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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Intrauterine exposure to bisphenol A promotes different effects in both neonatal and adult prostate of male and female gerbils (Meriones unguiculatus)

Substances that mimic endogenous hormones may alter the cell signaling that govern prostate development and predispose it to developing lesions in adult and senile life. Bisphenol A is able to mimic estrogens, and studies have demonstrated that low levels of exposure to this compound have caused alterations during prostate development. The aim of this study was to describe the prostate development in both male and female neonatal gerbils in normal conditions and under exposure to BPA during intrauterine life, and also to analyze whether the effects of intrauterine exposure to BPA remain in adulthood. Morphological, stereological, three-dimensional reconstruction, and immunohistochemical methods were employed. The results demonstrated that in 1-day-old normal gerbils, the female paraurethral glands and the male ventral lobe are morphologically similar, although its tissue components-epithelial buds (EB), periurethral mesenchyme (PeM), paraurethral mesenchyme (PaM) or ventral mesenchymal pad (VMP), and smooth muscle (SM)-have presented different immunolabeling pattern for androgen receptor (AR), and for proliferating cell nuclear antigen (PCNA). Moreover, we observed a differential response of male and female prostate to intrauterine BPA exposure. In 1-day-old males, the intrauterine exposure to BPA caused a decrease of AR-positive cells in the PeM and SM, and a decrease of the proliferative status in the EB. In contrast, no morphological alterations were observed in ventral prostate of adult males. In 1-day-old females, BPA exposure promoted an increase of estrogen receptor alpha (ERα) positive cells in PeM and PaM, a decrease of AR-positive cells in EB and PeM, besides a reduction of cell proliferation in EB. Additionally, the adult female prostate of BPA-exposed animals presented an increase of AR- and PCNA-positive cells. These results suggest that the prostate of female gerbils were more susceptible to the intrauterine BPA effects, since they became more proliferative in adult life. © 2015 Wiley Periodicals, Inc. Environ Toxicol, 2015.

Bisphenol-A promotes antiproliferative effects during neonatal prostate development in male and female gerbils

Conselho Nacional de Desenvolvimento Científico e Tecnol[ogico (CNPq)

Role of ATM and PTEN in prostatic carcinogenic dog prostate: validation of aCGH results

The canis lupus familiares is the only species besides human that spontaneously develop prostatic carcinoma (PCa). In addition, the metastatic sites are similar to those frequently reported in men. For these reasons, the dog is the best natural model to study the molecular mechanisms in PCa development providing a natural animal model for treatment by molecular targets. Previously, we investigated copy number alterations by arrayCGH (Canine Genome CGH Microarray 4x44K-G2519F, Agilent Technologies) in canine prostatic lesions: 3 benign prostatic hyperplasias (BPH), 4 proliferative inflammatory atrophies (PIA), and 14 PCa. Five histologically normal prostatic tissues were used as reference. Genomic alterations were evaluated using Genomic Workbench Standard Edition 5.0.14. This previous study revealed significant copy number losses of Atm and Pten exclusively in PCa. In the present study, ATM and PTEN immunoexpression were investigated using a tissue microarray (TMA) containing 149 canine prostatic paraffin-embedded lesions (BPH, PIA and PCa) collected from 67 animals. Immunohistochemical reactions were performed using the polyclonal rabbit antibody anti-PTEN (Santa Cruz Biotech, 1:50) and anti-ATM (Abcam, 1:50). The sections were developed with diaminobenzidine (DAB) and peroxidase. The immunohistochemical staining was assessed in each core by the distribution of positive cells for each antibody per lesion (score 1: <25% cells positive, 2: 26% to 50%, 3: being 51% and 75% and 4:> 75%) and intensity (1: weak, 2: moderate, 3: intense). Chi-square or Fisher exact test was used to determine the association between the categorical variables using GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA). Distribution of positive cells did not differ among lesions. PCa and PIA showed more samples with weak intensity for ATM when compared to normal prostatic tissue and BPH (PCa: p=0,032 and PIA: p=0,025). Benign prostatic hyperplasia and normal samples presented intense PTEN immunostaining than PCa (p=0,021) and PIA (p=0,0013). These results suggest that ATM and PTEN proteins expression in canine prostatic carcinoma are downregulated possibly by copy number losses. These findings are similar from those described in prostate carcinomas from human corroborating for the use of dogs as a natural model to study prostatic disease in men.

MMP-2 and TIMP-2 in male and female prostates of mongolian gerbil: effects of hormonal manipulation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The accuracy of pathological data for the prediction of insignificant prostate cancer

Introduction: The widespread screening programs prompted a decrease in prostate cancer stage at diagnosis, and active surveillance is an option for patients who may harbor clinically insignificant prostate cancer (IPC). Pathologists include the possibility of an IPC in their reports based on the Gleason score and tumor volume. This study determined the accuracy of pathological data in the identification of IPC in radical prostatectomy (RP) specimens. Materials and Methods: Of 592 radical prostatectomy specimens examined in our laboratory from 2001 to 2010, 20 patients harbored IPC and exhibited biopsy findings suggestive of IPC. These biopsy features served as the criteria to define patients with potentially insignificant tumor in this population. The results of the prostate biopsies and surgical specimens of the 592 patients were compared. Results: The twenty patients who had IPC in both biopsy and RP were considered real positive cases. All patients were divided into groups based on their diagnoses following RP: true positives (n = 20), false positives (n = 149), true negatives (n = 421), false negatives (n = 2). The accuracy of the pathological data alone for the prediction of IPC was 91.4%, the sensitivity was 91% and the specificity was 74%. Conclusion: The identification of IPC using pathological data exclusively is accurate, and pathologists should suggest this in their reports to aid surgeons, urologists and radiotherapists to decide the best treatment for their patients.

Prostate biopsy in patients with long-term use of indwelling bladder catheter: What is the rationale?

Objective: Acute urinary retention (AUR) is expected to occur in 2% to 39% men with benign prostatic hyperplasia. To date, no study has elucidated the effect of long-term use of indwelling bladder catheter on serum prostate specific antigen (PSA) levels and on the incidence of prostate cancer (CaP). The aim of the present study is to analyze the incidence of CaP in patients with long-term use of indwelling bladder catheter and determine some practice patterns on this issue. Materials and methods: The study comprised a retrospective analysis of data from 1,651 patients who had undergone transrectal ultrasound (TRUS)-guided prostate biopsy from July 2004 to June 2009. Among these patients, 198 (12%) were using an indwelling bladder catheter during the biopsy for at least 1 month. The incidence of CaP was recorded according to total PSA levels. Other variables such patient age, free/total PSA rate, PSA density, prostate volume, and duration of catheter use was also analyzed. Men with a digital rectal examination suspicious for cancer were not considered for analysis. Results: Median patient age was 71 years (37 to 89 years). Overall, 25% of patients presented a CaP diagnosis. CaP incidence according to the PSA levels was 0%, 18.9%, 24.5%, and 40.6% for patients with PSA <= 4.0, 4.1-10.0, 10.1-20.0, and >20.0 ng/ml, respectively. When prostate volume was analyzed together, we demonstrated that only 1 (2.4%) patient with PSA below 10.0 ng/ml and prostate volume >60 g had CaP. Median total PSA, PSA density, and prostate volume were statistically different between patients with and without CaP. Conclusions: Prostate biopsy should not be indicated for all patients with diagnosis of BPH and AUR who present an elevated PSA level. Patients with PSA below 10.0 ng/ml, and prostate volume >60 g should only undergo biopsy in selected cases. Patients with PSA >20.0 ng/ml and a prostate volume <= 60 g are at higher risk of CaP diagnosis. (C) 2012 Elsevier Inc. All rights reserved.

Surrogate endpoints for prostate cancer-specific mortality after radiotherapy and androgen suppression therapy in men with localised or locally advanced prostate cancer: an analysis of two randomised trials

Background Androgen suppression therapy and radiotherapy are used to treat locally advanced prostate cancer. 3 years of androgen suppression confers a small survival benefit compared with 6 months of therapy in this setting, but is associated with more toxic effects. Early identification of men in whom radiotherapy and 6 months of androgen suppression is insufficient for cure is important. Thus, we assessed whether prostate-specific antigen (PSA) values can act as an early surrogate for prostate cancer-specific mortality (PCSM). Methods We systematically reviewed randomised controlled trials that showed improved overall and prostate cancer-specific survival with radiotherapy and 6 months of androgen suppression compared with radio therapy alone and measured lowest PSA concentrations (PSA nadir) and those immediately after treatment (PSA end). We assessed a cohort of 734 men with localised or locally advanced prostate cancer from two eligible trials in the USA and Australasia that randomly allocated participants between Feb 2, 1996, and Dec 27, 2001. We used Prentice criteria to assess whether reported PSA nadir or PSA end concentrations of more than 0.5 ng/mL were surrogates for PCSM. Findings Men treated with radiotherapy and 6 months of androgen suppression in both trials were significantly less likely to have PSA end and PSA nadir values of more than 0.5 ng/mL than were those treated with radiotherapy alone (p<0.0001). Presence of candidate surrogates (ie, PSA end and PSA nadir values >0.5 ng/mL) alone and when assessed in conjunction with the randomised treatment group increased risk of PCSM in the US trial (PSA nadir p=0.0016; PSA end p=0.017) and Australasian trial (PSA nadir p<0.0001; PSA end p=0.0012). In both trials, the randomised treatment group was no longer associated with PCSM (p >= 0.20) when the candidate surrogates were included in the model. Therefore, both PSA metrics satisfied Prentice criteria for surrogacy. Interpretation After radiotherapy and 6 months of androgen suppression, men with PSA end values exceeding 0.5 ng/mL should be considered for long-term androgen suppression and those with localised or locally advanced prostate cancer with PSA nadir values exceeding 0.5 ng/mL should be considered for inclusion in randomised trials investigating the use of drugs that have extended survival in castration-resistant metastatic prostate cancer.

miR-21 may acts as an oncomir by targeting RECK, a matrix metalloproteinase regulator, in prostate cancer

Background: Prognosis of prostate cancer (PCa) is based mainly in histological aspects together with PSA serum levels that not always reflect the real aggressive potential of the neoplasia. The micro RNA (miRNA) mir-21 has been shown to regulate invasiveness in cancer through translational repression of the Metaloproteinase (MMP) inhibitor RECK. Our aim is to investigate the levels of expression of RECK and miR-21 in PCa comparing with classical prognostic factors and disease outcome and also test if RECK is a target of miR-21 in in vitro study using PCa cell line. Materials and methods: To determine if RECK is a target of miR-21 in prostate cancer we performed an in vitro assay with PCa cell line DU-145 transfected with pre-miR-21 and anti-miR-21. To determine miR-21 and RECK expression levels in PCa samples we performed quantitative real-time polymerase chain reaction (qRT-PCR). Results: The in vitro assays showed a decrease in expression levels of RECK after transfection with pre-miR-21, and an increase of MMP9 that is regulated by RECK compared to PCa cells treated with anti-miR-21. We defined three profiles to compare the prognostic factors. The first was characterized by miR-21 and RECK underexpression (N = 25) the second was characterized by miR-21 overexpression and RECK underexpression (N = 12), and the third was characterized by miR-21 underexpression and RECK overexpression (N = 16). From men who presented the second profile (miR-21 overexpression and RECK underexpression) 91.7% were staged pT3. For the other two groups 48.0%, and 46.7% of patients were staged pT3 (p = 0.025). Conclusions: Our results demonstrate RECK as a target of miR-21. We believe that miR-21 may be important in PCa progression through its regulation of RECK, a known regulator of tumor cell invasion.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Background: Given the established fact that obesity interferes with male reproductive functions, the present study aimed to evaluate sperm production in the testis and storage in the epididymis in a glutamate-induced model of obesity. Methods: Male rats were treated neonatally with monosodium glutamate (MSG) at doses of 4 mg/kg subcutaneously, or with saline solution (control group), on postnatal days 2, 4, 6, 8 and 10. On day 120, obesity was confirmed by the Lee index in all MSG-treated rats. After this, all animals from the two experimental groups were anesthetized and killed to evaluate body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology. Results: Significant reductions in absolute and relative weights of testis, epididymis, prostate and seminal vesicle were noted in MSG-treated animals. In these same animals plasma testosterone and follicle-stimulating hormone (FSH) concentrations were decreased, as well as sperm counts in the testis and epididymis and seminiferous epithelium height and tubular diameter. The sperm transit time was accelerated in obese rats. However, the number of Sertoli cells per seminiferous tubule and stereological findings on the epididymis were not markedly changed by obesity. Conclusions: Neonatal MSG-administered model of obesity lowers sperm production and leads to a reduction in sperm storage in the epididymis of adult male rats. The acceleration of sperm transit time can have implications for the sperm quality of these rats.

Is it safe to utilize in vitro reconstituted human oral epithelium? An oncogenetic pathway study

Cell therapy is a therapeutic strategy used to replace or repair damaged tissue. The epithelium transplantation of cultivated keratinocytes has been applied to several modalities of reconstruction, like oral, urethra and ocular surface. Life and death signals work coordinately to ensure cellular quality control and the viability of an organism. The aim of this study is to verify that culture conditions did not induce genetic mutations through the analysis of the key genes: pAKT, Pten, p53 and MDM2 and investigate the presence of the related proteins in human oral keratinocytes obtained by primary culture and in vitro cultivated. Formalin fixed and paraffin embedded tissues from the oral cavity were utilized as control for normal expression of the related markers and two oral squamous cell carcinoma cell lines provided the expression pattern of the proposed markers in the event of cellular transformation. Akt, PTEN, p53 and MDM2 immunohistochemistry and Western-Blotting analyzes were performed. The results showed the expression levels and intracellular localizations of the four proteins evaluated. These analyzes confirmed that the produced in vitro epithelium is bio-compatible for its utilization as reconstruction and reparatory tissue, however further analyses and additional research on other biomarkers should be performed to analyse the long term engraftment of transplantable primary culture of oral keratinocytes and the long term resistance to cellular transformation.

TWIST and p-Akt immunoexpression in normal oral epithelium, oral dysplasia and in oral squamous cell carcinoma

Objectives: The aim of this study was to evaluate the immunoexpression of TWIST and p-Akt proteins in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC), correlating their expressions with the histological features of the lesions. Study design: Immunohistochemical studies were carried out on 10 normal oral epithelium, 30 OL and 20 OSCC formalin-fixed, paraffin-embedded tissue samples. Immunoperoxidase reactions for TWIST and p-Akt proteins were applied on the specimens and the positivity of the reactions was calculated for 1000 epithelial cells. Results: Kruskal-Wallis and Dunn's post tests revealed a significant difference in TWIST and p-Akt immunoexpression among normal oral mucosa, OL and OSCC. In addition, a significant positive correlation was found between TWIST and p-Akt expressions according to the Pearson's correlation test. Conclusions: The results obtained in the current study suggest that TWIST and p-Akt may participate of the multi-step process of oral carcinogenesis since its early stages.

IS A VISIBLE (HYPOECHOIC) LESION AT BIOPSY AN INDEPENDENT PREDICTOR OF PROSTATE CANCER OUTCOME?

The aim of this study was to evaluate the prognostic implications of the sonographic appearance of prostate cancers. All patients with biopsy-proven prostate cancer between January 2003 and July 2004 (and at least 5 years of follow-up) were selected retrospectively. After exclusions, 101 patients constituted our study population and were divided into isoechoic (or nonvisible) and hypoechoic (or visible) lesion. The clinical outcomes of these two groups were compared. The outcomes for the two groups were significantly different (p < 0.01). For nonvisible lesions, 37 of the 41 patients (90.2%) had no disease relapse and 2 (4.9%) had biochemical failure. For the visible lesions, 37 of the 60 (61.6%) patients were free of recurrence, 7 (11.7%) had systemic metastases and 10 (16.7%) died of complications related to prostate cancer. Our data show that patients with nonvisible prostate cancer had significantly better outcomes than patients with visible lesions during a five-year period of evaluation. (E-mail: fmuglia@fmrp.usp.br) (c) 2012 World Federation for Ultrasound in Medicine & Biology.

Advanced prostate cancer as a cause of oncogenic osteomalacia: an underdiagnosed condition

Tumor-induced osteomalacia (TIO) is a paraneoplastic bone mineral disturbance related to fibroblast growth factor 23 (FGF23) overproduction by the tumor, usually from mesenchymal origin. Such condition leads to high phosphate renal wasting and, consequently, to cumbersome symptoms as weakness, bone pain, and fractures. Case report. We report a case of an advanced castration-refractory prostate cancer patient, which developed severe hypophosphatemia with elevated phosphate excretion fraction. TIO was suspected, and increased levels of FGF23 reinforced such diagnosis. The patient died 4 months after being diagnosed with TIO. This case suggests that TIO has a dismal prognosis in prostate cancer patients. The clinical oncology community must be aware about such disturbance that can be present in those patients with weakness, bone pain, and hypophosphatemia.

Underexpression of MMP-2 and its Regulators, TIMP2, MT1-MMP and IL-8, is Associated with Prostate Cancer

Objective: Extracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their regulators. The purpose of this study was to investigate whether MMP-2 and its specific regulators, TIMP-2, MT1-MMP and IL-8, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis and clinical outcome of prostate cancer (PCa). Materials and Methods: MMP-2, TIMP-2, MT1-MMP and IL-8 expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in freshly frozen malignant and benign tissue specimens collected from 79 patients with clinically localized PCa who underwent radical prostatectomies. The control group consisted of 11 patients with benign prostate hyperplasia (BPH). The expression profile of the MMP-2 and its regulators were compared using Gleason scores, pathological stage, pre-operative PSA levels and the final outcome of the PCa. Results: The analysis of 79 specimens of PCa revealed that MMP-2, TIMP-2, MT1-MMP and IL-8 were underexpressed at 60.0%, 72.2%, 62.0% and 65.8%, respectively, in malignant prostatic tissue in relation to BPH samples. Considering the prognostic parameters, we demonstrated that high Gleason score tumors (>= 7) over-expressed MMP-2 (p = 0.048) and TIMP-2 (p = 0.021), compared to low Gleason score tumors (< 7). Conclusion: We have demonstrated that MMP-2 and its regulators are underexpressed in PCa. Alternatively, overexpression of MMP-2 and TIMP-2 was related to higher Gleason score tumors. We postulate that alterations in metalloproteinase expression may be important in the control of tissue homeostasis related to prostate carcinogenesis and tumor behavior.