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.     

LRP16与 ERα相互作用增强ERα介导的转录活性

LRP16是一个雌激素（E2）通过受体α（ERα）诱导表达的靶基因,LRP16不仅促进人乳腺癌MCF-7细胞的增殖,而且促进细胞的侵袭生长,但对LRP16作用的分子机制尚不清楚.首先,检测到LRP16表达缺陷明显削弱了MCF-7细胞对E2的反应性增殖能力;采用Northern 印迹与Western印迹法,进一步检测到抑制LRP16表达,明显损害了ERα靶基因对E2诱导上调的反应性,这些基因包括了cyclin D1, c-myc, c-fos,MTA3, pS2和维甲酸受体α基因（RARα）等;以上结果提示,LRP16参与了ERα介导的信号途径.将ERα模式启动子报告基因3×ERE-TATA-luc,ERα以及LRP16表达载体共转染MCF-7与HeLa细胞.结果发现,LRP16增强了ERα对报告基因的转录激活,并呈现对LRP16的剂量依赖性;进而采用GST-pull down以及免疫共沉淀方法证实了LRP16与ERα之间的直接相互作用,该作用不依赖于E2的存在,但可被E2增强;采用哺乳动物双杂交方法进一步证实了ERα与LRP16相互作用位点存在于A/B区的激活功能域-１（AF1）.以上结果表明,LRP16是ERα的一个共激活因子,通过相互作用,LRP16增强了ERα介导转录活性.该研究为LRP16促进ERα阳性乳腺癌细胞增殖与侵袭生长提供了合理的分子解释.     

Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERα-induced proliferation of breast cancer cells

Estrogens, acting through estrogen receptor α (ERα), stimulate breast cancer proliferation, making ERα an attractive drug target. Since 384-well format screens for inhibitors of proliferation can be challenging for some cells, inhibition of luciferase-based reporters is often used as a surrogate end point. To identify novel small-molecule inhibitors of 17β-estradiol (E(2))-ERα-stimulated cell proliferation, we established a cell-based screen for inhibitors of E(2)-ERα induction of an estrogen response element (ERE)(3)-luciferase reporter. Seventy-five "hits" were evaluated in tiered follow-up assays to identify where hits failed to progress and evaluate their effectiveness as inhibitors of E(2)-ERα-induced proliferation of breast cancer cells. Only 8 of 75 hits from the luciferase screen inhibited estrogen-induced proliferation of ERα-positive MCF-7 and T47D cells but not control ERα-negative MDA-MB-231 cells. Although 12% of compounds inhibited E(2)-ERα-stimulated proliferation in only one of the ERα-positive cell lines, 40% of compounds were toxic and inhibited growth of all the cell lines, and ~37% exhibited little or no ability to inhibit E(2)-ERα-stimulated cell proliferation. Representative compounds were evaluated in more detail, and a lead ERα inhibitor was identified.     

The DHEA metabolite 7β-hydroxy-epiandrosterone exerts anti-estrogenic effects on breast cancer cell lines

7β-Hydroxy-epiandrosterone (7β-OH-EpiA), an endogenous androgenic derivative of dehydroepiandrosterone, has previously been shown to exert anti-inflammatory action in vitro and in vivo via a shift from prostaglandin E2 (PGE2) to 15-deoxy-Δ(12,14)-PGJ2 production. This modulation in prostaglandin production was obtained with low concentrations of 7β-OH-EpiA (1-100nM) and suggested that it might act through a specific receptor. Inflammation and prostaglandin synthesis is important in the development and survival of estrogen-dependent mammary cancers. Estrogen induced PGE2 production and cell proliferation via its binding to estrogen receptors (ERs) in these tumors. Our objective was to test the effects of 7β-OH-EpiA on the proliferation (by counting with trypan blue exclusion), cell cycle and cell apoptosis (by flow cytometry) of breast cancer cell lines MCF-7 (ERα+, ERβ+, G-protein coupled receptor 30: GPR30+) and MDA-MB-231 (ERα-, ERβ+, GPR30+) and to identify a potential target of this steroid in these cell lineages (by transactivations) and in the nuclear ER-negative SKBr3 cells (GPR30+) (by proliferation assays). 7β-OH-EpiA exerted anti-estrogenic effects in MCF-7 and MDA-MB-231 cells associated with cell proliferation inhibition and cell cycle arrest. Moreover, transactivation and proliferation with ER agonists assays indicated that 7β-OH-EpiA interacted with ERβ. Data from proliferation assays on the MCF-7, MDA-MB-231 and SKBr3 cell lines suggested that 7β-OH-EpiA may also act through the membrane GPR30 receptor. These results support that this androgenic steroid acts as an anti-estrogenic compound. Moreover, this is the first evidence that low doses of androgenic steroid exert antiproliferative effects in these mammary cancer cells. Further investigations are needed to improve understanding of the observed actions of endogenous 7β-OH-EpiA.     

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.     

Estrogen receptor alpha mediates epithelial to mesenchymal transition,expression of specific matrix effectors and functional properties of breast cancer cells

The 17β-estradiol (E2)/estrogen receptor alpha (ERα) signaling pathway is one of the most important pathways in hormone-dependent breast cancer. E2 plays pivotal roles in cancer cell growth, survival, and architecture as well as in gene expression regulatory mechanisms. In this study, we established stably transfected MCF-7 cells by knocking down the ERα gene (designated as MCF-7/SP10 + cells), using specific shRNA lentiviral particles, and compared them with the control cells (MCF-7/c). Interestingly, ERα silencing in MCF-7 cells strongly induced cellular phenotypic changes accompanied by significant changes in gene and protein expression of several markers typical of epithelial to mesenchymal transition (EMT). Notably, these cells exhibited enhanced cell proliferation, migration and invasion. Moreover, ERα suppression strongly affected the gene and protein expression of EGFR and HER2 receptor tyrosine kinases, and various extracellular matrix (ECM) effectors, including matrix metalloproteinases and their endogenous inhibitors (MMPs/TIMPs) and components of the plasminogen activation system. The action caused by E2 in MCF-7/c cells in the expression of HER2, MT1-MMP, MMP1, MMP9, uPA, tPA, and PAI-1 was abolished in MCF-7/SP10 + cells lacking ERα. These data suggested a regulatory role for the E2/ERα pathway in respect to the composition and activity of the extracellular proteolytic molecular network. Notably, loss of ERα promoted breast cancer cell migration and invasion by inducing changes in the expression levels of certain matrix macromolecules (especially uPA, tPA, PAI-1) through the EGFR–ERK signaling pathway.In conclusion, loss of ERα in breast cancer cells results in a potent EMT characterized by striking changes in the expression profile of specific matrix macromolecules highlighting the potential nodal role of matrix effectors in breast cancer endocrine resistance.     

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.     

HIF-1α acts downstream of TNF-α to inhibit vasodilator-stimulated phosphoprotein expression and modulates the adhesion and proliferation of breast cancer cells

Metastasis is the leading cause of death in breast cancer patients. Recent evidence suggests that inflammation-related cytokine tumor necrosis factor-alpha (TNF-α) is implicated in tumor invasion and metastasis, but the mechanism of its involvement remains elusive. In this study, we employed MCF-7 breast cancer cells as an experimental model to demonstrate that TNF-α inhibits breast cancer cell adhesion and cell proliferation through hypoxia inducible factor-1alpha (HIF-1α) mediated suppression of vasodilator-stimulated phosphoprotein (VASP). We observed that TNF-α treatment attenuated the adhesion and proliferation of MCF-7 cells it also dramatically increased HIF-1α expression and decreased VASP expression. Through a variety of approaches, including promoter assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP), we identified VASP as a direct target gene of HIF-1α. In addition, we confirmed that HIF-1α mediated the repression of VASP expression by TNF-α in MCF-7 cells. We also demonstrated that exogenous VASP expression or knockdown of HIF-1α relieved TNF-α induced inhibition of cell adhesion and proliferation. We identified a novel TNF-α/HIF-1α/VASP axis in which HIF-1α acts downstream of TNF-α to inhibit VASP expression and modulate the adhesion and proliferation of breast cancer cells. These data provide new insight into the potential anti-tumor effects of TNF-α.     

High levels of arachidonic acid and peroxisome proliferator-activated receptor-alpha in breast cancer tissues are associated with promoting cancer cell proliferation

Fatty acids are endogenous ligands of peroxisome proliferator-activated receptor-alpha (PPARα), which is linked to the regulation of fatty acid uptake, lipid metabolism and breast cancer cell growth. This study was designed to screen candidate fatty acids from breast cancer tissue and to investigate the effects of these candidate fatty acids on PPARα expression, cell growth and cell cycle progression in breast cancer cell lines. One breast cancer tissue and one reference tissue were each taken from 30 individual breasts to examine for fatty acid composition and PPARα expression. The cancer cell lines MDA-MB-231 (ER–), MCF-7 (ER++++) and BT-474 (ER++) were used to explore the mechanisms regulating cell proliferation. We found that arachidonic acid (AA) and PPARα were highly expressed in the breast cancer tissues. AA stimulated the growth of all three breast cancer cells in a time- and dose-dependent manner. The growth stimulatory effect of AA was associated with PPARα activation, and the most potent effect was found in MCF-7 cells. The stimulation of cell proliferation by AA was accompanied by the increased expression of cyclin E, a reduced population of G1 phase cells, and a faster G1/S phase transition. In contrast, AA had no effects on the levels of CDK2, CDK4, cyclin D1, p27, Bcl-2 and Bax. Our results demonstrate that high levels of AA and PPARα expression in human breast cancer tissues are associated with ER-overexpressed breast cancer cell proliferation, which is involved in activating PPARα, stimulating cyclin E expression, and promoting faster G1/S transition.