-cadherin expression could influence the localization of proteins cooperating in cell polarity, such as LKB1 and Cdc42. Surprisingly, immunofluorescence showed that, in adiponectin-treated MCF-7 cells, LKB1 and Cdc42 mostly colocalize in the nucleus, impairing their cytosolic cooperation in maintaining cell polarity. The orthotopic implantation of MCF-7 cells revealed an enhanced E-cadherin-mediated breast cancer growth induced by adiponectin. Moreover, tail vein injection of MCF-7 cells showed a higher metastatic burden in the lungs of mice receiving adiponectin-treated cells compared to control. From these findings it emerges that adiponectin treatment enhances E-cadherin expression, alters cell polarity and stimulates ERα-positive breast cancer cell growth in vitro and in vivo, sustaining higher distant metastatic burden.     

Expression of the tumor suppressor miR-206 is associated with cellular proliferative inhibition and impairs invasion in ERα-positive endometrioid adenocarcinoma

This study investigated the role of miR-206 in estrogen receptor-α (ERα)-positive endometrial endometrioid adenocarcinoma (EEC). We profiled miR-206 expression in 30 EEC clinical samples using qRT-PCR, and explored its relationship with ERα and clinical parameters. A luciferase reporter assay assessed the ERα targeting potential of miR-206. Functional analyses of miR-206 were performed in EEC cell lines. MiRNA-206 expression decreased in ERα-positive EECs, and its expression was negatively correlated with ERα. MiRNA-206 overexpression inhibited ERα-dependent proliferation, impaired invasiveness and induced cell cycle arrest of ERα-positive EEC cell lines. Therefore, aberrantly expressed miRNA-206 may be associated with the development of ERα-positive EEC.     

Stabilization of estrogen receptor α by USP37 contributes to the progression of breast cancer

Breast cancer is a major cause of cancer-related morbidity and mortality in women. Estrogen receptor-positive breast cancer accounts for roughly 70%-80% of breast tumors, and estrogen receptor alpha (ERα) has been considered as a key driver in promoting breast cancer progression. In the present study, we identified USP37 as a novel modulator in modulating ERα ubiquitination and stability. The expression of USP37 was upregulated in ERα-positive breast cancer and correlated with ERα protein level. High expression of USP37 was associated with unfavorable prognosis. USP37 depletion resulted in significantly decreased ERα protein level, ERα target genes expression as well as the estrogen response element activity in breast cancer cells. Further mechanistic study revealed the interaction between USP37 and ERα: USP37 regulated ERα signaling through modulating protein stability instead of gene expression, in which it stabilized ERα protein via inhibiting the K48-specific polyubiquitination process. Additionally, USP37 depletion led to growth inhibition and cell cycle arrest of ERα-positive breast cancer cells, which could be further rescued by ERα overexpression. Overall, our study proposed a novel post-translational mechanism of ERα in promoting breast cancer progression. Targeting USP37 may be proved to be a promising strategy for patients with ERα-positive breast cancer.     

MicroRNA ‘signature’ during estrogen-mediated mammary carcinogenesis and its reversal by ellagic acid intervention

Dysregulated miRNA expression has been associated with the development and progression of cancers, including breast cancer. The role of estrogen (E₂) in regulation of cell proliferation and breast carcinogenesis is well-known. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Investigation of the regulatory role of miRNAs is critical for understanding the effect of E₂ in human breast cancer, as well as developing strategies for cancer chemoprevention. In the present study we used the well-established ACI rat model that develops mammary tumors upon E₂ exposure and identified a ‘signature’ of 33 significantly modulated miRNAs during the process of mammary tumorigenesis. Several of these miRNAs were altered as early as 3 weeks after initial E₂ treatment and their modulation persisted throughout the mammary carcinogenesis process, suggesting that these molecular changes are early events. Furthermore, ellagic acid, which inhibited E₂-induced mammary tumorigenesis in our previous study, reversed the dysregulation of miR-375, miR-206, miR-182, miR-122, miR-127 and miR-183 detected with E₂ treatment and modulated their target proteins (ERα, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w and Bcl-2). This is the first systematic study examining the changes in miRNA expression associated with E₂ treatment in ACI rats as early as 3 week until tumor time point. The effect of a chemopreventive agent, ellagic acid in reversing miRNAs modulated during E₂-mediated mammary tumorigenesis is also established. These observations provide mechanistic insights into the new molecular events behind the chemopreventive action of ellagic acid and treatment of breast cancer.     

MicroRNAs Link Estrogen Receptor Alpha Status and Dicer Levels in Breast Cancer

To identify microRNAs (miRNAs) associated with estrogen receptor (ESR1) status, we profiled luminal A, ESR1+ breast cancer cell lines versus triple negative (TN), which lack ERα, progesterone receptor and Her2/neu. Although two thirds of the differentially expressed miRNAs are higher in ESR1+ breast cancer cells, some miRNAs, such as miR-222/221 and miR-29a, are dramatically higher in ESR1- cells (～100- and 16-fold higher, respectively). MiR-222/221 (which target ESR1 itself) and miR-29a are predicted to target the 3' UTR of Dicer1. Addition of these miRNAs to ESR1+ cells reduces Dicer protein, whereas antagonizing miR-222 in ESR1- cells increases Dicer protein. We demonstrate via luciferase reporter assays that these miRNAs directly target the Dicer1 3' UTR. In contrast, miR-200c, which promotes an epithelial phenotype, is 58-fold higher in the more well-differentiated ERα+ cells, and restoration of miR-200c to ERα- cells causes increased Dicer protein, resulting in increased levels of other mature miRNAs typically low in ESR1- cells. Together, our findings explain why Dicer is low in ERα negative breast cancers, since such cells express high miR-221/222 and miR-29a levels (which repress Dicer) and low miR-200c (which positively affect Dicer levels). Furthermore, we find that miR-7, which is more abundant in ERα+ cells and is estrogen regulated, targets growth factor receptors and signaling intermediates such as EGFR, IGF1R, and IRS-2. In summary, miRNAs differentially expressed in ERα+ versus ERα- breast cancers actively control some of the most distinguishing characteristics of the luminal A and TN subtypes, such as ERα itself, Dicer, and growth factor receptor levels.     

A novel taspine derivative, HMQ1611, inhibits breast cancer cell growth via estrogen receptor α and EGF receptor signaling pathways

Breast cancer is a common cancer with a leading cause of cancer mortality in women. Currently, the chemotherapy for breast cancer is underdeveloped. Here, we report a novel taspine derivative, HMQ1611, which has anticancer effects using in vitro and in vivo breast cancer models. HMQ1611 reduced cancer cell proliferation in four human breast cancer cell lines including MDA-MB-231, SK-BR-3, ZR-75-30, and MCF-7. HMQ1611 more potently reduced growth of estrogen receptor α (ERα)-positive breast cancer cells (ZR-75-30 and MCF-7) than ERα-negative cells (MDA-MB-231 and SK-BR-3). Moreover, HMQ1611 arrested breast cancer cell cycle at S-phase. In vivo tumor xenograft model, treatment of HMQ1611 significantly reduced tumor size and weight compared with vehicles. We also found that HMQ1611 reduced ERα expression and inhibited membrane ERα-mediated mitogen-activated protein kinase (MAPK) signaling following the stimulation of cells with estrogen. Knockdown of ERα by siRNA transfection in ZR-75-30 cells attenuated HMQ1611 effects. In contrast, overexpression of ERα in MDA-MB-231 cells enhanced HMQ1611 effects, suggesting that ERα pathway mediated HMQ1611's inhibition of breast cancer cell growth in ERα-positive breast cancer. HMQ1611 also reduced phosphorylation of EGF receptor (EGFR) and its downstream signaling players extracellular signal-regulated kinase (ERK)1/2 and AKT activation both in ZR-75-30 and MDA-MB-231 cells. These results showed that the novel compound HMQ1611 had anticancer effects, and partially via ERα and/or EGFR signaling pathways, suggesting that HMQ1611 may be a potential novel candidate for human breast cancer intervention.     

Estrogen receptor alpha mediates progestin-induced mammary tumor growth by interacting with progesterone receptors at the cyclin D1/MYC promoters

Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.     

A novel prenylflavone restricts breast cancer cell growth through AhR-mediated destabilization of ERα protein

There is concern that ingestion of dietary phytoestrogens may increase risk of estrogen receptor alpha (ERα)-positive breast cancer. The prenylflavone icaritin, a phytoestrogen consumed in East Asian societies for its perceived beneficial effects on bone health, stimulated the growth of breast cancer (MCF-7) cells at low concentrations. Although acting like an estrogenic ligand, icaritin exerted an unexpected suppressive effect on estrogen-stimulated breast cancer cell proliferation and gene expression at higher concentrations. Like estradiol, icaritin could dose-dependently destabilize ERα protein. However, destabilization of ERα by the estradiol/icaritin combination was profound and greater than that observed for either compound alone. Microarray gene expression analyses implicated aryl hydrocarbon receptor (AhR) signaling for this suppressive effect of icaritin. Indeed, icaritin was an AhR agonist that competitively reduced specific binding of a potent AhR agonist and increased expression of the AhR-regulated gene CYP1A1. When AhR was knocked down by small interfering RNA, the suppressive effect of icaritin on estradiol-stimulated breast cancer cell growth and gene expression was abolished, and ERα protein stability was partially restored. Similarly in an athymic nude mouse model, icaritin restricted estradiol-stimulated breast cancer xenograft growth and strongly reduced ERα protein levels. Overall, our data support the feasibility for the development of dual agonists like icaritin, which are estrogenic but yet, through activating AhR-signaling, can destabilize ERα protein to restrict ERα-positive breast cancer cell growth.     

Raptor localization predicts prognosis and tamoxifen response in estrogen receptor-positive breast cancer

Purpose

Deregulated PI3K/mTOR signals can promote the growth of breast cancer and contribute to endocrine treatment resistance. This report aims to investigate raptor and its intracellular localization to further understand its role in ER-positive breast cancer.

Methods

Raptor protein expression was evaluated by immunohistochemistry in 756 primary breast tumors from postmenopausal patients randomized to tamoxifen or no tamoxifen. In vitro, the MCF7 breast cancer cell line and tamoxifen-resistant MCF7 cells were studied to track the raptor signaling changes upon resistance, and raptor localization in ERα-positive cell lines was compared with that in ERα-negative cell lines.

Results

Raptor protein expression in the nucleus was high in ER/PgR-positive and HER2-negative tumors with low grade, features associated with the luminal A subtype. Presence of raptor in the nucleus was connected with ERα signaling, here shown by a coupled increase of ERα phosphorylation at S167 and S305 with accumulation of nuclear raptor. In addition, the expression of ERα-activated gene products correlated with nuclear raptor. Similarly, in vitro we observed raptor in the nucleus of ERα-positive, but not of ER-negative cells. Interestingly, raptor localized to the nucleus could still be seen in tamoxifen-resistant MCF7 cells. The clinical benefit from tamoxifen was inversely associated with an increase of nuclear raptor. High cytoplasmic raptor expression indicated worse prognosis on long-term follow-up.

Conclusion

We present a connection between raptor localization to the nucleus and ERα-positive breast cancer, suggesting raptor as a player in stimulating the growth of the luminal A subtype and a possible target along with endocrine treatment.

     

Arsenic trioxide re-sensitizes ERα-negative breast cancer cells to endocrine therapy by restoring ERα expression in vitro and in vivo

Approximately one-third of breast cancers lack estrogen receptor α (ERα) because of the hypermethylation of the CpG island in the receptor's promoter. These tumors are associated with poorer histological differentiation, a higher growth fraction, are rarely responsive to endocrine therapy and have a worse clinical outcome. Thus, re-expression of ERα in ERα-negative breast cancers may restore the sensitivity of antiestrogen therapy. The ERα-negative breast cancer cell line MDA-MB-435s was treated with different concentrations of arsenic trioxide (As2O3). MS-PCR was used to detect the change in the methylation status of ERα. RT-PCR, immunohistochemistry and Western blot analyses were used to detect changes in the mRNA and protein expression of DNA methyl-transferase-1 (DNMT1) and ERα. Cell proliferation was examined using the MTT assay. A xenograft model in nude mice was used to further examine the results we observed in vitro. The ERα gene was demethylated after As2O3 treatment of MDA-MB-435s cells. RT-PCR, immunohistochemistry and Western blot analyses revealed that DNMT1 expression was inhibited and ERα was re-expressed in a concentration-dependent manner after As2O3 treatment. The MTT assay showed that cell proliferation was significantly suppressed after exposure to different concentrations of As2O3. Addition of tamoxifen (TAM) further suppressed levels of cell proliferation. In vivo, the xenograft tumor volumes of As2O3-treated mice were smaller than those observed in untreated and TAM-treated mice. Treatment with a combination of As2O3+TAM resulted in further suppression. As2O3 can act as a demethylation agent to restore ERα expression in ERα-negative breast cancer cells and re-sensitize these cells to endocrine therapy in vitro and in vivo.     

Luteolin inhibits proliferation induced by IGF-1 pathway dependent ERα in human breast cancer MCF-7 cells

The growth of many breast tumors is stimulated by IGF-1, which activates signal transduction pathways inducing cell proliferation. ERα is important in this process. The aim of the study was to investigate relationships in vitro among inhibitory effects of luteolin on the growth of MCF-7 cells, IGF-1 pathway and ERα. Our results showed that luteolin could effectively block IGF-1-stimulated MCF-7 cell proliferation in a dose- and time- dependent manner and block cell cycle progression and induce apoptosis evidenced by the flow cytometric detection of sub-G1DNA content. Luteolin markedly decreased IGF-1-dependent IGF-1R and Akt phosphorylation without affecting Erk1/2 phosphorylation. Further experiments pointed out that ERα was directly involved in IGF-1 induced cell growth inhibitory effects of luteolin, which significantly decreased ERα expression. Knockdown of ERα in MCF-7 cells by an ERα-specific siRNA decreased the IGF-1 induced cell growth inhibitory effects of luteolin. ERα is thus a possible target of luteolin. These findings indicate that the inhibitory effect of luteolin on the growth of MCF-7 cells is via inhibiting IGF-1 mediated PI3K-Akt pathway dependent on ERα expression.     

Estrogen receptor splice variants as a potential source of false-positive estrogen receptor status in breast cancer diagnostics

It is well established that only estrogen receptor (ER)-positive tumors benefit from hormonal therapies. We hypothesized that a subgroup of breast cancer patients expresses estrogen receptor α (ERα), but fails to respond to hormonal therapy due to the expression of a non-functional receptor. We analyzed a series of 2,658 ERα-positive HER2-negative breast tumors for ERα and progesterone receptor (PR) status as determined by mRNA expression and for their molecular subtypes (Luminal type vs Basal type, assessed by BluePrint? molecular subtyping assay). In addition, we assessed the recurrence risk (low vs high) using the 70-gene MammaPrint? signature. We found that 55 out of 2,658 (2.1 %) tumors that are ERα positive by mRNA analysis also demonstrate a Basal molecular subtype, indicating that they lack expression of estrogen-responsive genes. These ERα-positive Basal-type tumors express significantly lower levels of both ERα and PR mRNA as compared to Luminal-type tumors (P < 0.0001) and almost invariably (94.5 %) have a high-risk MammaPrint? profile. Twelve of the MammaPrint? genes are directly ERα responsive, indicating that MammaPrint? assesses ERα function in breast cancer without considering ERα mRNA levels. We find a relatively high expression of the dominant negative ERα splice variant ERΔ7 in ERα-positive Basal-type tumors as compared to ERα-positive Luminal-type tumors (P < 0.0001). Expression of the dominant negative ERα variant ERΔ7 provides a rationale as to why tumors are of the Basal molecular subtype while staining ERα positive by immunohistochemistry. These tumors may lack a functional response to estrogen and consequently may not respond to hormonal therapy. Our data indicate that such patients are of MammaPrint? high recurrence risk and might benefit from adjuvant chemotherapy.