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Estradiol (E2) increases not only the cell growth but also the cancer stem cell (CSC) proportion in estrogen receptor (ER)-positive breast cancer cells. It has been suggested that the non-canonical hedgehog (Hh) pathway activated by E2 plays an important role in the regulation of CSC proportion in ER-positive breast cancer cells. We studied anti-CSC activity of a non-canonical Hh inhibitor GANT61 in ER-positive breast cancer cells. Effects of GANT61 on the cell growth, cell cycle progression, apoptosis and CSC proportion were investigated in four ER-positive breast cancer cell lines. CSC proportion was measured using either the mammosphere assay or CD44/CD24 assay. Expression levels of pivotal molecules in the Hh pathway were measured. Combined effects of GANT61 with antiestrogens on the anti-cell growth and anti-CSC activities were investigated. E2 significantly increased the cell growth and CSC proportion in all ER-positive cell lines. E2 increased the expression levels of glioma-associated oncogene (GLI) 1 and/or GLI2. GANT61 decreased the cell growth in association with a G1-S cell cycle retardation and increased apoptosis. GANT61 decreased the E2-induced CSC proportion measured by the mammosphere assay in all cell lines. Antiestrogens also decreased the E2-induced cell growth and CSC proportion. Combined treatments of GANT61 with antiestrogens additively enhanced anti-cell growth and/or anti-CSC activities in some ER-positive cell lines. In conclusion, the non-canonical Hh inhibitor GANT61 inhibited not only the cell growth but also the CSC proportion increased by E2 in ER-positive breast cancer cells. GANT61 enhanced anti-cell growth and/or anti-CSC activities of antiestrogens in ER-positive cell lines., (© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Triple negative (TN) breast cancer is defined as a subtype that is negative for estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). To clarify the characteristics of TN breast cancer, surveillance data of the Registration Committee of the Japanese Breast Cancer Society were analyzed., Method: Of 14,748 cases registered in 2004, 11,705 (79.4%) were examined for ER, PgR, and HER2. Of these, the most prevalent (53.8%) was a hormone-responsive subtype with ER positive/PgR positive/HER2 negative, followed by TN subtype (15.5%)., Results: The proportion of postmenopausal patients was relatively high in the TN subtype. This cancer was diagnosed at a slightly advanced stage and with more cases positive for lymph node metastases than other subtypes. Morphologically, the TN subtype was more frequently classified as solid-tubular carcinoma. Mucinous, tubular, or secretary carcinomas were frequently found in the hormone receptor positive/HER2 negative subtype, while squamous cell carcinoma, spindle cell carcinoma, and metaplastic carcinoma with bone/cartilage metaplasia were very frequently found in the TN group. Apocrine carcinoma was also found very frequently in the TN group. Selection of chemotherapy was not based on receptor subtypes, but was determined by the degree of tumor progression., Conclusions: Although TN types are similar to basal-like breast tumor, as determined by gene profiling, their diagnosis needs verification by determination of the level of epidermal growth factor receptor or cytokeratin 5/6 expression. TN type should be examined further for immunohistochemical features and analyzed for prognostic details in this cohort.

Taxanes are approved for the treatment of breast cancer that has spread to the lymph nodes, following surgery and doxorubicin containing chemotherapy. Taxanes have improved the survival of breast cancer patients, especially in estrogen receptor (ER) negative population in clinical settings. This time we examined the relationship between chemosensitivity to Taxanes and expresson of ERalpha in breast cancer cell lines. In vitro effects of paclitaxel in 4 ER-positive and 3 ER-negative breast cancer cell lines were investigated by MTT assay. We also investigated members of Bcl-2 family by Western blotting and RT-PCR to clarify their role in paclitaxel resistance both in ER-positive and in ER-negative cells. ER-negative cell lines were more sensitive to paclitaxel than ER-positive cells. ER-negative KPL-4 and ZR-75-30 cells, which were sensitive to paclitaxel, became resistant when they were treated with demethylation agent, 5-aza-2'-deoxycytidine. Analysis of proapoptotic (Bax) and antiapoptotic (Bcl-2) molecules suggested that Bcl-2 is likely to have a role in the resistance of ER-positive cells. Bcl-2 expression was increased in a time-dependent manner after treatment of ER-positive cell lines with estrogen (E2). On the other hand, Bcl-2 was not detected in ER-negative cell lines. However, no significant difference was detected for Bax mRNA levels before and after E2 treatment in ER-positive and negative cell lines. Activation of ER gene expression in ER-negative KPL-4 cells by 5-aza-2'-deoxycytidine resulted in up-regulation of Bcl-2 mRNA. To support our data, we examined paclitaxel sensitivity in ER-negative MDA-MB-231 and ER stable transfectant cells S30 and JM6. This experiment also showed ER-negative cells were sensitive to paclitaxel but ER-positive cells were resistant to it. These results suggest that ER influenced chemosensitivity to paclitaxel through regulation of Bcl-2 family and regulation of the pathway may be crucial to increase the efficacy of taxanes in ER-positive breast cancer.

The intrinsic subtype has demonstrated that breast cancers can be classified into biologically and clinically meaningful subgroups. Most breast tumors categorized as one of the intrinsic subtypes, i.e., basal-like, have an estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative phenotype, so-called triple-negative (TN) phenotype; however, TN breast cancer is not a synonym for basal-like subtype. TN breast cancers account for 10-20% of all breast cancers, and are more biologically aggressive than breast cancers of other subgroups. Tailored therapies, such as endocrine therapy and anti-HER2 therapy, are not applicable to TN breast cancer. To develop novel strategies against TN breast cancer, it is essential to understand the specific pathways driving the aggressive behavior of TN breast cancer. Preclinical and clinical studies have suggested that DNA-damaging agents and poly ADP-ribose polymerase inhibitors are active in TN breast cancer harboring BRCA1 dysfunction; anti-epidermal growth factor receptor (EGFR) antibodies and EGFR tyrosine kinase inhibitors are active in TN breast cancer with EGFR gene amplification; dasatinib is active in TN breast cancer with activated Src tyrosine kinases; inhibitors of a mammalian target of rapamycin are active in TN breast cancer with loss of PTEN tumor suppressor; antiangiogenic therapies enhance antitumor activity of chemotherapeutic agents in hypervascular TN breast cancer; and irinotecan, trabectedin, ixabepilone, and ABI-007 are active in TN breast cancer. A number of clinical trials are ongoing to clarify the antitumor activity of these challenging treatment strategies. Further biological characterization of TN breast cancer is needed to develop more specific treatment strategies against TN breast cancer.

Purpose: The sequential addition of tamoxifen (TAM) to chemotherapy seems superior to its concurrent addition in patients with breast cancer. This study was conducted to clarify the hypothesis that there are differential interactions among TAM and chemotherapeutic agents., Methods: Estrogen receptor (ER)-alpha-positive or -negative breast cancer cells were treated with 4-hydroxy TAM (4OHT), 5-fluorouracil (FU) and/or doxorubicin (Dox). Changes in the expression levels of genes related to sensitivity and resistance to TAM, 5-FU or Dox were tested., Results: Concurrent treatment of 4OHT with 5-FU but not with Dox additively inhibited the growth of ER-alpha-positive cells. 5-FU did not change the expression levels of any tested genes related to either sensitivity or resistance to TAM. Although Dox did not change the expression levels of any genes related to the sensitivity to TAM, Dox significantly increased the expression levels of some genes related to TAM resistance, Eph A-2, ER-beta, Fos and vascular endothelial growth factor. 4OHT significantly decreased thymidilate synthase (TS) activity., Conclusions: Although the antitumor effect of concurrent 4OHT and 5-FU was additive, that of concurrent 4OHT and Dox was less than additive in ER-alpha-positive cells. The increased expression of genes related to TAM resistance by Dox might be responsible for the interaction. Decreased TS activity by 4OHT might increase the antitumor activity of 5-FU. These findings may provide a preclinical rationale for concurrent use with 5-FU and TAM.

Estrogen plays important roles in the development and progression of breast cancer. However, one-third of breast cancers fail to respond to endocrine therapy and most endocrine-responsive breast cancers subsequently become resistant to endocrine therapy. A tremendous effort has been made to elucidate the mechanisms responsible for the development of endocrine-resistance in breast cancer. Since the main target molecule of estrogen in breast cancer is estrogen receptor (ER)-alpha, most studies have focused on investigating quantitative and qualitative changes in ER-alpha in endocrine-resistant breast cancer. Breast cancers expressing no ER-alpha fail to respond to endocrine therapy. Some breast cancers expressing ER-alpha also fail to respond to endocrine therapy and most breast cancers with acquired endocrine resistance retain ER-alpha expression, which suggests that the disappearance of ER-alpha in breast cancer cells is not a common cause of resistance to endocrine therapy. Recent molecular biological studies have shown evidence that qualitative and functional changes, such as gene mutations and phosphorylation of ER-alpha, cause endocrine resistance in breast cancer. In addition, it has been suggested that endocrine resistance could be induced by epigenetic changes, such as hypoxia, in breast cancer tissues. Understanding the precise mechanisms that underlie endocrine resistance may enable clinicians to develop new strategies for retarding or overcoming endocrine resistance in breast cancer.

It has recently been reported that the human myeloma cell line U266 proceeds to undergo apoptosis after cultivation with the antiestrogen tamoxifen, thus raising the possibility that antiestrogens may be candidates for use in myeloma therapy. To obtain basic information on the effects of antiestrogens on myeloma cells, we investigated the mRNA expression levels of estrogen receptor (ER)-alpha, ER-beta, and coactivators and corepressors in nine human myeloma cell lines and compared them with those of seven human breast cancer cell lines including four ER-positive and three ER-negative lines. The alterations in cell growth and mRNA expression of the target genes of ER or those of cytokines in the myeloma lines by estradiol or antiestrogens (tamoxifen and toremifene) were also investigated. In addition, effects on membrane Fas expression, appearance of apoptosis, and cell cycle perturbation were analyzed. It was revealed that ER-beta and corepressors were dominantly expressed in myeloma cells, and antiestrogens induced growth inhibition through apoptosis mediated by a Fas-related pathway and G1 arrest of the cell cycle in myeloma cell lines.

Recent studies have indicated that a complex machinery of transactivation of target genes by estrogen or antiestrogen through estrogen receptor (ER) exists. However, the substantial roles of ER-beta, coactivators, and corepressors in the development and progression of breast cancer remain to be elucidated. To obtain some clue to these roles, we screened the expression levels of ER-alpha, ER-beta, coactivators (SRC-1, TIF2, AIB1, CBP, and P/CAF) and corepressors (N-CoR and SMRT) in 6 normal mammary glands, 6 intraductal carcinomas, 22 invasive ductal carcinomas, and 7 breast cancer cell lines using a multiplex reverse transcription-PCR. ER-alpha mRNA expression levels significantly correlated with ER-alpha protein levels measured by enzyme immunoassay in the breast cancer tissues and cell lines. A significant correlation of expression levels was observed between ER-alpha and TIF2, AIB1, P/CAF, and N-CoR, and between ER-beta and AIB1 and CBP in the tissue samples. A significant correlation was also observed between ER-alpha and ER-beta and between ER-beta and CBP in the cell lines. The expression levels of ER-alpha, TIF2, and CBP were significantly higher in the intraductal carcinomas than those in the normal mammary glands. In addition, the expression levels of ER-alpha and N-CoR were significantly higher in the intraductal carcinomas than those in the invasive ductal carcinomas. These findings suggest a positive correlation of expression levels among ER-alpha and cofactors and among ER-beta and cofactors, an up-regulation of expression levels of ER-alpha and cofactors during the development of intraductal carcinomas from normal mammary glands, and a decrease in their expression levels during the progression of breast cancer.

Anti-oestrogen is effective for the treatment of oestrogen receptor (ER)-positive breast carcinomas, but most of these tumours become resistant to anti-oestrogen. It has been suggested that anti-oestrogen therapy may induce a HER2 signalling pathway in breast cancer cells and this may cause resistance to anti-oestrogen. Thus, it is conceivable that combined therapy with anti-oestrogen and anti-HER2 antibody might be more effective. In the present study, we investigated the effect of combined treatment with a humanized anti-HER2 monoclonal antibody, rhumAbHER2 (trastuzumab), and an anti-oestrogen, ICI 182,780, on the cell growth of three human breast cancer cell lines which respectively express different levels of ER and HER2. The combined treatment enhanced the growth inhibitory effect on ML-20 cells, which express a high level of ER and a moderate level of HER2, but showed no additive effect on either KPL-4 cells, which express no ER and a moderate level of HER2, or MDA-MB-231 cells, which express no ER and a low level of HER2. It is also suggested that both the antibody and anti-oestrogen induce a G1-S blockade and apoptosis. These findings indicate that combined treatment with anti-HER2 antibody and anti-oestrogen may be useful for the treatment of patients with breast cancer expressing both ER and HER2.

Both estrogen and progesterone receptors have been determined in 613 primary breast cancer patients treated by radical mastectomy. At the cut-off value of 5 fentomoles (fmol) cytosol protein/mg for both receptors, patients with estrogen or progesterone receptor-positive breast cancer showed significantly favorable disease-free, overall and post-recurrence survival curves to those of receptor-negative breast cancer patients. In the patient subgroups: premenopausal, stage III, more than four positive lymph node metastases, postoperative adjuvant tamoxifen therapy, a significantly favorable prognosis was recognized in either estrogen or progesterone receptor-positive patients. Both receptors are thought to be useful prognostic indicators for patients with advanced tumors or for those receiving postoperative adjuvant tamoxifen therapy. When the cut-off value was changed, the maximum significant difference in prognosis between receptor-positive and receptor-negative patients was observed at 5 fmol cytosol protein/mg for the estrogen receptor or 10 fmol/mg cytosol protein for the progesterone receptor. A more detailed examination should be made on the cut-off values of both receptors.

In order to investigate the mechanisms of the endocrine therapeutic agents and the applicability of combined endocrine therapies for breast cancers, we studied the effects of estrogen and the endocrine therapeutic agents on estrogen receptor (ER), progesterone receptor (PgR), and DNA synthesis in MCF-7 human breast cancer cells, which is known to be sensitive to estrogen. ER and PgR of MCF-7 cells were determined by whole cell uptake method. In brief, intact MCF-7 cells cultured in the multi-well plates were incubated with various concentrations of tritiated estradiol (E2) or promegestone (R5020) at 37 degrees C for 1 hour. The characteristics of the hormone binding were analyzed by Scatchard plots. MCF-7 cells had single class of ER (Kd: 2.1 +/- 0.2 X 10(-10) M, MBC: 9.0 +/- 1.5 X 10(3) sites/cell) and PgR (Kd: 7.2 +/- 1.1 X 10(-10) M, MBC: 3.1 +/- 0.5 X 10(4) sites/cell). When cultured in the presence of 10(-8) M or 10(-6) M of E2, tamoxifen (TAM), R5020 or medroxyprogesterone acetate (MPA) for 48 hrs, the numbers of binding sites of ER and PgR altered, but the affinities of either of them did not change. E2(10(-8) or 10(-6) M) increased about twice the number of PgR. Although treatment of 10(-8) M TAM, a non-steroidal antiestrogen, slightly increased the number of PgR, 10(-6) M TAM significantly decreased the number of PgR. Both of R5020 (10(-8) or 10(-6) M) and MPA (10(-8) or 10(-6) M), synthetic progestins, decreased the number of ER dose-dependently. On the other hand, E2 (10(-8) and 10(-6) M) and R5020 (10(-8) M) enhanced DNA synthesis, but 10(-6) M TAM or MPA inhibited DNA synthesis. The effects of single, sequential and coincidental treatment of these agents were compared. The sequential treatment of 10(-8) M E2 for 36 hrs and followed 10(-6) M MPA for 36 hrs ("10(-8) M E2----10(-6) M MPA") and "10(-6) M TAM----10(-6) M MPA" inhibited DNA synthesis of MCF-7 cells more efficiently than 10(-6) M TAM alone or 10(-6) M MPA alone for 72 hrs. However, "10(-6) M MPA----10(-6) M TAM" inhibited DNA synthesis less than "10(-6) M TAM----10(-6) M MPA".(ABSTRACT TRUNCATED AT 400 WORDS)