Peroxisome proliferator-activated receptor-γ and growth inhibition by its ligands in prostate cancer

Background: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is expressed in certain human cancers. Ligand-induced PPAR-γ activation can result in growth inhibition and differentiation in these cancer cells; however, the precise mechanism for the anti-proliferative effect of PPAR-γ ligands is not clear. Methods: In this study, we examined the expression of PPAR-γ in human prostate cancer and the effect of two PPAR-γ ligands, 15 deoxy-Δ^12,14-prostaglandin J2 (15d-PGJ2) and troglitazone, on prostate cancer cell growth. Results: PPAR-γ is frequently over-expressed in androgen independent prostate cancer cell lines and human prostate cancer tissues (22 of 47; 47%). Both 15d-PGJ2 and troglitazone inhibited proliferation and DNA synthesis of prostate cancer cell lines in a dose-dependent manner, and slightly increased the proportion of cells with S-phase DNA content. Prostate specific antigen (PSA) promoter reporter assays showed that troglitazone and 15d-PGJ2 down-regulated androgen stimulated reporter gene activity in prostate cancer cell lines LNCaP. Interestingly, LNCaP with troglitazone dramatically suppressed PSA protein expression without suppressing AR expression. Conclusions: Taken together, these results suggest that PPAR-γ ligands may be a useful therapeutic agent for the treatment of prostate cancer.     

Methylation and silencing of the retinoic acid receptor-β2 gene in cervical cancer

Background  

Expression of the retinoic acid receptor β2 (RAR-β2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-β2 expression remains obscure. We examined whether methylation of RAR-β2 gene could be responsible for this silencing in cervical SCC.     

Transient over-expression of estrogen receptor-�� in breast cancer cells promotes cell survival and estrogen-independent growth

Estrogen receptor-?? (ER??) positive breast cancer frequently responds to inhibitors of ER?? activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ER?? in ER??-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ER?? over-expression in ER??-positive breast cancer cells, we over-expressed ER?? in the MCF-7 breast cancer cell line using adenovirus gene transduction. ER?? over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ER?? transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ER??-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ER?? remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ER?? could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.     

Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses

Introduction

17β-estradiol (E₂) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mER^low) and high (mER^high) expression of the membrane form of estrogen receptor (mER)-α, and thus addressed the receptor subform involved in cAMP signaling.

Methods

MCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mER^high (mER-α-enriched) and mER^low (mER-α-depleted) populations. Unique (compared with previously reported) incubation conditions at 4°C were found to be optimal for demonstrating E₂-induced cAMP production. Time-dependent and dose-dependent effects of E₂ on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E₂-induced cell proliferation were assessed using the crystal violet assay.

Results

We demonstrated a rapid and transient cAMP increase after 1 pmol/l E₂ stimulation in mER^high cells; at 4°C these responses were much more reliable and robust than at 37°C (the condition most often used). The loss of cAMP at 37°C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mER^high cells than in mER^low cells, implicating mER-α levels in the process. ICI172,780 blocked the E₂-induced response and 17α-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E₂ dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E₂ concentrations in mER^high cells. Proliferation of mER^low cells was stimulated over the whole range of E₂concentrations, whereas the number of mER^high cells was greatly decreased at concentrations above 1 nmol/l, suggesting that estrogen over-stimulation can lead to cell death, as has previously been reported, and that mER-α participates. E₂-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses.

Conclusion

Rapid mER-α-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number.

     

Overexpression of estrogen receptor-α in human papillary thyroid carcinomas studied by laser- capture microdissection and molecular biology

The expression pattern of estrogen receptor (ER) isoforms in normal and tumor thyroid tissues is still controversial and poor defined, therefore, a more detailed study of the distribution of these molecules is needed. Most discrepancies might be due to the methods utilized. We studied the expression of ER isoforms in human papillary thyroid carcinoma (PTC), in fine-needle aspiration biopsy-derived specimens, and in cells, using more accurate techniques, such as laser-capture microdissection, real-time quantitative PCR, and Western blot. Laser-capture microdissection allowed us to isolate homogeneous cell populations from human PTC surgical samples. Tumor, peritumor, or normal host tissue of the same sample were separately dissected and analyzed by RT-PCR and Western blot. Estrogen receptor-α mRNA was more expressed in cancer-microdissected cells from human PTC, as compared with microdissected cells obtained from surrounding normal host tissue (450 vs 12, P = 0.001). A similar pattern was observed with Western blot for the ER-a protein. By contrast, ER-β mRNA expression was not detected among the microdissected tissue fractions. Fine-needle aspiration biopsy-derived specimens showed a similar expression pattern to ER. Moreover, human PTC cell line BCPAP and cancer stem cells from PTC, analyzed under hypoxic conditions, showed a hypoxia-driven increase in ER-α expression. In conclusion, ER-α might have an important role in human PTC, and its overexpression can be studied in routine needle aspirate as a possible marker of malignancy.     

Estrogen receptor-β protects against colitis-associated neoplasia in mice

Estrogen receptor-beta (ERβ) has been suggested to exert anti-inflammatory and anti-tumorigenic effects in the colon, providing a translational potential to prevent and/or treat inflammatory bowel disease (IBD) and its progression to colitis-associated colorectal cancer (CAC). However, the specific direct role of ERβ in CAC has not yet been tested. We assessed the effects of ERβ deficiency in the azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CAC model using ERβ knockout (βERKO) mice and wild-type (WT) littermates. These mice were injected with AOM followed by 1 week of DSS treatment, and sacrificed on weeks 9 or 16. βERKO mice developed more severe clinical colitis compared to WT mice, as evidenced by significantly higher disease activity index after DSS treatment, weight to length ratio of the colons, inflammation score and grade of dysplasia. ERβ-deficient colons presented greater number and size of polyps at weeks 9 and 16, respectively, and were characterized by a significant increase in interleukin (IL)-6, IL-17, tumor necrosis factor alpha and interferon-gamma mRNA levels. Furthermore, higher protein expression levels of nuclear factor-kappa B, inducible nitric oxide synthase, β-catenin, proliferating cell nuclear antigen, mucin-1 and significantly lower caveolin-1 and mucin-2 protein levels were shown in βERKO mice compared to WT mice. These data suggest a possible anti-inflammatory and anti-neoplastic mechanism of action of ERβ in CAC. These results demonstrate for the first time that ERβ provides protection in the AOM/DSS-induced CAC model in mice, suggesting a preventive and/or therapeutic potential for the use of ERβ-selective agonists in IBD.     

Functional activation of the estrogen receptor-α and aromatase by the HDAC inhibitor entinostat sensitizes ER-negative tumors to letrozole

Approximately 25% of breast cancers do not express the estrogen receptor-α (ERα) and consequently do not respond to endocrine therapy. In these tumors, ERα repression is often due to epigenetic modifications such as methylation and histone deacetylation. For this reason, we investigated the ability of the histone deacetylase inhibitor entinostat (ENT) to trigger reexpression of ERα and aromatase in breast cancer cells, with the notion that this treatment would restore sensitivity to the aromatase inhibitor (AI) letrozole. ENT treatment of tumor cells increased expression of ERα and aromatase, along with the enzymatic activity of aromatase, in a dose-dependent manner both in vitro and in vivo. Notably, ERα and aromatase upregulation resulted in sensitization of breast cancer cells to estrogen and letrozole. Tumor growth rate was significantly lower in tumor xenografts following treatment with ENT alone and in combination with letrozole than in control tumors (P > 0.001). ENT plus letrozole also prevented lung colonization and growth of tumor cells, with a significant reduction (P > 0.03) in both visible and microscopic foci. Our results show that ENT treatment can be used to restore the letrozole responsiveness of ER-negative tumors. More generally, they provide a strong rationale for immediate clinical evaluation of combinations of histone deacetylase and aromatase inhibitors to treat ER-negative and endocrine-resistant breast cancers.     

Endocrinology and hormone therapy in breast cancer: New insight into estrogen receptor-α function and its implication for endocrine therapy resistance in breast cancer

Estrogen and its receptor (ER) are critical for development and progression of breast cancer. This pathway is targeted by endocrine therapies that either block ER functions or deplete ER's estrogen ligand. While endocrine therapies are very effective, de novo and acquired resistance are still common. Laboratory and clinical data now indicate that bidirectional molecular crosstalk between nuclear or membrane ER and growth factor receptor pathways such as HER2/neu is involved in endocrine resistance. Preclinical data suggest that blockade of selected growth factor receptor signaling can overcome this type of resistance, and this strategy is already being tested in clinical trials     

Anterior gradient-2 plays a critical role in breast cancer cell growth and survival by modulating cyclin D1, estrogen receptor-α and survivin

Introduction  

Anterior-gradient 2 (AGR2) is an estrogen-responsive secreted protein. Its upregulation has been well documented in a number of cancers, particularly breast cancer, for which mixed data exist on the prognostic implications of AGR2 expression. Although emerging evidence indicates that AGR2 is associated with poor prognosis, its function and impact on cancer-relevant pathways have not been elucidated in breast cancer.     

Assessment of folate receptor-β expression in human neoplastic tissues

Over-expression of folate receptor alpha on cancer cells has been frequently exploited for delivery of folate-targeted imaging and therapeutic agents to tumors. Because limited information exists on expression of the beta isoform of the folate receptor in human cancers (FR-β), we have evaluated the immunohistochemical staining pattern of FR-β in 992 tumor sections from 20 different human cancer types using a new anti-human FR-β monoclonal antibody. FR-β expression was shown to be more pronounced in cells within the stroma, primarily macrophages and macrophage-like cells than cancer cells in every cancer type studied. Moreover, FR-β expression in both cancer and stromal cells was found to be statistically more prominent in females than males. A significant positive correlation was also observed between FR-β expression on stromal cells and both the stage of the cancer and the presence of lymph node metastases. Based on these data we conclude FR-β may constitute a good target for specific delivery of therapeutic agents to activated macrophages and that accumulation of FR-β positive macrophages in the stroma could serve as a useful indicator of a tumor''s metastatic potential.     

In vitro and in vivo evaluation of ⁶⁴Cu-radiolabeled KCCYSL peptides for targeting epidermal growth factor receptor-2 in breast carcinomas

Epidermal growth factor receptor-2 (EGFR-2) has been implicated in the pathogenesis of breast and other carcinomas. In this report, we tested the ability of a bacteriophage selected peptide KCCYSL, radiolabeled with ??Cu, to image EGFR-2 expressing breast tumors in vivo by positron emission tomography (PET). We evaluated and compared the in vivo tissue distribution and imaging properties of ??Cu-X-(Gly-Ser-Gly)-KCCYSL peptide (X?=?1,4,7,10, tetraazacyclododecane-N,N',N',N'-tetracetic acid, [DOTA] 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid [CB-TE2A], and 1,4,7-triazacyclononane-1,4,7-triacetic acid [NOTA] chelators) in a human breast cancer xenograft mouse model using dual modality PET and computed tomography (CT). The synthesized peptides DO3A-GSG-KCCYSL, CB-TE2A-GSG-KCCYSL, and NO2A-GSG-KCCYSL were purified, radiolabeled with ??Cu, and evaluated for human breast cancer cell (MDA-MB-435) binding, 50% inhibitory concentration, and serum stability. In vivo pharmacokinetic and small animal PET/CT imaging studies were performed using SCID mice bearing MDA-MB-435 xenografts. The radiolabeled peptides bound with an 50% inhibitory concentration of 42.0?±?10.2?nM (DO3A), 31?±?7.9?nM (CB-TE2A), and 44.2?±?6.6?nM (NO2A) to cultured MDA-MB-435 cells. All of the radiolabeled peptides were stable in vitro. The tumor uptake of DO3A, CB-TE2A, and NO2A peptides were 0.73?±?0.15 percent injected dose per gram (%ID/g), 0.64?±?0.08%ID/g, and 0.52?±?0.04%ID/g, respectively at 1 hour. Liver uptake for the ??Cu-DO3A-peptide (1.68?±?0.42%ID/g) was more than that of the ??Cu-CB-TE2A-peptide (0.52?±?0.02% ID/g) and ??Cu-NO2A-peptide (0.48?±?0.05%ID/g) at 2 hours. PET/CT studies revealed successful tumor uptake of ??Cu-peptides at 2 hours postinjection. In vivo kidney retention was observed with all of the radiolabeled peptides. The optimization of bifunctional chelators improves the pharmacokinetic properties of the ??Cu-labeled GSG-KCCYSL peptide, which enables the selection of a suitable peptide homolog as a PET imaging agent for EGFR-2 expressing breast carcinomas.     

Investigating the role of endogenous estrogens,hormone replacement therapy,and blockade of estrogen receptor-α activity on breast metabolic signaling

Breast Cancer Research and Treatment - Impaired DNA repair mechanism is one of the cancer hallmarks. Flap Endonuclease 1 (FEN1) is essential for genomic integrity. FEN1 has key roles during base...     

The effect of fenofibrate,a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo

Clinical and Translational Oncology - The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown...     

Withaferin a suppresses estrogen receptor-α expression in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of MCF-7 and MDA-MB-231 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo by causing apoptosis. However, the mechanism of WA-induced apoptosis is not fully understood. The present study was designed to systematically determine the role of tumor suppressor p53 and estrogen receptor-α (ER-α) in proapoptotic response to WA using MCF-7, T47D, and ER-α overexpressing MDA-MB-231 cells as a model. WA treatment resulted in induction as well as increased S15 phosphorylation of p53 in MCF-7 cells, but RNA interference of this tumor suppressor conferred modest protection at best against WA-induced apoptosis. WA-mediated growth inhibition and apoptosis induction in MCF-7 cells were significantly attenuated in the presence of 17β-estradiol (E2). Exposure of MCF-7 cells to WA resulted in a marked decrease in protein levels of ER-α (but not ER-β) and ER-α regulated gene product pS2, and this effect was markedly attenuated in the presence of E2. WA-mediated down-regulation of ER-α protein expression correlated with a decrease in its nuclear level, suppression of its mRNA level, and inhibition of E2-dependent activation of ERE2e1b-luciferase reporter gene. Ectopic expression of ER-α in the MDA-MB-231 cell line conferred partial but statistically significant protection against WA-mediated apoptosis, but not G2/M phase cell cycle arrest. Collectively, these results indicate that WA functions as an anti-estrogen, and the proapoptotic effect of this promising natural product is partially attenuated by p53 knockdown and E2-ER-α.