Maria Luisa Panno

Breast cancer: from estrogen to androgen receptor

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.

The G protein-coupled receptor 30 is up-regulated by hypoxia-inducible factor-1alpha (HIF-1alpha) in breast cancer cells and cardiomyocytes.

GPR30, also known as GPER, has been suggested to mediate rapid effects induced by estrogens in diverse normal and cancer tissues. Hypoxia is a common feature of solid tumors involved in apoptosis, cell survival, and proliferation. The response to low oxygen environment is mainly mediated by the hypoxia-inducible factor named HIF-1α, which activates signaling pathways leading to adaptive mechanisms in tumor cells. Here, we demonstrate that the hypoxia induces HIF-1α expression, which in turn mediates the up-regulation of GPER and its downstream target CTGF in estrogen receptor-negative SkBr3 breast cancer cells and in HL-1 cardiomyocytes. Moreover, we show that HIF-1α-responsive elements located within the promoter region of GPER are involved in hypoxia-dependent transcription of GPER, which requires the ROS-induced activation of EGFR/ERK signaling in both SkBr3 and HL-1 and cells. Interestingly, the apoptotic response to hypoxia was prevented by estrogens through GPER in SkBr3 cells. Taken together, our data suggest that the hypoxia-induced expression of GPER may be included among the mechanisms involved in the anti-apoptotic effects elicited by estrogens, particularly in a low oxygen microenvironment.

PEG-templated mesoporous silica nanoparticles exclusively target cancer cells

Mesoporous silica nanoparticles (MSNs) have been proposed as DNA and drug delivery carriers, as well as efficient tools for fluorescent cell tracking. The major limitation is that MSNs enter cells regardless of a target-specific functionalization. Here we show that non functionalized MSNs, synthesized using a PEG surfactant-based interfacial synthesis procedure, do not enter cells, while a highly specific, receptor mediated, cellular internalization of folic acid (FOL) grafted MSNs (MSN-FOL), occurs exclusively in folate receptor (FR) expressing cells. Neither the classical clathrin pathway nor macropinocytosis is involved in the MSN endocytic process, while fluorescent MSNs (MSN-FITC) enter cells through aspecific, caveolae-mediated, endocytosis. Moreover, internalized particles seem to be mostly exocytosed from cells within 96 h. Finally, cisplatin (Cp) loaded MSN-FOL were tested on cancerous FR-positive (HeLa) or normal FR-negative (HEK293) cells. A strong growth arrest was observed only in HeLa cells treated with MSN-FOL-Cp. The results presented here show that our mesoporous nanoparticles do not enter cells unless opportunely functionalized, suggesting that they could represent a promising vehicle for drug targeting applications.

Combined Low Doses of PPARγ and RXR Ligands Trigger an Intrinsic Apoptotic Pathway in Human Breast Cancer Cells

Ligand activation of peroxisome proliferator-activated receptor (PPAR)gamma and retinoid X receptor (RXR) induces antitumor effects in cancer. We evaluated the ability of combined treatment with nanomolar levels of the PPARgamma ligand rosiglitazone (BRL) and the RXR ligand 9-cis-retinoic acid (9RA) to promote antiproliferative effects in breast cancer cells. BRL and 9RA in combination strongly inhibit of cell viability in MCF-7, MCF-7TR1, SKBR-3, and T-47D breast cancer cells, whereas MCF-10 normal breast epithelial cells are unaffected. In MCF-7 cells, combined treatment with BRL and 9RA up-regulated mRNA and protein levels of both the tumor suppressor p53 and its effector p21(WAF1/Cip1). Functional experiments indicate that the nuclear factor-kappaB site in the p53 promoter is required for the transcriptional response to BRL plus 9RA. We observed that the intrinsic apoptotic pathway in MCF-7 cells displays an ordinated sequence of events, including disruption of mitochondrial membrane potential, release of cytochrome c, strong caspase 9 activation, and, finally, DNA fragmentation. An expression vector for p53 antisense abrogated the biological effect of both ligands, which implicates involvement of p53 in PPARgamma/RXR-dependent activity in all of the human breast malignant cell lines tested. Taken together, our results suggest that multidrug regimens including a combination of PPARgamma and RXR ligands may provide a therapeutic advantage in breast cancer treatment.

Interaction between estrogen receptor alpha and insulin/IGF signaling in breast cancer.

Estrogens and insulin/Insulin like growth factor 1 (IGF-I) have potent positive effects on the proliferation of mammary epithelial cells and estrogen-dependent breast cancer cells. A cooperative crosstalk between estrogens and insulin/IGF-I signaling pathways exists and it plays a critical role in breast carcinogenesis, tumor cell proliferation, differentiation and survival through the modulation of multiple biological events. The biological effects of estrogens are mainly mediated by the activation of estrogen receptor (ERalpha) whose activity is deeply influenced by the insulin/IGF-I signaling pathway. On the other hand, estrogens enhance insulin signaling by increasing the expression and/or the functional activity of some proteins involved in the insulin/IGF-I pathway. This review will focus on the critical node of the IGF-I network involved in the crosstalk with ERalpha and implicated in breast cancer development and progression.

Resveratrol, through NF-Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38MAPK/CK2 signaling in human breast cancer cells

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long‐term benefits of hormonal therapy. In the present study, we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation, including MCF‐7 tamoxifen‐resistant cells (IC50 values for viability were in the 30–45 μM range). We show that Res, through p38MAPK phosphorylation, causes induction of p53, which recruits at the estrogen receptor α (ERα) proximal promoter, leading to an inhibition of ERα expression in terms of mRNA and protein content. These events appear specifically p53 dependent, since they are drastically abrogated with p53‐targeting siRNA. Coimmunoprecipitation assay showed specific interaction between p53, the Sin3A corepressor, and histone deacetylase 1 (HDAC1), which was phosphorylated. The enhancement of the tripartite complex p53/Sin3A/HDAC1, together with NF‐Y on Res treatment, was confirmed by chromatin immunoprecipitation analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF‐7 tamoxifen‐resistant cells at a higher extent than parental MCF‐7 cells addresses how Res may be considered a useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.—De Amicis, F., Giordano, F., Vivacqua, A., Pellegrino, M., Panno, M. L., Tramontano, D., Fuqua, S. A. W., Andò, S. Resveratrol, through NF‐Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expression via p38MAPK/CK2 signaling in human breast cancer cells. FASEB J. 25, 3695–3707 (2011). www.fasebj.org

Estrogen receptor beta (ERβ) produces autophagy and necroptosis in human seminoma cell line through the binding of the Sp1 on the phosphatase and tensin homolog deleted from chromosome 10 (PTEN) promoter gene.

Testicular germ cell tumors are the most common tumor in male and the least studied. We focused on human seminoma using the TCAM2 cell line. Through ERβ, 10 nM estradiol (E2) was able to induce PTEN gene expression and promoter transactivation. Transient transfections, ChIP and EMSA assays evidenced the 5′-flanking region of PTEN gene promoter E2-responsive. The ERβ binding to the Sp1 on PTEN promoter decreased cell survival. The presence of ERβ or PTEN is necessary to induce the loss of cell survival upon E2, addressing their cooperation in this action. pAKT and AKT expression decreased under E2 and DPN, while known apoptotic markers appeared to be unchanged. The PI3K/AKT pathway inhibition also leads to autophagy: E2 and DPN enhanced the expression of autophagy-related markers such as PI3III, Beclin 1, AMBRA and UVRAG. MDC and TEM assays confirmed E2-induced autophagy. The absence of DNA fragmentation, caspase 9 and PARP1 cleavages suggested that necroptosis and/or parthanatos may occur. FACS analysis, LDH assay and RIP1 expression attested this hypothesis. Our study reveals a unique mechanism through which ERβ/PTEN signaling induces cell death in TCAM2 by autophagy and necroptosis. These data, supporting estrogen-dependency of human seminoma, propose ERβ ligands for therapeutic use in the treatment of this pathological condition.

A cross-talk between the androgen receptor and the epidermal growth factor receptor leads to p38MAPK-dependent activation of mTOR and cyclinD1 expression in prostate and lung cancer cells

In androgen sensitive LNCaP prostate cancer cells, the proliferation induced by the epidermal growth factor (EGF) involves a cross-talk between the EGF receptor (EGFR) and the androgen receptor (AR). In lung cancer the role of the EGF-EGFR transduction pathway has been documented, whereas androgen activity has received less attention. Here we demonstrate that in LNCaP and A549 non-small cell lung cancer (NSCLC), AR and EGFR are required for either 5alpha-dihydrotestosterone (DHT) or EGF-stimulated cell growth. Only EGF activated ERK signaling and up-regulated early gene expression, while DHT triggered the expression of classical AR-responsive genes with the exception of the EGF-induced PSA transcript in A549 cells. DHT and EGF up-regulated cyclinD1 (CD1) at both mRNA and protein levels in A549 cells, while in LNCaP cells each mitogen increased only CD1 protein expression. In both cell contexts, CD1 up-regulation was prevented by selective inhibitors as well as by knock-down of either AR or EGFR and also inhibiting p38MAPK and the mammalian target of rapamycin (mTOR) pathways. Interestingly, p38MAPK and mTOR repression prevented the activation of the mTOR target ribosomal p70S6 kinase induced by DHT and EGF, indicating that p38MAPK acts as an upstream mTOR regulator. In addition, the proliferative effects promoted by both DHT and EGF in LNCaP and A549 cancer cells were no longer observed blocking either p38MAPK or mTOR activity. Hence, our data suggest that p38MAPK-dependent activation of the mTOR/CD1 pathway may represent a mechanism through which AR and EGFR cross-talk contributes to prostate and lung cancer progression.

Farnesoid X Receptor, through the Binding with Steroidogenic Factor 1-responsive Element, Inhibits Aromatase Expression in Tumor Leydig Cells

The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that regulates bile acid homeostasis. It is expressed in the liver and the gastrointestinal tract, but also in several non-enterohepatic tissues including testis. Recently, FXR was identified as a negative modulator of the androgen-estrogen-converting aromatase enzyme in human breast cancer cells. In the present study we detected the expression of FXR in Leydig normal and tumor cell lines and in rat testes tissue. We found, in rat Leydig tumor cells, R2C, that FXR activation by the primary bile acid chenodeoxycholic acid (CDCA) or a synthetic agonist GW4064, through a SHP-independent mechanism, down-regulates aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. Transient transfection experiments, using vector containing rat aromatase promoter PII, evidenced that CDCA reduces basal aromatase promoter activity. Mutagenesis studies, electrophoretic mobility shift, and chromatin immunoprecipitation analysis reveal that FXR is able to compete with steroidogenic factor 1 in binding to a common sequence present in the aromatase promoter region interfering negatively with its activity. Finally, the FXR-mediated anti-proliferative effects exerted by CDCA on tumor Leydig cells are at least in part due to an inhibition of estrogen-dependent cell growth. In conclusion our findings identify for the first time the activators of FXR as negative modulators of the aromatase enzyme in Leydig tumor cell lines.

Progesterone Receptor B Recruits a Repressor Complex to a Half-PRE Site of the Estrogen Receptor α Gene Promoter

In the present study, we demonstrate that elevated levels of the progesterone receptor (PR)-B isoform in breast cancer cells induces down-regulation of estrogen receptor (ER) alpha mRNA and protein content, causing concomitant repression of the estrogen-regulated genes insulin receptor substrate 1, cyclin D1, and pS2, addressing a specific effect of PR/PR-B on ERalpha gene transcription. ERalpha gene promoter activity was drastically inhibited by PR-B overexpression. Promoter analysis revealed a transcriptionally responsive region containing a half-progesterone response element (PRE) site located at -1757 bp to -1752 bp. Mutation of the half-PRE down-regulated the effect induced by PR/PR-B overexpression. Moreover chromatin immunoprecipitation analyses revealed an increase of PR bound to the ERalpha-regulatory region encompassing the half-PRE site, and the recruitment of a corepressor complex containing nuclear receptor corepressor (NCoR) but not silencing mediator of retinoid and thyroid hormone receptor and DAX1, concomitantly with hypoacetylation of histone H4 and displacement of RNA polymerase II. Furthermore, NCoR ablation studies demonstrated the crucial involvement of NCoR in the down-regulatory effects due to PR-B overexpression on ERalpha protein and mRNA. We also demonstrated that the ERalpha regulation observed in MCF-7 cells depended on PR-B expression because PR-B knockdown partially abrogates the feedback inhibition of ERalpha levels after estrogenic stimulus. Our study provides evidence for a mechanism by which overexpressed PR-B is able to actively repress ERalpha gene expression.