Protein kinase D1 regulates ERα‐positive breast cancer cell growth response to 17β‐estradiol and contributes to poor prognosis in patients

About 70% of human breast cancers express and are dependent for growth on estrogen receptor α (ERα), and therefore are sensitive to antiestrogen therapies. However, progression to an advanced, more aggressive phenotype is associated with acquisition of resistance to antiestrogens and/or invasive potential. In this study, we highlight the role of the serine/threonine‐protein kinase D1 (PKD1) in ERα‐positive breast cancers. Growth of ERα‐positive MCF‐7 and MDA‐MB‐415 human breast cancer cells was assayed in adherent or anchorage‐independent conditions in cells overexpressing or depleted for PKD1. PKD1 induces cell growth through both an ERα‐dependent manner, by increasing ERα expression and cell sensitivity to 17β‐estradiol, and an ERα‐independent manner, by reducing cell dependence to estrogens and conferring partial resistance to antiestrogen ICI 182,780. PKD1 knockdown in MDA‐MB‐415 cells strongly reduced estrogen‐dependent and independent invasion. Quantification of PKD1 mRNA levels in 38 cancerous and non‐cancerous breast cell lines and in 152 ERα‐positive breast tumours from patients treated with adjuvant tamoxifen showed an association between PKD1 and ERα expression in 76.3% (29/38) of the breast cell lines tested and a strong correlation between PKD1 expression and invasiveness (P < 0.0001). In tamoxifen‐treated patients, tumours with high PKD1 mRNA levels (n = 77, 50.66%) were significantly associated with less metastasis‐free survival than tumours with low PKD1 mRNA expression (n = 75, 49.34%; P = 0.031). Moreover, PKD1 mRNA levels are strongly positively associated with EGFR and vimentin levels (P < 0.0000001). Thus, our study defines PKD1 as a novel attractive prognostic factor and a potential therapeutic target in breast cancer.     

ChIP‐Seq of ERα and RNA polymerase II defines genes differentially responding to ligands

We used ChIP‐Seq to map ERα‐binding sites and to profile changes in RNA polymerase II (RNAPII) occupancy in MCF‐7 cells in response to estradiol (E2), tamoxifen or fulvestrant. We identify 10 205 high confidence ERα‐binding sites in response to E2 of which 68% contain an estrogen response element (ERE) and only 7% contain a FOXA1 motif. Remarkably, 596 genes change significantly in RNAPII occupancy (59% up and 41% down) already after 1 h of E2 exposure. Although promoter proximal enrichment of RNAPII (PPEP) occurs frequently in MCF‐7 cells (17%), it is only observed on a minority of E2‐regulated genes (4%). Tamoxifen and fulvestrant partially reduce ERα DNA binding and prevent RNAPII loading on the promoter and coding body on E2‐upregulated genes. Both ligands act differently on E2‐downregulated genes: tamoxifen acts as an agonist thus downregulating these genes, whereas fulvestrant antagonizes E2‐induced repression and often increases RNAPII occupancy. Furthermore, our data identify genes preferentially regulated by tamoxifen but not by E2 or fulvestrant. Thus (partial) antagonist loaded ERα acts mechanistically different on E2‐activated and E2‐repressed genes.     

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.     

Furanodienone inhibits cell proliferation and survival by suppressing ERα signaling in human breast cancer MCF-7 cells

Estrogen receptor alpha (ERα) plays an important role in the development and progression of breast cancer and thus the attenuation of ERα activities is a promising treatment strategy. Furanodienone is one of the main bioactive chemical components of Rhizoma Curcumae which is commonly used in Chinese medicine for the treatment of cancer. In this study, we investigated the effects of furanodienone on human breast cancer MCF‐7, T47D, and MDA‐MB‐231 cells. Our results showed that furanodienone could inhibit MCF‐7, T47D, and MDA‐MB‐231 cells proliferation in a dose (10–160 µM) dependent manner. ERα‐negative MDA‐MB‐231 cells were less sensitive to furanodienone than ERα‐positive MCF‐7 and T47D cells. Furanodienone could effectively block 17β‐estradiol (E2)‐stimulated MCF‐7 cell proliferation and cell cycle progression and induce apoptosis evidenced by the flow cytometric detection of sub‐G1 DNA content and the appearance of apoptotic nuclei after DAPI staining. Furanodienone specifically down‐regulated ERα protein and mRNA expression levels without altering ERβ expression. Furanodienone treatment inhibited E2‐stimulation of estrogen response element (ERE)‐driven reporter plasmid activity and ablated E2‐targeted gene (e.g., c‐Myc, Bcl‐2, and cyclin D1) expression which resulted in the inhibition of cell cycle progression and cell proliferation, and in the induction of apoptosis. Knockdown of ERα in MCF‐7 cells by ERα‐specific siRNA decreased the cell growth inhibitory effect of furanodienone. These findings suggest that effects of furanodienone on MCF‐7 cells are mediated, at least in part, by inhibiting ERα signaling. J. Cell. Biochem. 112: 217–224, 2011. © 2010 Wiley‐Liss, Inc.     

NDV‐D90 inhibits 17β‐estradiol‐mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV‐D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β‐estradiol (E2). We found that NDV‐D90 significantly killed the tumor cell lines MCF‐7 and BT549 in a time‐ and dose‐dependent manner. We also found that NDV‐D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV‐D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF‐7 cells (ER‐positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis‐related proteins through ERα‐and GPER‐independent processes. NDV‐D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER‐negative cells), possibly by impairing E2‐mediated GPER expression. Furthermore, NDV‐D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV‐D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER‐positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.     

NF‐κB and estrogen receptor α interactions: Differential function in estrogen receptor‐negative and ‐positive hormone‐independent breast cancer cells

Estrogen receptor (ER)‐positive breast cancer cells have low levels of constitutive NF‐κB activity while ER negative (?) cells and hormone‐independent cells have relatively high constitutive levels of NF‐κB activity. In this study, we have examined the aspects of mutual repression between the ERα and NF‐κB proteins in ER+ and ER? hormone‐independent cells. Ectopic expression of the ERα reduced cell numbers in ER+ and ER? breast cancer cell lines while NF‐κB‐binding activity and the expression of several NF‐κB‐regulated proteins were reduced in ER? cells. ER overexpression in ER+/E2‐independent LCC1 cells only weakly inhibited the predominant p50 NF‐κB. GST‐ERα fusion protein pull downs and in vivo co‐immunoprecipitations of NF‐κB:ERα complexes showed that the ERα interacts with p50 and p65 in vitro and in vivo. Inhibition of NF‐κB increased the expression of diverse E2‐regulated proteins. p50 differentially associated directly with the ER:ERE complex in LCC1 and MCF‐7 cells by supershift analysis while p65 antibody reduced ERα:ERE complexes in the absence of a supershift. ChIP analysis demonstrated that NF‐κB proteins are present on an endogenous ERE. Together these results demonstrate that the ER and NF‐κB undergo mutual repression, which may explain, in part, why expression of the ERα in ER? cells does not confer growth signaling. Secondly, the acquisition of E2‐independence in ER+ cells is associated with predominantly p50:p50 NF‐κB, which may reflect alterations in the ER in these cells. Since the p50 homodimer is less sensitive to the presence of the ER, this may allow for the activation of both pathways in the same cell. J. Cell. Biochem. 107: 448–459, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

Estrogen receptor α promotes Cav1.2 ubiquitination and degradation in neuronal cells and in APP/PS1 mice

Cav1.2 is the pore‐forming subunit of L‐type voltage‐gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen‐mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17β‐estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin–proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29‐deubiquitinating enzyme TRAF‐binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane‐permeable PEST peptides prevented ERα‐mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα‐induced Cav1.2 degradation involved K29‐linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.     

Generation and characterization of PDGFRα‐GFPCreERt2 knock‐In mouse line

Platelet‐derived growth factor (PDGF) and its receptor play an important role in embryogenesis. PDGF receptor α (PDGFRα) is expressed specifically in the embryonic day 7.5 (E7.5) mesoderm and in the E9.5 neural crest among other tissues. PDGFRα‐expressing cells and their descendants are involved in the formation of various tissues. To trace PDGFRα‐expressing cells in vivo, we generated a knock‐in mouse line that expressed a fusion protein of green fluorescent protein (GFP), Cre recombinase (Cre), and mutated estrogen receptor ligand‐binding domain (ER^T2) under the control of the PDGFRα promoter. In these mice, Cre activity in PDGFRα‐expressing cells could be induced by tamoxifen treatment. Taken together, our results suggest that the knock‐in mouse line generated here could be useful for studying PDGFRα‐expressing cells and their descendants in vivo at various stages of development. genesis 53:329–336, 2015. © 2015 Wiley Periodicals, Inc.