Estrogenic Activity of Mycoestrogen (3β,5α,22E)-Ergost-22-en-3-ol via Estrogen Receptor α-Dependent Signaling Pathways in MCF-7 Cells

Armillariella tabescens (Scop.) Sing., a mushroom of the family Tricholomataceae, has been used in traditional oriental medicine to treat cholecystitis, improve bile secretion, and regulate bile-duct pressure. The present study evaluated the estrogen-like effects of A. tabescens using a cell-proliferation assay in an estrogen-receptor-positive breast cancer cell line (MCF-7). We found that the methanol extract of A. tabescens fruiting bodies promoted cell proliferation in MCF-7 cells. Using bioassay-guided fractionation of the methanol extract and chemical investigation, we isolated and identified four steroids and four fatty acids from the active fraction. All eight compounds were evaluated by E-screen assay for their estrogen-like effects in MCF-7 cells. Among the tested isolates, only (3β,5α,22E)-ergost-22-en-3-ol promoted cell proliferation in MCF-7 cells; this effect was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of (3β,5α,22E)-ergost-22-en-3-ol was evaluated using Western blot analysis to detect the expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), Akt, and estrogen receptor α (ERα). We found that (3β,5α,22E)-ergost-22-en-3-ol induced an increase in phosphorylation of ERK, PI3K, Akt, and ERα. Together, these experimental results suggest that (3β,5α,22E)-ergost-22-en-3-ol is responsible for the estrogen-like effects of A. tabescens and may potentially aid control of estrogenic activity in menopause.

PGRMC1 Promotes Progestin-Dependent Proliferation of Breast Cancer Cells by Binding Prohibitins Resulting in Activation of ERα Signaling

In previous studies, we reported that progesterone receptor membrane component 1 (PGRMC1) is implicated in progestin signaling and possibly associated with increased breast cancer risk upon combined hormone replacement therapy. To gain mechanistic insight, we searched for potential PGRMC1 interaction partners upon progestin treatment by co-immunoprecipitation and mass spectrometry. The interactions with the identified partners were further characterized with respect to PGRMC1 phosphorylation status and with emphasis on the crosstalk between PGRMC1 and estrogen receptor α (ERα). We report that PGRMC1 overexpression resulted in increased proliferation of hormone receptor positive breast cancer cell lines upon treatment with a subgroup of progestins including norethisterone and dydrogesterone that promote PGRMC1-phosphorylation on S181. The ERα modulators prohibitin-1 (PHB1) and prohibitin-2 (PHB2) interact with PGRMC1 in dependency on S181-phosphorylation upon treatment with the same progestins. Moreover, increased interaction between PGRMC1 and PHBs correlated with decreased binding of PHBs to ERα and subsequent ERα activation. Inhibition of either PGRMC1 or ERα abolished this effect. In summary, we provide strong evidence that activated PGRMC1 associates with PHBs, competitively removing them from ERα, which then can develop its transcriptional activities on target genes. This study emphasizes the role of PGRMC1 in a key breast cancer signaling pathway which may provide a new avenue to target hormone-dependent breast cancer.

Schisandrol A Exhibits Estrogenic Activity via Estrogen Receptor α-Dependent Signaling Pathway in Estrogen Receptor-Positive Breast Cancer Cells

The aim of this study was to examine the estrogen-like effects of gentiopicroside, macelignan, γ-mangostin, and three lignans (schisandrol A, schisandrol B, and schisandrin C), and their possible mechanism of action. Their effects on the proliferation of the estrogen receptor (ER)-positive breast cancer cell line (MCF-7) were evaluated using Ez-Cytox reagents. The expression of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), AKT, and estrogen receptor α (ERα) was measured by performing Western blot analysis. 17β-estradiol (E2), also known as estradiol, is an estrogen steroid and was used as a positive control. ICI 182,780 (ICI), an ER antagonist, was used to block the ER function. Our results showed that, except for gentiopicroside, all the compounds promoted proliferation of MCF-7 cells, with schisandrol A being the most effective; this effect was better than that of E2 and was mitigated by ICI. Consistently, the expression of ERK, PI3K, AKT, and ERα increased following treatment with schisandrol A; this effect was slightly better than that of E2 and was mitigated by ICI. Taken together, the ERα induction via the PI3K/AKT and ERK signaling pathways may be a potential mechanism underlying the estrogen-like effects of schisandrol A. This study provides an experimental basis for the application of schisandrol A as a phytoestrogen for the prevention of menopausal symptoms.

The Novel Oral mTORC1/2 Inhibitor TAK-228 Reverses Trastuzumab Resistance in HER2-Positive Breast Cancer Models

The use of anti-HER2 therapies has significantly improved clinical outcome in patients with HER2-positive breast cancer, yet a substantial proportion of patients acquire resistance after a period of treatment. The PI3K/AKT/mTOR pathway is a good target for drug development, due to its involvement in HER2-mediated signalling and in the emergence of resistance to anti-HER2 therapies, such as trastuzumab. This study evaluates the activity of three different PI3K/AKT/mTOR inhibitors, i.e., BEZ235, everolimus and TAK-228 in vitro, in a panel of HER2-positive breast cancer cell lines with primary and acquired resistance to trastuzumab. We assess the antiproliferative effect and PI3K/AKT/mTOR inhibitory capability of BEZ235, everolimus and TAK-228 alone, and in combination with trastuzumab. Dual blockade with trastuzumab and TAK-228 was superior in reversing the acquired resistance in all the cell lines. Subsequently, we analyse the effects of TAK-228 in combination with trastuzumab on the cell cycle and found a significant increase in G0/G1 arrest in most cell lines. Likewise, the combination of both drugs induced a significant increase in apoptosis. Collectively, these experiments support the combination of trastuzumab with PI3K/AKT/mTOR inhibitors as a potential strategy for inhibiting the proliferation of HER2-positive breast cancer cell lines that show resistance to trastuzumab.

Sequential usage of imidazole followed by tamoxifen to enhance the anticancer effect of tamoxifen in estrogen receptor-positive breast cancer cell lines.

e15057 Background: In women, breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death worldwide. Tamoxifen is the most commonly used drug for the endocrine treatment of breast cancer. It reduces the risk of recurrence and death from breast cancer when given to estrogen-receptor-positive breast cancer patients. It has recently been shown that imidazoles, an antifungal drug, possess anticancer potentials, and it can be a novel therapeutic in cancer treatment. However, the effects of combined treatment with imidazole and tamoxifen are unknown in estrogen receptor-positive breast cancer cell lines. Our study aimed to investigate the effects of imidazole and tamoxifen combination in estrogen receptor-positive breast cancer cell lines. Methods: MCF7 cell line, an estrogen receptor-positive breast cancer cell line, was used in this study. Following 24 hours 50 mM imidazole (molecular grade) treatment, 15µM tamoxifen was treated to MCF-7 cells by 72 hours. As control groups, following for 24-hour imidazole alone treated, only medium treated cells for 72 hours were used. MTT assay was performed for the determination of cell viability. Apoptosis was shown using acridine orange/ethidium bromide staining. The cellular morphological alterations were observed on bright-field microscopy using Giemsa staining. Cell migration status was determined using by in vitro scratch assay. Results: MTT assay results showed that tamoxifen alone treatment for 72 hours decreased cell viability by 18 percent (p < 0.001). On the other hand, cell viability is not affected by imidazole alone treatment for 24 hours compared with the control group (p > 0.05). However, it was calculated that 24 hours of imidazole followed by tamoxifen for 72 hours decreased cell viability up to 42 percent (p < 0.001). Tamoxifen alone group, compared with combined treatment with tamoxifen and imidazole, observed an increase of apoptotic cell numbers in combined treatment with tamoxifen and imidazole group, statistically significantly (p < 0.01). It was observed that cellular morphology was affected by combined treatment with tamoxifen and imidazole. Giemsa staining results showed that MCF 7 cells changed their cellular morphology, lost cell-cell contact, differentiated from parental morphology and cellular morphology, and appeared unhealthy. Parallel to these findings, a decrease in cell migration was observed in the combined-treated group compared to the tamoxifen alone group (p < 0.01). Conclusions: Our results showed that sequential usage of imidazole followed by tamoxifen has an enhanced anticancer effect of tamoxifen in estrogen receptor-positive breast cancer cell lines. These results allow us to establish a new hormonal treatment for patients with breast cancer.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer

Background Around 15–20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. Methods RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. Results RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. Conclusions Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.