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.     

Autophagy facilitates the progression of ERα-positive breast cancer cells to antiestrogen resistance

A major impediment to the successful treatment of estrogen receptor α (ERα)-positive breast cancer is the development of antiestrogen resistance. Tamoxifen, the most commonly used antiestrogen, exerts its pharmacological action by binding to ERα and blocking the growth- promoting action of estrogen-bound ERα in breast cancer cells. Tamoxifen treatment primarily induces cytostasis (growth arrest) and the surviving breast cancer cells commonly acquire tamoxifen resistance. Numerous clinically-relevant mechanisms of acquired antiestrogen resistance have been identified by in vitro studies. Our recent studies (Mol Cancer Ther 2008: 7:2977-87) now demonstrate that autophagy (also referred to as macroautophagy) is critical to the development of antiestrogen resistance. Under conditions of compromised autophagy, including treatments with pharmacological inhibitors and RNAi targeting of the beclin 1 gene, the cytotoxicity (death-inducing effects) of the antiestrogen 4-hydroxytamoxifen (4-OHT) was significantly increased. 4-OHT is an active metabolite of tamoxifen commonly used for in vitro studies. A step-wise drug selection protocol, using 4-OHT as the selecting drug, established antiestrogen-resistant breast cancer cell lines. Analysis of a representative resistant cell line showed an increased ability of the cells to sustain high levels of antiestrogen-induced autophagy without progression to death. Importantly, blockade of autophagosome function in the 4-OHT-treated, antiestrogen-resistant cells induced a robust death response. These data provide strong evidence that autophagy is a key mechanism of cell survival during antiestrogen challenge and progression to antiestrogen resistance. We discuss the potential benefit of blocking autophagosome function to significantly reduce the emergence of antiestrogen-resistant breast cancer cells.     

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)     

Estrogen receptor alpha mediated induction of the transforming growth factor alpha gene by estradiol and 4-hydroxytamoxifen in MDA-MB-231 breast cancer cells

The selective estrogen receptor modulator, 4-hydroxytamoxifen (4-OHT) is a full agonist at the transforming growth factor (TGF) alpha gene in ER negative breast cancer cells stably transfected with ER alpha cDNA (Levenson et al., Br. J. Cancer 77 (1998) 1812-1819). E(2) and 4-OHT increase TGF alpha mRNA and protein in a concentration dependent manner. The responses to E(2) and 4-OHT are blocked by the pure antiestrogen ICI 182,780, which does not induce TGF alpha. Transfected MDA-MB-231 cells contain functional ER alpha but no ER beta function was detected. Neo transfected cells that did not express ER alpha or cells stably transfected with the DNA binding domain mutant C202R/E203V which prevents gene activation did not induce TGF alpha mRNA after either E(2) or 4-OHT treatment. An examination of the time course for either 10 nM E(2) or 1 microM 4-OHT for MDA-MB-231 cells stably transfected with cDNA for ER alpha showed increases in TGF alpha mRNA within 2 or 3 h respectively. Cells pretreated with cycloheximide (1 microg/ml) showed induced TGF alpha mRNA in response to E(2) or 4-OHT but TGF alpha mRNA induction was blocked by actinomycin D (1 microg/ml). We conclude that both E(2) and 4-OHT induce TGF alpha by direct interaction of ER alpha with DNA and that ER beta is not involved in the estrogen-like response to 4-OHT in the MDA-MB-231 cells.     

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.     

Elevated expression of the estrogen receptor prevents the down-regulation of p21Waf1/Cip1 in hormone dependent breast cancer cells

Expression of an estrogen receptor alpha (ER) transgene in hormone independent breast cancer and normal breast epithelial cells arrests cell cycling when estradiol is added. Although endogenously expressed ER does not typically affect estradiol-induced cell cycling of hormone dependent breast cancer cells, we observed that elevated expression of a green fluorescent protein fused to ER (GFP-ER) hindered entry of estrogen treated MCF-7 cells into S phase of the cell cycle. In analyses of key cell-cycle regulating proteins, we observed that GFP-ER expression had no affect on the protein levels of cyclin D1, cyclin E, or p27, a cyclin dependent kinase (Cdk) inhibitor. However, at 24 h, p21 (Waf1, Cip1; a Cdk2 inhibitor) protein remained elevated in the high GFP-ER expressing cells but not in non-GFP-ER expressing cells. Elevated expression of p21 inhibited Cdk2 activity, preventing cells from entering S phase. The results show that elevated levels of ER prevented the down-regulation of p21 protein expression, which is required for hormone responsive cells to enter S phase.     

Estrogen utilization of IGF-1-R and EGF-R to signal in breast cancer cells

As breast cancer cells develop secondary resistance to estrogen deprivation therapy, they increase their utilization of non-genomic signaling pathways. Our prior work demonstrated that estradiol causes an association of ERα with Shc, Src and the IGF-1-R. In cells developing resistance to estrogen deprivation (surrogate for aromatase inhibition) and to the anti-estrogens tamoxifen, 4-OH-tamoxifen, and fulvestrant, an increased association of ERα with c-Src and the EGF-R occurs. At the same time, there is a translocation of ERα out of the nucleus and into the cytoplasm and cell membrane. Blockade of c-Src with the Src kinase inhibitor, PP-2 causes relocation of ERα into the nucleus. While these changes are not identical in response to each anti-estrogen, ERα binding to the EGF-R is increased in response to 4-OH-tamoxifen when compared with tamoxifen. The changes in EGF-R interactions with ERα impart an enhanced sensitivity of tamoxifen-resistant cells to the inhibitory properties of the specific EGF-R tyrosine kinase inhibitor, AG 1478. However, with long term exposure of tamoxifen-resistant cells to AG 1478, the cells begin to re-grow but can now be inhibited by the IGF-R tyrosine kinase inhibitor, AG 1024. These data suggest that the IGF-R system becomes the predominant signaling mechanism as an adaptive response to the EGF-R inhibitor. Taken together, this information suggests that both the EGF-R and IGF-R pathways can mediate ERα signaling.To further examine the effects of fulvestrant on ERα function, we examined the acute effects of fulvestrant, on non-genomic functionality. Fulvestrant enhanced ERα association with the membrane IGF-1-receptor (IGF-1-R). Using siRNA or expression vectors to knock-down or knock-in selective proteins, we further demonstrated that the ERα/IGF-1-R association is Src-dependent. Fulvestrant rapidly induced IGF-1-R and MAPK phosphorylation. The Src inhibitor PP2 and IGF-1-R inhibitor AG1024 greatly blocked fulvestrant-induced ERα/IGF-1-R interaction leading to a further depletion of total cellular ERα induced by fulvestrant and further enhanced fulvestrant-induced cell growth arrest. More dramatic was the translocation of ERα to the plasma membrane in combination with the IGF-1-R as shown by confocal microscopy. Taken in aggregate, these studies suggest that secondary resistance to hormonal therapy results in usage of both IGF-R and EGF-R for non-genomic signaling.     

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.     

Docosahexaenoic acid induces proteasome-dependent degradation of estrogen receptor α and inhibits the downstream signaling target in MCF-7 breast cancer cells

About two thirds of breast cancers in women are hormone-dependent and require estrogen for growth, its effects being mainly mediated through estrogen receptor α (ERα). Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) have opposite effects on carcinogenesis, with DHA suppressing and AA promoting tumor growth both in vitro and in vivo. However, the mechanism is not clear. Here, we examined whether the effect is mediated through changes in ERα distribution. MCF-7 cells, an ERα-positive human breast cancer cell line, was cultured in estrogen-free medium containing 0, 10 or 60 μM DHA or AA, then were stimulated with estradiol. DHA supplementation resulted in down-regulation of ERα expression (particularly in the extranuclear fraction), a reduction in phosphorylated MAPK, a decrease in cyclin D1 levels and an inhibition in cell viability. In contrast, AA had no such effects. The DHA-induced decrease in ERα expression resulted from proteasome-dependent degradation and not from decreased ERα mRNA expression. We propose that breast cancer cell proliferation is inhibited by DHA through proteasome-dependent degradation of ERα, reduced cyclin D1 expression and inhibition of MAPK signaling.     

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G₂/M phases, whereas the G₁ phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G₂/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G₂/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G₂/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.     

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.     

A naringenin–tamoxifen combination impairs cell proliferation and survival of MCF-7 breast cancer cells

Since over 60% of breast cancers are estrogen receptor positive (ER+), many therapies have targeted the ER. The ER is activated by both estrogen binding and phosphorylation. While anti-estrogen therapies, such as tamoxifen (Tam) have been successful they do not target the growth factor promoting phosphorylation of the ER. Other proliferation pathways such as the phosphatidylinositol-3 kinase, (PI3K) and the mitogen-activated protein kinase (MAPK) pathways are activated in breast cancer cells and are associated with poor prognosis. Thus targeting multiple cellular proliferation and survival pathways at the onset of treatment is critical for the development of more effective therapies. The grapefruit flavanone naringenin (Nar) is an inhibitor of both the PI3K and MAPK pathways. Previous studies examining either Nar or Tam used charcoal-stripped serum which removed estrogen as well as other factors. We wanted to use serum containing medium in order to retain all the potential inducers of cell proliferation so as not to exclude any targets of Nar. Here we show that a Nar–Tam combination is more effective than either Tam alone or Nar alone in MCF-7 breast cancer cells. We demonstrate that a Nar–Tam combination impaired cellular proliferation and viability to a greater extent than either component alone in MCF-7 cells. Furthermore, the use of a Nar–Tam combination requires lower concentrations of both compounds to achieve the same effects on proliferation and viability. Nar may function by inhibiting both PI3K and MAPK pathways as well as localizing ERα to the cytoplasm in MCF-7 cells. Our results demonstrate that a Nar–Tam combination induces apoptosis and impairs proliferation signaling to a greater extent than either compound alone. These studies provide critical information for understanding the molecular mechanisms involved in cell proliferation and apoptosis in breast cancer cells.     

Glucose-derived acetate and ACSS2 as key players in cisplatin resistance in bladder cancer

Cisplatin is an important chemotherapeutic agent against metastatic bladder cancer, but resistance often limits its usage. With the recent recognition of lipid metabolic alterations in bladder cancers, we studied the metabolic implications of cisplatin resistance using cisplatin-sensitive (T24S) and resistant (T24R) bladder cancer cells. Real-time live metabolomics revealed that T24R cells consume more glucose, leading to higher production of glucose-derived acetate and fatty acids. Along with the activation of general metabolic regulators, enzymes involved in acetate usage (ACSS2) and fatty acid synthesis (ACC) and a precursor for fatty acid synthesis (acetyl-CoA) were elevated in T24R cells. Consistently, metabolic analysis with ¹³C isotope revealed that T24R cells preferred glucose to acetate as the exogenous carbon source for the increased fatty acid synthesis, contrary to T24S cells. In addition, ACSS2, rather than the well-established ACLY, was the key enzyme that supplies acetyl-CoA in T24R cells through glucose-derived endogenous acetate. The relevance of ACSS2 in cisplatin resistance was further confirmed by the abrogation of resistance by an ACSS2 inhibitor and, finally, by the higher expression of ACSS2 in the patient tissues with cisplatin resistance. Our results may help improve the treatment options for chemoresistant bladder cancer patients and provide possible vulnerability targets to overcome the resistance.