Arsenic induces functional re-expression of estrogen receptor α by demethylation of DNA in estrogen receptor-negative human breast cancer

Estrogen receptor α (ERα) is a marker predictive for response of breast cancers to endocrine therapy. About 30% of breast cancers, however, are hormone- independent because of lack of ERα expression. New strategies are needed for re-expression of ERα and sensitization of ER-negative breast cancer cells to selective ER modulators. The present report shows that arsenic trioxide induces reactivated ERα, providing a target for therapy with ER antagonists. Exposure of ER-negative breast cancer cells to arsenic trioxide leads to re-expression of ERα mRNA and functional ERα protein in in vitro and in vivo. Luciferase reporter gene assays and 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays show that, upon exposure to arsenic trioxide, formerly unresponsive, ER-negative MDA-MB-231 breast cancer cells become responsive to ER antagonists, 4-hydroxytamoxifen and ICI 182,780. Furthermore, methylation- specific PCR and bisulfite-sequencing PCR assays show that arsenic trioxide induces partial demethylation of the ERα promoter. A methyl donor, S-adenosylmethionine (SAM), reduces the degree of arsenic trioxide-induced re-expression of ERα and demethylation. Moreover, Western blot and ChIP assays show that arsenic trioxide represses expression of DNMT1 and DNMT3a along with partial dissociation of DNMT1 from the ERα promoter. Thus, arsenic trioxide exhibits a previously undefined function which induces re-expression ERα in ER-negative breast cancer cells through demethylation of the ERα promoter. These findings could provide important information regarding the application of therapeutic agents targeting epigenetic changes in breast cancers and potential implication of arsenic trioxide as a new drug for the treatment of ER-negative human breast cancer.     

Point mutation of estrogen receptor (ER) in the ligand-binding domain changes the pharmacology of antiestrogens in ER-negative breast cancer cells stably expressing complementary DNAs for ER.

The antiestrogen tamoxifen is used in the treatment of hormone-responsive breast cancer. However, therapeutic failure has frequently been observed in both patients and animal models after long term treatment. We have studied the effect of a point mutation that leads to the substitution of Val for Gly at codon 400 in the ligand-binding domain of the estrogen receptor (ER) on estrogenic and antiestrogenic activities of 4-hydroxytamoxifen (4-OHT) and its derivatives. Stable ER transfectants derived from MDA-MB-231 CL10A, an ER-negative breast cancer cell line, have been used in these studies. 4-OHT and its fixed ring derivatives showed more estrogen-like activity in ER transfectants than in MCF-7, an ER-positive breast cancer cell line. In this study, 4-OHT was a partial agonist of cell growth in the transfectant S30 cells, which express the wild-type ER. However, it was a full agonist in the mutant ER transfectant ML alpha 2H, which expressed ER with Val at codon 400. The increased estrogenic activity of 4-OHT in ML alpha 2H cells was not due to the preferential isomerization of trans 4-OHT to cis 4-OHT, since the nonisomerizable fixed ring trans 4-OHT was a partial agonist for cell growth in S30 cells and was a full agonist in ML alpha 2H cells. Transient transfection using a reporter plasmid containing an estrogen response element demonstrated that fixed ring trans 4-OHT had estrogenic activity in ML alpha 2H cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photodynamic cell-kill analysis of breast tumor cells with a tamoxifen-pyropheophorbide conjugate

We hypothesized that estrogen receptor (ER) in hormone-sensitive breast cancer cells could be targeted for selective photodynamic killing of tumor cell with antiestrogen-porphyrin conjugates by combining the over-expression of ER in hormone-sensitive breast cancer cells and tumor-retention property of porphyrin photosensitizers. In this study we describe that a tamoxifen (TAM)-pyropheophorbide conjugate that specifically binds to ER alpha, caused selective cell-kill in MCF-7 breast cancer cells upon light exposure. Therefore, it is a potential candidate for ER-targeted photodynamic therapy of cancers (PDT) of tissues and organs that respond to estrogens/antiestrogens.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Induction of estrogen-regulated genes differs in immortal and tumorigenic human mammary epithelial cells expressing a recombinant estrogen receptor.

Studies on estrogen receptor (ER)-positive human breast cancer cell lines have shown that estrogen treatment positively modulates the expression of the genes encoding transforming growth factor-alpha (TGF alpha), 52-kDa cathepsin-D, and pS2. To determine whether these genes would be similarly regulated by estrogens in normal human mammary epithelial cells, we stably transfected immortal nontumorigenic human mammary epithelial cells with an ER-encoding expression vector. ER-negative tumor cells were also transfected for comparison. Levels of TGF alpha and 52-kDa cathepsin-D mRNA were enhanced by estrogen treatment of both ER-transfected immortal and tumorigenic cells, demonstrating that the ER by itself is sufficient to elicit estrogenic regulation of the expression of these genes. In contrast, expression of the pS2 gene was detected only in the ER-transfected tumor cells. The ER in both cell lines is capable of recognizing the pS2 promoter, however, since estrogen enhanced the activity of an introduced pS2-CAT reporter plasmid in transient expression analyses. These and other experiments with somatic cell hybrids between the immortal cells and ER+/pS2+ MCF-7 tumor cells, where pS2 gene expression is extinguished, support the conclusion that the immortal nontumorigenic cells encode gene products that block endogenous pS2 expression. These results also imply that such repressors are not active in the tumor cells.     

Epigenetic reprogramming of breast cancer cells by valproic acid occurs regardless of estrogen receptor status

Estrogen receptors (ERs) are a recognized prognostic factor and therapeutic target in breast cancer. The loss of ER expression relates to poor prognosis, poor clinical outcome and impairs the use of anti-estrogenic treatment. Histone deacetylase inhibitors are candidate drugs for cancer therapy. Among them, valproic acid (VPA) is a long used and safe anti-epileptic drug. We studied the biological consequences of the chromatin remodeling action of VPA in a normal human mammary epithelial cell line and in ERalpha-positive and ERalpha-negative breast cancer cell lines. In these cells and regardless of their ER status, VPA-induced cell differentiation, as shown by increased milk lipids production, decreased expression of the CD44 antigen and growth arrest in the G(0)-G(1) phase of the cell cycle. These effects were accompanied by decreased Rb phosphorylation, hyperacetylation of the p21(WAF1/CIP1) gene promoter and increased p21 protein expression. Only in breast cancer cells, cyclin B1 expression was decreased and the cells accumulated also in G(2). ERalpha expression decreased in ERalpha-positive, increased in ERalpha-negative and was unchanged in normal mammary epithelial cells, as did the expression of progesterone receptor, a physiological ERalpha target. VPA decreased the expression of the invasiveness marker pS2 in ERalpha-positive breast cancer cells, but did not cause its re-expression in ERalpha-negative cells. Overall, these data suggest that in both ERalpha-positive and -negative malignant mammary epithelial cells VPA reprograms the cells to a more differentiated and "physiologic" phenotype that may improve the sensitivity to endocrine therapy and/or chemotherapy in breast cancer patients.     

Estrogen receptors: new perspectives in breast cancer management

The imbalance between proliferative and differentiative estrogenic effect, caused by quantitative and qualitative alteration of the estrogen receptor (ER) expression, may play a determinant role in mammary neoplastic transformation. Our studies demonstrate that ER levels are significantly higher in human mammary neoplastic tissues when compared to perineoplastic tissues and that increased ER expression is associated with ER gene hypomethylation. During progressive multifactorial carcinogene, ER overexpression may represent an early step in neoplastic transformation. In fact, high levels of ER represent good markers of differentiation and can predict the likelihood of benefiting from anti-estrogen therapy. Nevertheless, about 35% of ER-positive breast cancers are resistant to endocrine therapy and 10% of ER-negative tumors behave as hormone-sensitive tumors. Recent studies on ER mRNA variants, which naturally occur in human breast tumors, demonstrated mutations, deletions and alternative splicings, yielding deletions of exons 3, 4, 5 and 7. ER variants exhibited altered functions or changed the responsiveness to hormonal therapy. Analysis of these variants could be a useful parameter to better predict tumor responsiveness to anti-estrogen therapy. Recently, a regain of hormonal responsiveness by ER-negative breast cancer cells has been reported following ER gene transfection. However, estradiol treatment inhibits rather than stimulates cell growth as well as the metastatic and invasive potential of the ER gene transduced cells. Transfer of the ER gene may be considered as a new therapeutic approach in the management of hormone-independent breast cancer.     

Identification of a Gene (GPR30) with Homology to the G-Protein-Coupled Receptor Superfamily Associated with Estrogen Receptor Expression in Breast Cancer

Using the technique of differential cDNA library screening, a cDNA clone was isolated from an estrogen receptor (ER)-positive breast carcinoma cell line (MCF7) cDNA library based upon the overexpression of this gene compared to an ER-negative cell line (MDA-MB-231). Sequence analysis of this clone determined that it shared significant homology to G-protein-coupled receptors. This receptor, GPCR-Br, was abundantly expressed in the ER-positive breast carcinoma cell lines MCF7, T-47D, and MDA-MB-361. Expression was absent or minimal in the ER-negative breast carcinoma cell lines BT-20, MDA-MB-231, and HBL-100. GPCR-Br was ubiquitously expressed in human tissues examined but was most abundant in placenta. GPCR-Br expression was examined in 11 primary breast carcinomas. GPCR-Br was detected in all 4 ER-positive tumors and only 1 of 7 ER-negative tumors. Based upon PCR analysis in hybrid cell lines, the gene for GPCR-Br (HGMW-approved symbol GPR30) was mapped to chromosome 7p22. The pattern of expression of GPCR-Br indicates that this receptor may be involved in physiologic responses specific to hormonally responsive tissues.     

Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.     

Different proliferation patterns in breast cancer: AgNOR measurements in ER-negative and ER-positive tumor cells.

The relation between estrogen receptors (ER) and argyrophilic nucleolar organizer regions (AgNORs) in situ within human breast cancer cells was analyzed. For AgNOR measurements in 49 invasive breast carcinomas, a new reproducible staining method for dual demonstration of ER and AgNORs was applied. Quantitative AgNOR variables were determined in ER-positive and ER-negative tumor cells by digital image analysis. The relationships between AgNOR parameters of ER-positive and ER-negative cells and other prognostic factors of breast cancer [Bloom-Richardson-Grading and growth fraction (Ki-67 index)] were investigated. A higher AgNOR content in ER-negative cells and a special clustering phenomenon in ER-positive tumor cells were found. Correlation with other criteria of malignant potential could be exclusively demonstrated for ER-negative cells. ER-negative cells of breast cancer can be characterized as the more malignant and possibly prognosis-dictating cell fraction. Thus, ER-negative cells probably contribute more to the progression of the tumor disease and furthermore to the prognosis than ER-positive cells. We recommend measurement AgNORs exclusively in ER-negative cells of breast cancer.