Therapy for Cancer of the Breast: Current Status of Steroid Hormone Receptors

Estrogen receptor (ER) assays in human breast cancer tissue have proved useful in selecting patients for endocrine therapies. The absence of ER indicates hormone independent tumors and precludes the use of endocrine therapy. Patients with positive tumor ER respond to endocrine therapy at nearly twice the rate of those patients chosen by clinical criteria, although about a third of ER positive tumors in patients still do not respond. Recently, research has been directed toward increasing the accuracy of the ER assay in the ER positive group. The absolute tumor ER value and the presence of progesterone receptor appear promising in this regard. The significance of nuclear estrogen receptor is being studied. Finally, the ER status of a primary breast tumor appears to be a marker for the length of time until recurrence after mastectomy, and for survival. The ER assay may prove valuable in planning new adjuvants in the treatment of breast cancer.     

Estrogen receptor beta in breast cancer—Diagnostic and therapeutic implications

More than 10 years have passed since the discovery of the second estrogen receptor, estrogen receptor β (ERβ). It is now evident that ERα is not the only ER in breast cancer cells; in fact, ERβ is expressed in the majority of breast cancers although at lower levels than in the normal breast. In addition, ERβ is expressed in breast cancer infiltrating lymphocytes, fibroblasts and endothelial cells, all known to influence tumor growth. By overexpressing or knocking-out ERβ in breast cancer cell lines, several researchers have investigated its function with respect to proliferation and tumor growth. It appears that ERβ is anti-proliferative, in many ways antagonising the function of ERα. Furthermore, phytoestrogens have a binding-preference for ERβ and several epidemiological studies indicate a breast cancer preventing effect of this class of compounds. Tamoxifen is one of the standard, adjuvant treatments for ERα positive breast cancer, classically thought to mediate its effect through ERα. However, in several recent studies, ERβ has been described as a potential marker for tamoxifen response. In summary, experimental, epidemiological as well as diagnostic studies point towards ERβ as an important factor in breast cancer, opening up the possibility for novel ERβ-selective therapies in the treatment of breast cancer.     

TWIST represses estrogen receptor-alpha expression by recruiting the NuRD protein complex in breast cancer cells

Loss of estrogen receptor α (ERα) expression and gain of TWIST (TWIST1) expression in breast tumors correlate with increased disease recurrence and metastasis and poor disease-free survival. However, the molecular and functional regulatory relationship between TWIST and ERα are unclear. In this study, we found TWIST was associated with a chromatin region in intron 7 of the human ESR1 gene coding for ERα. This association of TWIST efficiently recruited the nucleosome remodeling and deacetylase (NuRD) repressor complex to this region, which subsequently decreased histone H3K9 acetylation, increased histone H3K9 methylation and repressed ESR1 expression in breast cancer cells. In agreement with these molecular events, TWIST expression was inversely correlated with ERα expression in both breast cancer cell lines and human breast ductal carcinomas. Forced expression of TWIST in TWIST-negative and ERα-positive breast cancer cells such as T47D and MCF-7 cells reduced ERα expression, while knockdown of TWIST in TWIST-positive and ERα-negative breast cancer cells such as MDA-MB-435 and 4T1 cells increased ERα expression. Furthermore, inhibition of histone deacetylase (HDAC) activity including the one in NuRD complex significantly increased ERα expression in MDA-MB-435 and 4T1 cells. HDAC inhibition together with TWIST knockdown did not further increase ERα expression in 4T1 and MDA-MB-435 cells. These results demonstrate that TWIST/NuRD represses ERα expression in breast cancer cells. Therefore, TWIST may serve as a potential molecular target for converting ERα-negative breast cancers to ERα-positive breast cancers, allowing these cancers to restore their sensitivity to endocrine therapy with selective ERα antagonists such as tamoxifen and raloxifene.     

Serine protease PRSS23 is upregulated by estrogen receptor α and associated with proliferation of breast cancer cells

Serine protease PRSS23 is a newly discovered protein that has been associated with tumor progression in various types of cancers. Interestingly, PRSS23 is coexpressed with estrogen receptor α (ERα), which is a prominent biomarker and therapeutic target for human breast cancer. Estrogen signaling through ERα is also known to affect cell proliferation, apoptosis, and survival, which promotes tumorigenesis by regulating the production of numerous downstream effector proteins.In the present study, we aimed to clarify the correlation between and functional implication of ERα and PRSS23 in breast cancer. Analysis of published breast cancer microarray datasets revealed that the gene expression correlation between ERα and PRSS23 is highly significant among all ERα-associated proteases in breast cancer. We then assessed PRSS23 expression in 56 primary breast cancer biopsies and 8 cancer cell lines. The results further confirmed the coexpression of PRSS23 and ERα and provided clinicopathological significance. In vitro assays in MCF-7 breast cancer cells demonstrated that PRSS23 expression is induced by 17β-estradiol-activated ERα through an interaction with an upstream promoter region of PRSS23 gene. In addition, PRSS23 knockdown may suppress estrogen-driven cell proliferation of MCF-7 cells.Our findings imply that PRSS23 might be a critical component of estrogen-mediated cell proliferation of ERα-positive breast cancer cells. In conclusion, the present study highlights the potential for PRSS23 to be a novel therapeutic target in breast cancer research.     

Phosphorylation by p38 mitogen-activated protein kinase promotes estrogen receptor α turnover and functional activity via the SCF(Skp2) proteasomal complex

The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCF(Skp2) proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers.     

Aromatase inhibitors and xenograft studies

Aromatase inhibitors (AIs) have become the front-line choice for treatment of ER+ breast cancer. Nevertheless, although patients are responsive initially, they may acquire resistance and become unresponsive to further treatment. In addition, approximately 25% of breast cancers do not express the estrogen receptor (ERα) and consequently, are innately resistant to endocrine therapy. We have investigated the mechanisms associated with this lack of treatment response using xenograft models. We found that in cells and tumors that acquired resistance to the AI letrozole therapy, expression of the ER was reduced whereas growth factor signally was enhanced, including a marked increase in HER2 expression. Treatment with trastuzumab (HER2 antibody) resulted in a significant down-regulation of HER2 and p-MAPK as well as restoration of ERα expression. Thus, when trastuzumab was added to letrozole treatment at the time of tumor progression, there was significantly prolonged tumor suppression compared to trastuzumab or letrozole alone. This suggests that inhibition of both HER2 and ERα signaling pathways are required for overcoming resistance and restoring treatment sensitivity. ER negative tumors are innately resistant to endocrine therapy. Repression of the ERα has been found to be due to epigenetic modifications such as increased methylation and histone deacetylation. We found that entinostat (ENT), a histone deacetylase inhibitor (HDACi), activated not only expression of ERα but also aromatase in MDA-MB-231 ER-negative breast cancer cells, resulting in their ability to respond to estrogen and letrozole. Treatment with ENT in combination with letrozole significantly reduced tumor growth rate in xenografts compared to control tumors (p < 0.001). ENT plus letrozole treatment also prevented the colonization and growth of MDA-MB-231 cells in the lung with a significant reduction (p < 0.03) in both visible and microscopic foci. These results provide a strong indication for possible use of AIs in combination with HDAC inhibitors for the treatment of ER-negative breast cancer.     

Aromatase resistance mechanisms in model systems in vivo

Aromatase inhibitors (AIs) have now been shown to be more effective than the anti-estrogen (AE) tamoxifen and have few side effects in ER+ breast cancer patients. However, some patients may not respond and resistance to treatment may develop in others. To investigate the mechanisms involved in the loss of sensitivity of the tumors to AIs, we have studied athymic mice with tumors grown from human estrogen receptor (ER) positive breast cancer cells (MCF-7) stably transfected with aromatase (MCF-7Ca). Treatment with letrozole upregulated Her-2 after four weeks despite continued responsiveness of tumor growth to letrozole. Furthermore, the level of Her-2 protein in letrozole refractory tumors was found to be six fold higher than the control tumors. Cells isolated from these tumors also had increased levels of Her-2 along with lower expression of ERα and aromatase and apparent estradiol independent growth. When Her-2 was inhibited by trastuzumab (antibody against Her-2) ERα levels in the cells were restored indicating that Her-2 is a negative regulator of ERα. This interaction between Her-2 and ER suggests that inhibition of both the Her-2 and estrogen signaling pathways is required to prolong the responsiveness of the tumors to endocrine therapies. Thus, when treatment with trastuzumab and letrozole was combined, ER was restored and tumor growth markedly inhibited compared to treatment with either drug alone. These findings demonstrate that tumor cells under the stress of treatment can adapt and utilize alternate pathways. Thus, when letrozole treatment was stopped, tumor Her-2 levels declined and ER levels were restored to those of hormone sensitive tumors. A second course of letrozole treatment inhibited tumors growth to the same extent and for as long as the initial treatment. These and other strategies to restore aromatase and ERα resulting in sensitivity to hormone therapy could be of substantial benefit to patients who have acquired resistance to AIs.     

Estradiol-dependent regulation of angiopoietin expression in breast cancer cells

Angiopoietin-1 (Ang-1) is a ligand for Tie-2 receptors and a promoter of angiogenesis. Angiogenesis plays an important role in breast cancer, as it is one of the critical events required for tumors to grow and metastasize. In this study, we investigated the influence of estradiol (E2) on the expression of angiopoietins in breast cancer cell lines. Ang-1 mRNA and protein expressions were significantly higher in estrogen receptor-negative (ERα-) breast cancer cells than in estrogen receptor-positive (ERα+) cells. Exposure of ERα+ cells to E2 resulted in further reductions of Ang-1 levels. In mouse mammary pads inoculated with breast cancer cells, both tumor size and Ang-1 production were significantly lower in ERα+ cell-derived xenografts, as compared to those derived from ERα- cells. Reduction of circulating levels of E2 by ovariectomy eliminated this response. Overall, these results indicate that Ang-1 mRNA and protein expressions: (1) negatively correlate with the level of ERα in breast cancer cell lines; (2) are downregulated by E2 in an ERα dependent manner; and (3) positively correlate with the degree of angiogenesis in vivo. We conclude that Ang-1 is an important modulator of growth and progression of ERα- breast cancers.     

Design,synthesis, biological evaluation and molecular docking studies of novel 3-aryl-4-anilino-2H-chromen-2-one derivatives targeting ERα as anti-breast cancer agents

The estrogen receptor (ER) has played an important role in breast cancer development and progression and is a central target for anticancer drug discovery. In order to develop novel selective ERα modulators (SERMs), we designed and synthesized 18 novel 3-aryl-4-anilino-2H-chromen-2-one derivatives based on previously reported lead compounds. The biological results indicated that most of the compounds presented potent ERα binding affinity and possessed better anti-proliferative activities against MCF-7 and Ishikawa cell lines than the positive control tamoxifen. The piperidyl substituted compounds such as 16d and 18d demonstrated strong ERα binding affinities and excellent anti-proliferative activities respectively. Compound 18d displayed the most potent ERα binding affinity with RBA value of 2.83%, while 16d exhibited the best anti-proliferative activity against MCF-7 cells with IC₅₀ value of 4.52 ± 2.47 μM. Further molecular docking studies were also carried out to investigate binding pattern of the newly synthesized compounds with ERα. All these results together with the structure–activity relationships (SARs) indicated that these 3-aryl-4-anilino-2H-chromen-2-one derivatives with basic side chain could serve as promising leads for further optimization as novel SERMs.