56 research outputs found

    PLEKHS1 drives PI3Ks and remodels pathway homeostasis in PTEN-null prostate

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    The PIP3/PI3K network is a central regulator of metabolism and is frequently activated in cancer, commonly by loss of the PIP3/PI(3,4)P2 phosphatase, PTEN. Despite huge research investment, the drivers of the PI3K network in normal tissues and how they adapt to overactivation are unclear. We find that in healthy mouse prostate PI3K activity is driven by RTK/IRS signaling and constrained by pathway feedback. In the absence of PTEN, the network is dramatically remodeled. A poorly understood YXXM- and PIP3/PI(3,4)P2-binding PH domain-containing adaptor, PLEKHS1, became the dominant activator and was required to sustain PIP3, AKT phosphorylation, and growth in PTEN-null prostate. This was because PLEKHS1 evaded pathway-feedback and experienced enhanced PI3K- and Src-family kinase-dependent phosphorylation of Y258XXM, eliciting PI3K activation. hPLEKHS1 mRNA and activating Y419 phosphorylation of hSrc correlated with PI3K pathway activity in human prostate cancers. We propose that in PTEN-null cells receptor-independent, Src-dependent tyrosine phosphorylation of PLEKHS1 creates positive feedback that escapes homeostasis, drives PIP3 signaling, and supports tumor progression

    Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats

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    Abstract The Developmental Origins of Health and Disease (DOHaD) concept has provided the framework to assess how early life experiences can shape health and disease throughout the life course. While maternal malnutrition has been proposed as a risk factor for the developmental programming of prostate cancer (PCa), the molecular mechanisms remain poorly understood. Using RNA-seq data, we demonstrated deregulation of miR-206-Plasminogen (PLG) network in the ventral prostate (VP) of young maternally malnourished offspring. RT-qPCR confirmed the deregulation of the miR-206-PLG network in the VP of young and old offspring rats. Considering the key role of estrogenic signaling pathways in prostate carcinogenesis, in vitro miRNA mimic studies also revealed a negative correlation between miR-206 and estrogen receptor α (ESR1) expression in PNT2 cells. Together, we demonstrate that early life estrogenization associated with the deregulation of miR-206 networks can contribute to the developmental origins of PCa in maternally malnourished offspring. Understanding the molecular mechanisms by which early life malnutrition affects offspring health can encourage the adoption of a governmental policy for the prevention of non-communicable chronic diseases related to the DOHaD concept

    Reversal of diabetic-induced myopathy by swimming exercise in pregnant rats:a translational intervention study

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    Gestational diabetes mellitus (GDM) plus rectus abdominis muscle (RAM) myopathy predicts long-term urinary incontinence (UI). Atrophic and stiff RAM are characteristics of diabetes-induced myopathy (DiM) in pregnant rats. This study aimed to determine whether swimming exercise (SE) has a therapeutic effect in mild hyperglycemic pregnant rats model. We hypothesized that SE training might help to reverse RAM DiM. Mild hyperglycemic pregnant rats model was obtained by a unique subcutaneous injection of 100 mg/kg streptozotocin (diabetic group) or citrate buffer (non-diabetic group) on the first day of life in Wistar female newborns. At 90 days of life, the rats are mated and randomly allocated to remain sedentary or subjected to a SE protocol. The SE protocol started at gestational day 0 and consisted of 60 min/day for 6 days/week in a period of 20 days in a swim tunnel. On day 21, rats were sacrificed, and RAM was collected and studied by picrosirius red, immunohistochemistry, and transmission electron microscopy. The SE protocol increased the fiber area and diameter, and the slow-twitch and fast-twitch fiber area and diameter in the diabetic exercised group, a finding was also seen in control sedentary animals. There was a decreased type I collagen but not type III collagen area and showed a similar type I/type III ratio compared with the control sedentary group. In conclusion, SE during pregnancy reversed the RAM DiM in pregnant rats. These findings may be a potential protocol to consider in patients with RAM damage caused by GDM

    miR-18a-5p Is Involved in the Developmental Origin of Prostate Cancer in Maternally Malnourished Offspring Rats: A DOHaD Approach

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    The Developmental Origins of Health and Disease (DOHaD) concept correlates early life exposure to stressor conditions with the increased incidence of non-communicable chronic diseases, including prostate cancer (PCa), throughout the life span. However, the molecular mechanisms involved in this process remain poorly understood. In this study, the deregulation of two miRNAs (rno-miR-18a-5p and rno-miR-345-3p) was described in the ventral prostate VP of old rats born to dams fed with a low protein diet (LPD) (6% protein in the diet) during gestational and lactational periods. Integrative analysis of the (VP) transcriptomic and proteomic data revealed changes in the expression profile of 14 identified predicted targets of these two DE miRNAs, which enriched terms related to post-translational protein modification, metabolism of proteins, protein processing in endoplasmic reticulum, phosphonate and phosphinate metabolism, the calnexin/calreticulin cycle, metabolic pathways, N-glycan trimming in the ER and the calnexin/calreticulin cycle, hedgehog ligand biogenesis, the ER-phagosome pathway, detoxification of reactive oxygen species, antigenprocessing-cross presentation, RAB geranylgeranylation, collagen formation, glutathione metabolism, the metabolism of xenobiotics by cytochrome P450, and platinum drug resistance. RT-qPCR validated the deregulation of the miR-18a-5p/P4HB (prolyl 4-hydroxylase subunit beta) network in the VP of older offspring as well as in the PNT-2 cells transfected with mimic miR-18a-5p. Functional in vitro studies revealed a potential modulation of estrogen receptor α (ESR1) by miR-18a-5p in PNT-2 cells, which was also confirmed in the VP of older offspring. An imbalance of the testosterone/estrogen ratio was also observed in the offspring rats born to dams fed with an LPD. In conclusion, deregulation of the miR-18a-5p/P4HB network can contribute to the developmental origins of prostate cancer in maternally malnourished offspring, highlighting the need for improving maternal healthcare during critical windows of vulnerability early in life

    Mmp-2 And Mmp-9 activities and Timp-1 and Timp-2 expression in the prostatic tissue of two ethanol-preferring rat models

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    We investigated whether chronic ethanol intake is capable of altering the MMP-2 and MMP-9 activities and TIMP-2 and TIMP-1 expression in the dorsal and lateral prostatic lobes of low (UChA) and high (UChB) ethanol-preferring rats. MMP-2 and MMP9 activities and TIMP-1 and TIMP-2 expression were significantly reduced in the lateral prostatic lobe of the ethanol drinking animals. Dorsal prostatic lobe was less affected showing no significant alterations in these proteins, except for a reduction in the TIMP-1 expression in UChA rats. These important findings demonstrate that chronic ethanol intake impairs the physiological balance of the prostate extracellular matrix turnover, through downregulation of MMPs, which may contribute to the development of prostatic diseases. Furthermore, since these proteins are also components of prostate secretion, the negative impact of chronic ethanol intake on fertility may also involve reduction of MMPs and TIMPs in the seminal fluid2015COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informação2011/03394-4; 2011/13713-

    Fibrosis-related gene expression in the prostate is modulated by doxazosin treatment

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    Aims To gain new insights into the molecular mechanisms of action of doxazosin, we investigated the prostatic stroma ultrastructure and the expression of genes involved with fibrosis, such as collagen type I and III (COL1A1 and COL3A1, respectively) and TGF-beta 1, in the rat ventral prostate. Main methods Adult Wistar rats were treated with doxazosin (25 mg/kg/day), and the ventral prostates were excised at 7 and 30 days after treatment. Untreated rats were controls. Ventral prostates were subjected to ultrastructural, immunohistochemical, biochemical and molecular analyses. Key findings Doxazosin-treated prostates showed thickened bundles of collagen fibrils, activated fibroblasts, enlarged neurotransmitter vesicles and increased tissue immunostaining for collagen type I and type III when compared to untreated prostates. After 7 and 30 days of doxazosin treatment mRNA expression of COL1A1 and COL3A1 was significantly increased and reduced, respectively, compared to the control group. TGF-beta 1 mRNA and protein levels were increased after 7 days of doxazosin treatment, whereas only mRNA levels remained increased after 30 days of treatment. Significance Our data suggest that relaxation of smooth muscle cells by alpha-blockers interferes with the mechanical dynamics of the prostatic stroma extracellular matrix components, generating a pro-fibrotic effect probably via the TGF-beta 1 signaling pathway. Long term treatment with doxazosin may also lead to a reduced turnover of extracellular matrix components. Our results add to a better understanding of the molecular mechanisms behind the effects of alpha-blockade on prostatic histoarchitecture and the response to treatment for benign prostatic hyperplasia9125-2612811287CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçãosem informação06/60114-6; 06/60115-2The authors thank the Electron Microscopy Center (Institute of Biosciences, UNESP, Botucatu, SP) for the transmission electron microscopy protocol procedures and for the use of Phillips electron microscope. This work was funded by FAPESP (São Paulo State Research Foundation; grants 06/60114-6, to S.L.F. and 06/60115-2, to F.K.D); by FUNDUNESP (Foundation for Development of Univ Estadual Paulista), by CNPq, and by CAPES do Brazil. This paper represents part of the PhD thesis presented by F.K.D. to the University of Campinas—UNICAMP, Brazi

    Prostate telocytes change their phenotype in response to castration or testosterone replacement

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    Telocytes are CD34-positive cells with a fusiform cell body and long, thin cytoplasmic projections called telopodes. These cells were detected in the stroma of various organs, including the prostate. The prostate is a complex gland capable of undergoing involution due to low testosterone levels; and this condition can be reversed with testosterone replacement. Telocyte function in the mature prostate remains to be dermined, and it is not known whether telocytes can take place in tissue remodeling during prostate involution and regrowth. The present study employed structural, ultrastructural and immunohistochemical methods to investigate the telocyte's phenotypes in the ventral prostate (VP) from control (CT), castrated (CS) and testosterone replacement (TR) groups of adult male Wistar rats. Telocytes were found in the subepithelial, perimuscular and interstitical regions around glandular acini. Telocytes from CT animals have condensed chromatin and long and thin telopodes. In CS group, telocytes appeared quiescent and exhibited layers of folded up telopodes. After TR, telocytes presented loose chromatin, abundant rough endoplasmic reticulum and enlarged telopodes, closely associated with bundles of collagen fibrils. We called these cells "telocytes with a synthetic phenotype". As testosterone levels and glandular morphology returned toward to the CT group parameters, after 10 days ofTR, these telocytes progressively switched to the normal phenotype. Our results demonstrate that telocytes exhibit phenotypic plasticity upon androgen manipulation and interact with fibroblast and smooth muscle cells to maintain glandular architecture in control animals and during tissue remodeling after hormonal manipulation9CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305391/2014-3; 306900/2016-5; 305840/2015-0; 310663/2018-02002/11102-4; 2014/26660-0; 2017/01063-

    Epigenetic and oncogenic regulation of SLC16A7 (MCT2) results in protein over-expression, impacting on signalling and cellular phenotypes in prostate cancer

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    Felisbino S. received a fellowship from the Sao Paulo Research Foundation (FAPESP) ref. 2013/08830-2 and 2013/06802-1. Anne Y Warren research time funded by: Cambridge Biomedical Research Centre.Monocarboxylate Transporter 2 (MCT2) is a major pyruvate transporter encoded by the SLC16A7 gene. Recent studies pointed to a consistent overexpression of MCT2 in prostate cancer (PCa) suggesting MCT2 as a putative biomarker and molecular target. Despite the importance of this observation the mechanisms involved in MCT2 regulation are unknown. Through an integrative analysis we have discovered that selective demethylation of an internal SLC16A7/MCT2 promoter is a recurrent event in independent PCa cohorts. This demethylation is associated with expression of isoforms differing only in 5'-UTR translational control motifs, providing one contributing mechanism for MCT2 protein overexpression in PCa. Genes co-expressed with SLC16A7/MCT2 also clustered in oncogenic-related pathways and effectors of these signalling pathways were found to bind at the SLC16A7/MCT2 gene locus. Finally, MCT2 knock-down attenuated the growth of PCa cells. The present study unveils an unexpected epigenetic regulation of SLC16A7/MCT2 isoforms and identifies a link between SLC16A7/MCT2, Androgen Receptor (AR), ETS-related gene (ERG) and other oncogenic pathways in PCa. These results underscore the importance of combining data from epigenetic, transcriptomic and protein level changes to allow more comprehensive insights into the mechanisms underlying protein expression, that in our case provide additional weight to MCT2 as a candidate biomarker and molecular target in PCa.Publisher PDFPeer reviewe

    Abstract A123: Preclinical evaluation of dual mTOR inhibitor, AZD2014, in prostate cancer

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    Abstract Background: An estimated 220,800 cases and 27,540 deaths from prostate cancer (PCa) will occur in the USA during 2015. Altered PI3K/AKT/mTOR signalling contributes to prostate cancer progression and transition to androgen-independent disease, for example one study reported 42% of primary and 100% of metastatic PCa tumours exhibited mutations, altered expression or copy number variations within this pathway. First generation mTOR inhibitors (preferentially inhibit mTORC1), have had limited anti-cancer effect in patients with PCa, possibly due to negative feedback activation of the AKT pathway via mTORC2. The dual mTORC1/2 inhibitor, AZD2014, may overcome this liability. Using a genetically engineered PTEN conditional mouse model (Ptenloxp/loxp;PB-Cre4), we have investigated the effects of AZD2014. The studies complement a clinical trial (NCT02064608) of AZD2014, given to men before radical prostatectomy and are timed for when invasive prostate carcinomas develop in the model around 10-14 months prior to onset of resistance to castration through AKT pathway activation. AZD2014, 15mg/kg daily, oral (with or without castration) or vehicle were administered for 14 days. Results: AZD2014 was well tolerated with no overt toxicity observed. Pharmacokinetic (PK) analysis revealed mean concentrations of 4.4±2.1μM of AZD2014 in the plasma samples collected 4 hours after day 14 dose. AZD2014 alone or combined with castration inhibited mTORC1 and mTORC2 measured by reductions in p4EBP1(Thr37/46) by approximately 48%±27% (p<0.001) and 37%±11% (p<0.001); pS6(Ser235/236) by 74%±43% (p<0.001) and 44%±13% (p<0.001) and pAKT(Ser473) by 36%±8% (p<0.001) and 20%±3% (p<0.01) as compared to vehicle-treated mice. AZD2014 treatment was anti-proliferative; Ki67 was significantly reduced in AZD2014-treated mice (70%±45%, p<0.001) or AZD2014 plus castration (42%±16%, p<0.001). Apoptosis was detected with cleaved caspase 3 and increased by 3.3-fold (p<0.001) in both AZD2014 or AZD2014 plus castration groups and 2-fold (p<0.001) in the castration only group, respectively. In all cases, 10 mice were used in each group and 80-120 randomly chosen images were analysed using Aperio automatic quantitative algorithms. Tumour volumes (ultrasound imaging) were reduced by 51% (p<0.05) comparing AZD2014 plus castration against control. HRMAS 1H NMR spectroscopy was used on tumour tissue to determine changes in metabolites following treatment and identified that the total choline to creatine ratio (t-Cho/Cr) was reduced by 40% in AZD2014-treated mice tumour samples (p<0.05) as compared to control-treated mice. Conclusions: Short term (14 days) treatment with AZD2014 with or without castration was associated with both pharmacodynamic and anti-tumour effects. The t-Cho/Cr ratio, previously reported as positively correlated with Gleason score in PCa patients, might be, in addition to our standard mTOR PD markers, utilised as a non-invasive biomarker of AZD2014 activity. The primary and phenotypic biomarker effects of monotherapy with AZD2014 in this relevant genetically engineered mouse model of prostate cancer will be compared with paired biopsies from the ongoing exploratory window study in the prostate cancer patients prior to prostatectomy, and may inform potential novel combination approaches that are translatable to the clinic. Citation Format: Chiranjeevi Sandi, Antonio Ramos-Montoya, Sergio L. Felisbino, Sarah Jurmeister, Basetti Madhu, Karan Wadhwa, John R. Griffiths, Frances M. Richards, Duncan I. Jodrell, David E. Neal, Sabina Cosulich, Barry Davies, Simon Pacey. Preclinical evaluation of dual mTOR inhibitor, AZD2014, in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A123.This is the accepted manuscript. The final version is available at http://mct.aacrjournals.org/content/14/12_Supplement_2/A123.short
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