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.     

CIS3/SOCS-3 suppresses erythropoietin (EPO) signaling by binding the EPO receptor and JAK2

The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin (EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing Tyr(401) in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr(401) region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2. In the N-terminal region of CIS3, the amino acid Gly(45) was critical for binding to the EPOR but not to JAK2, while Leu(22) was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of STAT5, which also binds to Tyr(401), reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.     

Cryptotanshinone inhibition of mammalian target of rapamycin pathway is dependent on oestrogen receptor alpha in breast cancer

Cryptotanshinone (CPT) has been demonstrated to inhibit proliferation and mammalian target of rapamycin (mTOR) pathway in MCF‐7 breast cancer cells. However, the same results are unable to be repeated in MDA‐MB‐231 cells. Given the main difference of oestrogen receptor α (ERα) between two types of breast cancer cells, It is possibly suggested that CPT inhibits mTOR pathway dependent on ERα in breast cancer. CPT could significantly inhibit cell proliferation of ERα‐positive cancer cells, whereas ERα‐negative cancer cells are insensitive to CPT. The molecular docking results indicated that CPT has a high affinity with ERα, and the oestrogen receptor element luciferase reporter verified CPT distinct anti‐oestrogen effect. Furthermore, CPT inhibits mTOR signalling in MCF‐7 cells, but not in MDA‐MB‐231 cells, which is independent on binding to the FKBP12 and disrupting the mTOR complex. Meanwhile, increased expression of phosphorylation AKT and insulin receptor substrate (IRS1) induced by insulin‐like growth factor 1 (IGF‐1) was antagonized by CPT, but other molecules of IGF‐1/AKT/mTOR signalling pathway such as phosphatase and tensin homolog (PTEN) and phosphatidylinositol‐4,5‐bisphosphate 3‐kinase (PI3K) were negatively affected. Finally, the MCF‐7 cells transfected with shERα for silencing ERα show resistant to CPT, and p‐AKT, phosphorylation of p70 S6 kinase 1 (p‐S6K1) and eukaryotic initiation factor 4E binding protein 1 (4E‐BP1) were partially recovered, suggesting ERα is required for CPT inhibition of mTOR signalling. Overall, CPT inhibition of mTOR is dependent on ERα in breast cancer and should be a potential anti‐oestrogen agent and a natural adjuvant for application in endocrine resistance therapy.     

Mitogen-activated protein kinase plays an essential role in the erythropoietin-dependent proliferation of CTLL-2 cells

Erythropoietin (EPO) and its receptor (EPOR) are required for development of erythrocytes. It has been shown that the ectopic expression of EPOR confers EPO-dependent proliferation on an interleukin 3 (IL3)-dependent cell line, Ba/F3, whereas the IL2-dependent T cell line, CTLL-2 expressing the EPOR (T-ER), fails to proliferate in response to EPO. However, the molecular basis of the EPO unresponsiveness in CTLL-2 has not been clarified. We found that the expression level of JAK2 in T-ER cells was much lower than that in Ba/F3 cells. Therefore, we examined the effects of forced expression of JAK2 in T-ER cells. In T-ER transformants expressing JAK2 (T-JER), EPO induced tyrosine phosphorylation of the EPOR, JAK2, and STAT5, and consequently STAT5-responsive genes including bcl-X and cis1 were normally induced. Furthermore, T-JER cells were resistant to apoptosis until at least 72 h after switching from IL2 to EPO. Although T-JER cells could not continuously proliferate in the presence of EPO, additional expression of JAK2 in T-JER (T-JJER) to a level similar to that in Ba/F3 cells supported long term proliferation in response to EPO. JAK2 was equally co-immunoprecipitated with the EPOR among T-JER, T-JJER, and Ba/F3 cells expressing the EPOR (BF-ER). However, EPO-dependent mitogen-activated protein (MAP) kinase activation was observed in T-JJER and BF-ER cells but not in T-JER cells. EPO-dependent long term proliferation of T-JER cells was conferred by expression of the constitutively activated form of MEK1. Our results suggest that MAP kinase activation is, at least in part, an important component for mitotic signal from the EPOR, and CTLL-2 cells probably lack signaling molecule(s) in JAK2 and the Ras-MAP kinase pathway.     

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.     

Evidences that estrogen receptor α interferes with adiponectin effects on breast cancer cell growth

Adiponectin, the most abundant protein secreted by adipose tissue, exhibits insulin-sensitizing, anti-inflammatory, antiatherogenic, and antiproliferative properties. In addition, it appears to play an important role also in the development and progression of several obesity-related malignancies, including breast cancer.

Here, we demonstrated that adiponectin induces a dichotomic effect on breast cancer growth. Indeed, it stimulates growth in ERα+ MCF-7 cells while inhibiting proliferation of ERα? MDA-MB-231 cells. Notably, only in MCF-7 cells adiponectin exposure exerts a rapid activation of MAPK phosphorylation, which is markedly reduced when knockdown of the ERα gene occurred. In addition, adiponectin induces rapid IGF-IR phosphorylation in MCF-7 cells, and the use of ERα siRNA prevents this effect. Moreover, MAPK activation induced by adiponectin was reversed by IGF-IR siRNA. Coimmunoprecipitation studies show the existence of a multiprotein complex involving AdipoR1, APPL1, ERα, IGF-IR, and c-Src that is responsible for MAPK signaling activation in ERα+ positive breast cancer cells. It is well known that in addition to the rapid effects through non-genomic mechanisms, ERα also mediates nuclear genomic actions. In this concern, we demonstrated that adiponectin is able to transactivate ERα in MCF-7 cells. We showed the classical features of ERα transactivation: nuclear localization, downregulation of mRNA and protein levels, and upregulation of estrogen-dependent genes. Thus, our study clarifies the molecular mechanism through which adiponectin modulates breast cancer cell growth, providing evidences on the cell-type dependency of adiponectin action in relationship to ERα status.     

STAT3, HIF-1alpha, EPO and EPOR - signaling proteins in human primary ductal breast cancers

STAT3 upregulates expression of HIF-1 induced EPO. Receptor EPOR was reported to activate STAT3. Our study was aimed at demonstration of tissue immunoreactivities of those proteins and determination of their relationships in reference to clinicopathological variables of breast cancers. We detected STAT3, HIF-1alpha, EPO and EPOR in specimens of 76 human, female, ductal breast cancers by immunohistochemistry. STAT3 was detected in 38 of 76 cancers (50%). HIF-1alpha was found in 55 cases (72%). EPO positive tumors comprised 89% of all the cancers (68 cases). EPOR was also visualized in 55 cases (72%). Anti-HIF-1alpha and anti-STAT3 stained nuclei and cytoplasm of breast cancer cells in diffuse and finely granular fashion. Strong membranous expressions of EPO and EPOR were distributed in cytoplasmic and membranous granularity or diffuse staining. STAT3 correlated with HIF-1 in general (r=0.4012, p<0.0001) and in different patients' subgroups. STAT3 was significantly associated with EPO and EPOR in all the cancers (r=0.2370, p=0.039 and r=0.3336, p=0.003, respectively). Besides a correlation between STAT3 and EPOR in node negative ones, STAT3 wasn't related to EPO and EPOR in remaining subgroups. HIF-1alpha correlated with EPO and EPOR in most of analyzed groups. Immunoreactivity to EPO generally was associated with EPOR (r=0.3520, p=0.002). Statistically analyzed distributions of the proteins reflected functional dependences among STAT3, HIF-1alpha, EPO and EPOR in cellular signal conduction.     

EPO/EPOR 在非小性细胞肺癌发展中的作用研究

目的：研究促红细胞生成素（erythropoietin, EPO）及其受体（EPOR）在非小性细胞肺癌中的生物学作用。方法：收集27 例非小 性细胞肺癌（NSCLC）,免疫组织化学方法检测肺癌组织中EPO 和EPOR的表达;观察人源重组EPO（rhEPO）对HCC15 和 HCC1819 细胞活力和细胞周期的影响;分析缺氧对NSCLC细胞EPO 及EPOR 表达的影响。结果：27例非小细胞肺癌的组织标 本中13 例表达EPO,表达率为48 %,25 例表达EPOR,表达率为92 %。rhEPO明显增加了高表达EPOR 的HCC1819 细胞克隆 数,而对低表达EPOR 的HCC15 细胞的克隆形成没有影响。rhEPO增强了HCC1819 的细胞活力,但以siRNA干涉HCC1819 EPOR后,EPO对HCC1819 细胞活力增强作用消失。rhEPO 明显增加了HCC1819 细胞的细胞周期。缺氧促进了HCC1819 细胞的 EPO 的表达,增强了细胞活力。结论：EPO 和EPOR在非小性细胞肺癌中表达增高,EPO 通过EPOR 促进了NSCLC 细胞的增殖, 缺氧诱导了NSCLC 细胞EPO的表达。     

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.     

Mutations in the Trp-Ser-X-Trp-Ser motif of the erythropoietin receptor abolish processing, ligand binding, and activation of the receptor.

The erythropoietin receptor (EPOR) is a member of the newly identified cytokine receptor superfamily. A common sequence motif, Trp-Ser-X-Trp-Ser (WSXWS), near the transmembrane domain is highly conserved in this family. To determine the function of this motif, we constructed deletion and insertion mutations in this part of the EPOR and introduced them into an interleukin-3 (IL-3)-dependent hematopoietic Ba/F3 cell line. Cells expressing the wild-type EPOR displayed 1,500 erythropoietin (EPO)-binding sites/cell with a single affinity of about 300 pM and proliferate in the presence of IL-3 or EPO. Ba/F3 cells expressing receptors mutated in the WSXWS motif displayed little EPO binding on the cell surface and did not grow in the presence of EPO. The mutant receptors were retained in the endoplasmic reticulum (ER) and, as such, were unable to bind EPO. A single Gly insertion between the two WS sequences caused defects in receptor structure and function similar to mutations lacking all or part of the WSXWS motif. The EPOR can be activated, resulting in proliferation independent of EPO either by an Arg129 to Cys point mutation or by association with the Friend spleen focus-forming virus (SFFV) envelope glycoprotein gp55. Introduction of the point mutation (Arg129 to Cys) did not activate any of the receptors mutated in the WSXWS motif. Moreover, gp55 did not activate the mutant receptors in Ba/F3 cells. Our study indicates that the WSXWS motif is critical for protein folding, ligand-binding, and signal transduction.     

Erythropoietin is a hypoxia inducible factor-induced protective molecule in experimental autoimmune neuritis

Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3⁺/CD4⁺ regulatory T cells and decrease of IFN-γ⁺/CD4⁺ Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies.     

Predictive features of ligand‐specific signaling through the estrogen receptor

Some estrogen receptor‐α (ERα)‐targeted breast cancer therapies such as tamoxifen have tissue‐selective or cell‐specific activities, while others have similar activities in different cell types. To identify biophysical determinants of cell‐specific signaling and breast cancer cell proliferation, we synthesized 241 ERα ligands based on 19 chemical scaffolds, and compared ligand response using quantitative bioassays for canonical ERα activities and X‐ray crystallography. Ligands that regulate the dynamics and stability of the coactivator‐binding site in the C‐terminal ligand‐binding domain, called activation function‐2 (AF‐2), showed similar activity profiles in different cell types. Such ligands induced breast cancer cell proliferation in a manner that was predicted by the canonical recruitment of the coactivators NCOA1/2/3 and induction of the GREB1 proliferative gene. For some ligand series, a single inter‐atomic distance in the ligand‐binding domain predicted their proliferative effects. In contrast, the N‐terminal coactivator‐binding site, activation function‐1 (AF‐1), determined cell‐specific signaling induced by ligands that used alternate mechanisms to control cell proliferation. Thus, incorporating systems structural analyses with quantitative chemical biology reveals how ligands can achieve distinct allosteric signaling outcomes through ERα.     

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.