Disruption of 3D MCF-12A breast cell cultures by estrogens - An in vitro model for ER-mediated changes indicative of hormonal carcinogenesis

Copyright @ 2012 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and 85 reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel University Open Access Publishing Fund.Introduction: Estrogens regulate the proliferation of normal and neoplastic breast epithelium. Although the intracellular mechanisms of estrogens in the breast are largely understood, little is known about how they induce changes in the structure of the mammary epithelium, which are characteristic of breast cancer. In vitro three dimensional (3D) cultures of immortalised breast epithelial cells recapitulate features of the breast epithelium in vivo, including formation of growth arrested acini with hollow lumen and basement membrane. This model can also reproduce features of malignant transformation and breast cancer, such as increased cellular proliferation and filling of the lumen. However, a system where a connection between estrogen receptor (ER) activation and disruption of acini formation can be studied to elucidate the role of estrogens is still missing. Methods/Principal Findings: We describe an in vitro 3D model for breast glandular structure development, using breast epithelial MCF-12A cells cultured in a reconstituted basement membrane matrix. These cells are estrogen receptor (ER)α, ERβ and G-protein coupled estrogen receptor 1 (GPER) competent, allowing the investigation of the effects of estrogens on mammary gland formation and disruption. Under normal conditions, MCF-12A cells formed organised acini, with deposition of basement membrane and hollow lumen. However, treatment with 17β-estradiol, and the exogenous estrogens bisphenol A and propylparaben resulted in deformed acini and filling of the acinar lumen. When these chemicals were combined with ER and GPER inhibitors (ICI 182,780 and G-15, respectively), the deformed acini recovered normal features, such as a spheroid shape, proliferative arrest and luminal clearing, suggesting a role for the ER and GPER in the estrogenic disruption of acinar formation. Conclusion: This new model offers the opportunity to better understand the role of the ER and GPER in the morphogenesis of breast glandular structure as well as the events implicated in breast cancer initiation and progression.This study was partly funded by a School of Pharmacy Studentship

Testicular cancer and cryptorchidism

The failure of testicular descent or cryptorchidism is the most common defect in newborn boys. The descent of the testes during development is controlled by insulin-like 3 peptide and steroid hormones produced in testicular Leydig cells, as well as by various genetic and developmental factors. While in some cases the association with genetic abnormalities and environmental causes has been shown, the etiology of cryptorchidism remains uncertain. Cryptorchidism is an established risk factor for infertility and testicular germ cell tumors (TGCT). Experimental animal models suggest a causative role for an abnormal testicular position on the disruption of spermatogenesis however the link between cryptorchidism and TGCT is less clear. The most common type of TGCT in cryptorchid testes is seminoma, believed to be derived from pluripotent prenatal germ cells. Recent studies have shown that seminoma cells and their precursor carcinoma in situ cells express a number of spermatogonial stem cell (SSC) markers suggesting that TGCTs might originate from adult stem cells. We review here the data on changes in the SSC somatic cell niche observed in cryptorchid testes of mouse models and in human patients. We propose that the misregulation of growth factors' expression may alter the balance between SSC self-renewal and differentiation and shift stem cells towards neoplastic transformation

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Arfl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis

Editorial: Sphingolipid metabolism and cancer

Cancer; Metabolism; SphingolipidCáncer; Metabolismo; EsfingolípidoCàncer; Metabolisme; Esfingolípi

NOTCH1 Gain of Function in Germ Cells Causes Failure of Spermatogenesis in Male Mice

NOTCH1 is a member of the NOTCH receptor family, a group of single-pass trans-membrane receptors. NOTCH signaling is highly conserved in evolution and mediates communication between adjacent cells. NOTCH receptors have been implicated in cell fate determination, as well as maintenance and differentiation of stem cells. In the mammalian testis expression of NOTCH1 in somatic and germ cells has been demonstrated, however its role in spermatogenesis was not clear. To study the significance of NOTCH1 in germ cells, we applied a cre/loxP approach in mice to induce NOTCH1 gain- or loss-of function specifically in male germ cells. Using a Stra8-icretransgene we produced mice with conditional activation of the NOTCH1 intracellular domain (NICD) in germ cells. Spermatogenesis in these mutants was progressively affected with age, resulting in decreased testis weight and sperm count. Analysis of downstream target genes of NOTCH1 signaling showed an increased expression of Hes5, with a reduction of the spermatogonial differentiation marker, Neurog3 expression in the mutant testis. Apoptosis was significantly increased in mouse germ cells with the corresponding elevation of pro-apoptotic Trp53 and Trp63genes\u27 expression. We also showed that the conditional germ cell-specific ablation of Notch1 had no effect on spermatogenesis or male fertility. Our data suggest the importance of NOTCH signaling regulation in male germ cells for their survival and differentiation

Blebbistain, a Myosin II Inhibitor, as a Novel Strategy to Regulate Detrusor Contractility in a Rat Model of Partial Bladder Outlet Obstruction

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40–45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ∼50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5’ myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3’ MHC SM1 and essential light chain isoform MLC17b also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB

INPP4B protects from metabolic syndrome and associated disorders

A high fat diet and obesity have been linked to the development of metabolic dysfunction and the promotion of multiple cancers. The causative cellular signals are multifactorial and not yet completely understood. In this report, we show that Inositol Polyphosphate-4-Phosphatase Type II B (INPP4B) signaling protects mice from diet-induced metabolic dysfunction. INPP4B suppresses AKT and PKC signaling in the liver thereby improving insulin sensitivity. INPP4B loss results in the proteolytic cleavage and activation of a key regulator in de novo lipogenesis and lipid storage, SREBP1. In mice fed with the high fat diet, SREBP1 increases expression and activity of PPARG and other lipogenic pathways, leading to obesity and non-alcoholic fatty liver disease (NAFLD). Inpp4b−/− male mice have reduced energy expenditure and respiratory exchange ratio leading to increased adiposity and insulin resistance. When treated with high fat diet, Inpp4b−/− males develop type II diabetes and inflammation of adipose tissue and prostate. In turn, inflammation drives the development of high-grade prostatic intraepithelial neoplasia (PIN). Thus, INPP4B plays a crucial role in maintenance of overall metabolic health and protects from prostate neoplasms associated with metabolic dysfunction

Activation of relaxin family receptor 1 from different mammalian species by relaxin peptide and small-molecule agonist ML290

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing

A novel androgen-regulated isoform of the TSC2 tumour suppressor gene increases cell proliferation

TSC2 (Tuberous sclerosis complex 2) is an important tumour suppressor gene, mutations within which are linked to the development of tuberous sclerosis and implicated in multiple tumour types. TSC2 protein complexes with TSC1 and blocks the ability of the Rheb (Ras homolog enriched in brain) GTPase to activate mTOR (mammalian target of rapamycin), a crucial signal transducer which regulates protein synthesis and cell growth. Here, we report the characterisation of a novel isoform of TSC2 which is under direct control of the ligand-activated androgen receptor. TSC2 isoform A (TSC2A) is derived from an internal androgen-regulated alternative promoter and encodes a 508-amino acid cytoplasmic protein corresponding to the C-terminal region of full-length TSC2, lacking the interaction domain for TSC1 and containing an incomplete interaction domain required for Rheb inactivation. Expression of TSC2A is induced in response to androgens and full-length TSC2 is co-ordinately down-regulated, indicating an androgen-driven switch in TSC2 protein isoforms. In contrast to the well-characterised suppressive effect on cell proliferation of full-length TSC2 protein, both LNCaP and HEK293 cells over-expressing TSC2 isoform A proliferate more rapidly (measured by MTT assays) and have increased levels of cells in S-phase (measured by both Edu staining and FACS analysis). Our work indicates, for the first time, a novel role for this well-known tumour suppressor gene, which encodes an activator of cell proliferation in response to androgen stimulation

Androgen receptor footprint on the way to prostate cancer progression

The prostate gland is exquisitely sensitive to androgen receptor (AR) signaling. AR signaling is obligatory for prostate development and changes in AR levels, its ligands or shifts in AR mode of action are reflected in the physiology of the prostate. The AR is intimately linked to prostate cancer biology through the regulation of epithelial proliferation, suppression of apoptosis and the development of castration-resistant disease. Thus, AR is the primary therapeutic target in various prostate diseases such as BPH and cancer. Although some tumors lose AR expression, most retain the AR and have elevated levels and/or shifts in activity that are required for tumor progression and metastasis. New AR inhibitors currently in clinical trials with higher receptor affinity and specificity may improve prostate cancer patient outcome. Several events play an important role in initiation, primary tumor development and metastatic spread. Androgen receptor activity and promoter specificity change due to altered coregulator expression. Changes in epigenetic surveillance alter the AR cistrome. Both systemic and local inflammation increases with PCa progression affecting AR levels, activity, and requirement for ligand. Our current understanding of AR biology suggest that global androgen suppression may drive the development of castration-resistant disease and therefore the question remains: Does effective inhibition of AR activity mark the end of the road for PCa or only a sharp turn toward a different type of malignancy