Rocco Malivindi

Evidence that leptin through STAT and CREB signaling enhances cyclin D1 expression and promotes human endometrial cancer proliferation.

Obesity is a risk factor for endometrial cancer in pre‐ and post‐menopausal women. Leptin, an adipocyte‐derived hormone, in addition to the control weight homeostasis, is implicated in multiple biological actions. A recent study demonstrated that leptin promotes endometrial cancer growth and invasiveness through STAT/MAPK and Akt pathways, but the molecular mechanism involved in such processes still needs to be elucidated. In an attempt to understand the role of leptin in regulating endometrial cancer cells proliferation, we have demonstrated that leptin treatment reduced the numbers of cells in G0/G1‐phase while increased cell population in S‐phase. This effect is associated with an up‐regulation of cyclin D1 together with a down‐regulation of cyclin‐dependent kinase inhibitor p21WAF1/Cip1. Mutagenesis studies, eletrophoretic mobility shift, and chromatin immunoprecipitation analysis revealed that signal transducers and activators of transcription 3 (STAT3) and cyclic AMP‐responsive element (CRE) binding protein motifs, within cyclin D1 promoter, were required for leptin‐induced cyclin D1 expression in Ishikawa endometrial cancer cells. Silencing of STAT3 and CREB gene expression by RNA interference reversed the up‐regulatory effect of leptin on cyclin D1 expression and cells proliferation. These results support the hypothesis that STAT3 and CREB play an important role in leptin signaling pathway that leads to the proliferation of Ishikawa cells, thus establishing a direct association between obesity and endometrial tumorogenesis. J. Cell. Physiol. 218: 490–500, 2009.

Insulin-Like Growth Factor-I, Regulating Aromatase Expression through Steroidogenic Factor 1, Supports Estrogen-Dependent Tumor Leydig Cell Proliferation

The aim of this study was to investigate the role of estrogens in Leydig cell tumor proliferation. We used R2C rat Leydig tumor cells and testicular samples from Fischer rats with a developed Leydig tumor. Both experimental models express high levels of aromatase and estrogen receptor alpha (ERalpha). Treatment with exogenous 17beta-estradiol (E(2)) induced proliferation of R2C cells and up-regulation of cell cycle regulators cyclin D1 and cyclin E, the expression of which was blocked by addition of antiestrogens. These observations led us to hypothesize an E(2)/ERalpha-dependent mechanism for Leydig cell tumor proliferation. In determining the molecular mechanism responsible for aromatase overexpression, we found that total and phosphorylated levels of transcription factors cyclic AMP-responsive element binding protein and steroidogenic factor 1 (SF-1) were higher in tumor samples. Moreover, we found that tumor Leydig cells produce high levels of insulin-like growth factor I (IGF-I), which increased aromatase mRNA, protein, and activity as a consequence of increased total and phosphorylated SF-1 levels. Specific inhibitors of IGF-I receptor, protein kinase C, and phosphatidylinositol 3-kinase determined a reduction in SF-1 expression and in IGF-I-dependent SF-1 recruitment to the aromatase PII promoter. The same inhibitors also inhibited aromatase expression and activity and, consequently, R2C cell proliferation. We can conclude that one of the molecular mechanisms determining Leydig cell tumorigenesis is an excessive estrogen production that stimulates a short autocrine loop determining cell proliferation. In addition, cell-produced IGF-I amplifies estrogen signaling through an SF-1-dependent up-regulation of aromatase expression. The identification of this molecular mechanism will be helpful in defining new therapeutic approaches for Leydig cell tumors.

Rapid Estradiol/ERα Signaling Enhances Aromatase Enzymatic Activity in Breast Cancer Cells

In situ estrogen production by aromatase conversion from androgens plays an important role in breast tumor promotion. Here, we show that 17beta-estradiol (E2) can rapidly enhance aromatase enzymatic activity through an increase of aromatase protein phosphorylation in breast cancer cell lines. In vivo labeling experiments and site-directed mutagenesis studies demonstrated that phosphorylation of the 361-tyrosine residue is crucial in the up-regulation of aromatase activity under E2 exposure. Our results demonstrated a direct involvement of nonreceptor tyrosine-kinase c-Src in E2-stimulated aromatase activity because inhibition of its signaling abrogated the up-regulatory effects induced by E2 on aromatase activity as well as phosphorylation of aromatase protein. In addition, from our data it emerges that aromatase is a target of cross talk between growth factor receptors and estrogen receptor alpha signaling. These findings show, for the first time, that tyrosine phosphorylation processes play a key role in the rapid changes induced by E2 in aromatase enzymatic activity, revealing the existence of a short nongenomic autocrine loop between E2 and aromatase in breast cancer cells.

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.

Estrogens and PTP1B Function in a Novel Pathway to Regulate Aromatase Enzymatic Activity in Breast Cancer Cells

Local estrogen production by aromatase is an important mechanism of autocrine stimulation in hormone-dependent breast cancer. We have previously shown that 17-β estradiol (E(2)) rapidly enhances aromatase enzymatic activity through an increase of tyrosine protein phosphorylation controlled by the activity of the c-Src kinase in breast cancer cells. Here, we investigated the protein tyrosine phosphatase PTP1B (protein tyrosine phosphatase 1B) as a potential regulator of aromatase activity. We demonstrated a specific association between PTP1B and aromatase at protein-protein level and a reduction of aromatase activity in basal and E(2)-treated MCF-7 and ZR75 breast cancer cells when PTP1B was overexpressed. Indeed, a specific tyrosine phosphatase inhibitor increased basal and E(2)-induced enzymatic activity as well as tyrosine phosphorylation status of the purified aromatase protein. Moreover, E(2) through phosphatidylinositol 3 kinase/Akt activation caused a significant decrease of PTP1B catalytic activity along with an increase in its serine phosphorylation. Concomitantly, the phosphatidylinositol 3 kinase inhibitor LY294002 or a dominant negative of Akt was able to reduce the E(2) stimulatory effects on activity and tyrosine phosphorylation levels of aromatase. Taken together, our results suggest that E(2) can impair PTP1B ability to dephosphorylate aromatase, and thus it increases its enzymatic activity, creating a positive feedback mechanism for estradiol signaling in breast cancer.

Estrogen related receptor α (ERRα) a promising target for the therapy of adrenocortical carcinoma (ACC)

The pathogenesis of the adrenocortical cancer (ACC) involves integration of molecular signals and the interplay of different downstream pathways (i.e. IGFII/IGF1R, β-catenin, Wnt, ESR1). This tumor is characterized by limited therapeutic options and unsuccessful treatments. A useful strategy to develop an effective therapy for ACC is to identify a common downstream target of these multiple pathways. A good candidate could be the transcription factor estrogen-related receptor alpha (ERRα) because of its ability to regulate energy metabolism, mitochondrial biogenesis and signalings related to cancer progression. In this study we tested the effect of ERRα inverse agonist, XCT790, on the proliferation of H295R adrenocortical cancer cell line. Results from in vitro and in vivo experiments showed that XCT790 reduced H295R cell growth. The inhibitory effect was associated with impaired cell cycle progression which was not followed by any apoptotic event. Instead, incomplete autophagy and cell death by a necrotic processes, as a consequence of the cell energy failure, induced by pharmacological reduction of ERRα was evidenced. Our results indicate that therapeutic strategies targeting key factors such as ERRα that control the activity and signaling of bioenergetics processes in high-energy demanding tumors could represent an innovative/alternative therapy for the treatment of ACC.

Inhibition of leydig tumor growth by farnesoid X receptor activation: The in vitro and in vivo basis for a novel therapeutic strategy

Leydig cell tumors (LCTs) are the most common tumors of the gonadal stroma and represent about 3% of all testicular neoplasms. In most cases, LCTs are benign; however, if the tumor is malignant, no effective treatments are currently available. We have recently reported that farnesoid X receptor (FXR) is expressed in R2C Leydig tumor cells, and it reduces the estrogen‐dependent cell proliferation by negatively regulating aromatase expression. Here, we demonstrated that treatment with GW4064, a specific FXR agonist, markedly reduced Leydig tumor growth in vivo by inhibiting proliferation and inducing apoptosis. Indeed, the tumors from GW4064‐treated mice exhibited a decrease in the expression of the proliferation marker Ki‐67 and aromatase along with an increase in the apoptotic nuclei. FXR activation induced an enhanced poly(ADP‐ribose) polymerase cleavage, a marked DNA fragmentation and a strong increase in TUNEL‐positive R2C cells also in vitro. Moreover, in both in vivo and in vitro models, FXR ligands upregulated mRNA and protein levels of p53 and of its downstream effector p21WAF1/Cip1. Functional experiments showed that FXR ligands upregulated p53 promoter activity and this occurred through an increased binding of FXR/nuclear factor‐kB (NF‐kB) complex to the NF‐kB site located within p53 promoter region as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation analysis. Taken together, results from our study show, for the first time, that treatment with FXR ligands induces Leydig tumor regression in vivo, suggesting that activation of FXR may represent a promising therapeutic strategy for LCTs.

Activated FXR Inhibits Leptin Signaling and Counteracts Tumor-promoting Activities of Cancer-Associated Fibroblasts in Breast Malignancy

Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. Herein, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Interestingly, GW4064-treated tumors exhibited decreased levels of leptin-regulated proteins along with a strong staining intensity for SOCS3. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment.

Androgens inhibit aromatase expression through DAX-1: insights into the molecular link between hormone balance and Leydig cancer development.

Leydig cell tumors (LCTs) of the testis are steroid-secreting tumors associated with various steroid biosynthetic abnormalities and endocrine dysfunctions. Despite their overall rarity, LCTs are still of substantial interest owing to the paucity of information regarding their exact nature and malignant potential. In the present study, we disclose the ability of androgens to inhibit Leydig tumor cell proliferation by opposing to self-sufficient in situ estrogen production. In rat Leydig tumor cells, R2C, androgen treatment significantly decreases the expression and the enzymatic activity of cytocrome P450 aromatase, responsible for the local conversion of androgens into estrogens. This inhibitory effect relies on androgen receptor (AR) activation and involves negative regulation of the CYP19 gene transcriptional activity through the nuclear orphan receptor DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1). Ligand-activated AR up-regulates the expression of DAX-1 and promotes its increased recruitment within the steroidogenic factor-1 site-containing region of the aromatase proximal promoter II in association with the nuclear receptor corepressor. The biological relevance in LCTs of the newly highlighted functional interplay between AR, DAX-1, and aromatase is underlined by our in vivo observations, revealing a marked down-regulation of AR and DAX-1 expression and a strong increase in aromatase levels in testes tissues from old Fischer rats with spontaneously developed Leydig cell neoplasia, compared with normal testes tissues from younger animals. In elucidating a mechanism by which androgens modulate the growth of Leydig tumor cells, our finding support the hypothesis that maintaining the adequate balance between androgen and estrogens may represent the key for blocking estrogen-secreting Leydigioma development, opening new prospects for therapeutic intervention.