Upregulation of shelterin and CST genes and longer telomeres are associated with unfavorable prognostic characteristics in prostate cancer.

INTRODUCTION: Search for new clinical biomarkers targets in prostate cancer (PC) is urgent. Telomeres might be one of these targets. Telomeres are the extremities of linear chromosomes, essential for genome stability and control of cell divisions. Telomere homeostasis relies on the proper functioning of shelterin and CST complexes. Telomeric dysfunction and abnormal expression of its components are reported in most cancers and are associated with PC. Despite this, there are only a few studies about the expression of the main telomere complexes and their relationship with PC progression. We aimed to evaluate the role of shelterin (POT1, TRF2, TPP1, TIN2, and RAP1) and CST (CTC1, STN1, and TEN1) genes and telomere length in the progression of PC. METHODS: We evaluated genetic alterations of shelterin and CST by bioinformatics in samples of localized (n = 499) and metastatic castration-resistant PC (n = 444). We also analyzed the expression of the genes using TCGA (localized PC n = 497 and control n = 152) and experimental approaches, with surgical specimens (localized PC n = 81 and BPH n = 10) and metastatic cell lines (LNCaP, DU145, PC3 and PNT2 as control) by real-time PCR. Real-time PCR also determined the telomere length in the same experimental samples. All acquired data were associated with clinical parameters. RESULTS: Genetic alterations are uncommon in PC, but POT1, TIN2, and TEN1 showed significantly more amplifications in the metastatic cancer. Except for CTC1 and TEN1, which are differentially expressed in localized PC samples, we did not detect an expression pattern relative to control and cell lines. Nevertheless, except for TEN1, the upregulation of all genes is associated with a worse prognosis in localized PC. We also found that increased telomere length is associated with disease aggressiveness in localized PC. CONCLUSION: The upregulation of shelterin and CST genes creates an environment that favors telomere elongation, giving selective advantages for localized PC cells to progress to more aggressive stages of the disease.

The Effect of Gene Editing by CRISPR-Cas9 of miR-21 and the Indirect Target MMP9 in Metastatic Prostate Cancer.

Prostate cancer (PCa) has a high prevalence and represents an important health problem, with an increased risk of metastasis. With the advance of CRISPR-Cas9 genome editing, new possibilities have been created for investigating PCa. The technique is effective in knockout oncogenes, reducing tumor resistance. MMP9 and miR-21 target genes are associated with PCa progression; therefore, we evaluated the MMP-9 and miR-21 targets in PCa using the CRISPR-Cas9 system. Single guide RNAs (sgRNAs) of MMP9 and miR-21 sequences were inserted into a PX-330 plasmid, and transfected in DU145 and PC-3 PCa cell lines. MMP9 and RECK expression was assessed by qPCR, WB, and IF. The miR-21 targets, integrins, BAX and mTOR, were evaluated by qPCR. Flow cytometry was performed with Annexin5, 7-AAD and Ki67 markers. Invasion assays were performed with Matrigel. The miR-21 CRISPR-Cas9-edited cells upregulated RECK, MARCKS, BTG2, and PDCD4. CDH1, ITGB3 and ITGB1 were increased in MMP9 and miR-21 CRISPR-Cas9-edited cells. Increased BAX and decreased mTOR were observed in MMP9 and miR-21 CRISPR-Cas9-edited cells. Reduced cell proliferation, increased apoptosis and low invasion in MMP9 and miR-21 edited cells was observed, compared to Scramble. CRISPR-Cas9-edited cells of miR-21 and MMP9 attenuate cell proliferation, invasion and stimulate apoptosis, impeding PCa evolution.

Overexpression of miR-17-5p may negatively impact p300/CBP factor-associated inflammation in a hypercholesterolemic advanced prostate cancer model.

BACKGROUND: Previously, we demonstrated that cholesterol triggers the increase in p300/CBP-associated factor (PCAF), targeted by miR-17-5p. The p300, IL-6, PCAF, and miR-17-5p genes have important and contradictory roles in inflammation and prostate cancer (PCa). This study aimed to demonstrate the potential anti-inflammatory effect of miR-17-5 in an advanced PCa model with diet-induced hypercholesterolemia. METHODS AND RESULTS: In vitro, using the PC-3 cell line, we show that induction of miR-17-5p reduces p300 and PCAF expression, increases apoptosis, and decreases cell migration. Furthermore, we demonstrate that supplementing this same cell with cholesterol (2 µg/mL) triggers increased p300, IL-6, and PCAF. In vivo, after establishing the hypercholesterolemic (HCOL) model, xenografts were treated with miR-17-5p. Increased expression of this miR after intratumoral injections attenuated tumor growth in the control and HCOL animals and reduced cell proliferation. CONCLUSION: Our results demonstrate that inducing miR-17-5p expression suppresses tumor growth and inflammatory mediator expression. Further studies should be conducted to fully explore the role of miR-17-5p and the involvement of inflammatory mediators p300, PCAF, and IL-6.

Intratumoral Restoration of miR-137 Plus Cholesterol Favors Homeostasis of the miR-137/Coactivator p160/AR Axis and Negatively Modulates Tumor Progression in Advanced Prostate Cancer.

MicroRNAs (miRNAs) have gained a prominent role as biomarkers in prostate cancer (PCa). Our study aimed to evaluate the potential suppressive effect of miR-137 in a model of advanced PCa with and without diet-induced hypercholesterolemia. In vitro, PC-3 cells were treated with 50 pmol of mimic miR-137 for 24 h, and gene and protein expression levels of SRC-1, SRC-2, SRC-3, and AR were evaluated by qPCR and immunofluorescence. We also assessed migration rate, invasion, colony-forming ability, and flow cytometry assays (apoptosis and cell cycle) after 24 h of miRNA treatment. For in vivo experiments, 16 male NOD/SCID mice were used to evaluate the effect of restoring miR-137 expression together with cholesterol. The animals were fed a standard (SD) or hypercholesterolemic (HCOL) diet for 21 days. After this, we xenografted PC-3 LUC-MC6 cells into their subcutaneous tissue. Tumor volume and bioluminescence intensity were measured weekly. After the tumors reached 50 mm3, we started intratumor treatments with a miR-137 mimic, at a dose of 6 µg weekly for four weeks. Ultimately, the animals were killed, and the xenografts were resected and analyzed for gene and protein expression. The animals' serum was collected to evaluate the lipid profile. The in vitro results showed that miR-137 could inhibit the transcription and translation of the p160 family, SRC-1, SRC-2, and SRC-3, and indirectly reduce the expression of AR. After these analyses, it was determined that increased miR-137 inhibits cell migration and invasion and impacts reduced proliferation and increased apoptosis rates. The in vivo results demonstrated that tumor growth was arrested after the intratumoral restoration of miR-137, and proliferation levels were reduced in the SD and HCOL groups. Interestingly, the tumor growth retention response was more significant in the HCOL group. We conclude that miR-137 is a potential therapeutic miRNA that, in association with androgen precursors, can restore and reinstate the AR-mediated axis of transcription and transactivation of androgenic pathway homeostasis. Further studies involving the miR-137/coregulator/AR/cholesterol axis should be conducted to evaluate this miR in a clinical context.

Cholesterol Triggers Nuclear Co-Association of Androgen Receptor, p160 Steroid Coactivators, and p300/CBP-Associated Factor Leading to Androgenic Axis Transactivation in Castration-Resistant Prostate Cancer.

BACKGROUND/AIMS: Cholesterol modulates intratumoral androgenic signaling in prostate cancer; however, the molecular mechanisms underlying these changes in castration-resistant prostate cancer (CRPC) are not fully elucidated. Herein, we investigated the effect of cholesterol on androgen receptor (AR) coactivators expression and tumorigenesis in vitro and in vivo. METHODS: Herein, we monitored the expression of AR coactivators (SRC-1, 2, 3 and PCAF) genes in PC-3 cells exposed to 2µg/mL of cholesterol for 8 hours by qPCR. We also performed cell migration at 0, 8, 24, 48 and 72h and flow cytometry assays (viability, apoptosis, and cell cycle) after a 24h exposure. Immunofluorescence assay was performed to evaluate the protein expression of the AR coactivators. Additionally, in vivo experiments were conducted using 22 male NOD/SCID mice. Mice were fed a standard (Control) or hypercholesterolemic (HCOL) diet for 21 days and then subcutaneously implanted with PC-3 cells. The tumor volume was calculated every two days, and after four weeks, the tumors were resected, weighed, and the serum lipid profile was measured. We also measured the intratumoral lipid profile and AR coactivators gene and protein expression by qPCR and Western Blot, respectively. Intratumor testosterone and dihydrotestosterone (DHT) concentrations were determined using ELISA. RESULTS: Cholesterol up-regulated the gene expression of coactivators SRC-1, SRC-2, SRC-3and PCAF, increasing AR expression in PC-3 cells. Next, cholesterol-supplemented PC-3 cells exhibited increased cell migration and altered cell cycle phases, leading to changes in proliferation and reduced apoptosis. We found that SRC-1, SRC-2, SRC-3 and PCAF proteins co-localized in the nucleus of cholesterol-supplemented cells and co-associate with AR. In the in vivo model, the hypercholesterolemic (HCOL) group displayed higher serum total and intratumoral cholesterol levels, increased testosterone and dihydrotestosterone concentrations, and up-regulated AR coactivator expression. The tumor volume of the HCOL group was significantly higher than the control group. CONCLUSION: Our findings revealed that increased nuclear translocation of the coactivators leads to up-regulated AR gene and protein expression, potentially influencing tumor progression. Studies targeting cholesterol-modulated changes in AR coactivator expression may provide insights into the molecular mechanisms associated with the CRPC phenotype.

Can we use Ki67 expression to predict prostate cancer aggressiveness?

INTRODUCTION: specialists have an urge for biomarkers that can discriminate indolent prostate cancer from aggressive tumors. Ki67 is a proliferation marker, and its expression is associated with the aggressiveness of several cancers. OBJECTIVE: analyze the expression of Ki67 in prostate cancer samples correlating with the aggressiveness of the disease. METHODS: Ki67 mRNA levels were determined utilizing data from a TCGA cohort (Tumor(n)=492 and control(n)=52). The protein expression was determined on 94 biopsies from patients by immunohistochemical assay. RESULTS: in mRNA, the Ki67 upregulation is associated with cancer tissue (p<0.0001) and worst disease-free survival (p=0.035). The protein upregulation is associated with increase of the ISUP score (p<0.0001), cancer stage (p=0.05), biochemical recurrence (p=0.0006) and metastasis (p<0.0001). We also show a positive correlation between Ki67 expression and ISUP score (r=0.5112, p<0.0001) and disease risk stratification (r=0.3388, p=0.0009). Ki67 expression is a factor independently associated with biochemical recurrence (p=0.002) and metastasis (p<0.0001). Finally, the patients with high Ki67expression shows better survival regarding biochemical recurrence (p=0.008) and metastasis (p=0.056). Patients with high Ki67 expression are 2.62 times more likely to develop biochemical recurrence (p=0.036). CONCLUSION: Ki67 upregulation is associated with prostate cancer aggressiveness.

Combined spinal and general anesthesia attenuate tumor promoting effects of surgery. An experimental animal study.

Background: Radical prostatectomy, a standard management approach for localized Prostate Cancer (PC), may cause a stress response associated with immune modulating effects. Regional anesthesia was hypothesized to reduce the immune effects of surgery by minimizing the neuroendocrine surgical stress response, thus mitigating tumor cells dissemination. Our primary objective was to investigate whether the use of spinal blocks attenuates PC tumor cells dissemination on an animal model. We also assessed the number of circulating NK cells and the amount of inflammatory and anti-inflammatory cytokines. Materials and methods: A subcutaneous tumor model, with PC-3M cell line transfected with a luciferase-producing gene (PC-3M-luc-C6) was used. After proper tumor establishment and before tumors became metastatic, animals were submitted to tumor excision surgeries under general or combined (general and spinal) anesthesia. A control group was only anesthetized with general anesthesia. Results: The subcutaneous tumor model with PC-3M-luc-C6 cells was effective in causing distant metastasis after 35 days. The number of circulating tumor cells increased in animals that underwent surgery under general anesthesia alone compared to the group submitted to combined anesthesia. Interleukin 6 levels were different in all groups, with increase in the general anesthesia group. Conclusion: Our results suggest that combination of spinal and general anesthesia may attenuate the suppression of innate tumor immunity and it might be related to a reduction in the neuroendocrine response to surgery. Institutional protocol number: Animal Ethics Committee 1332/2019.

Pan-cancer analysis reveals that CTC1-STN1-TEN1 (CST) complex may have a key position in oncology.

Telomere dysfunction is one of the hallmarks of cancer, which puts telomere-associated genes in a prominent position in oncology. The CTC1-STN1-TEN1 (CST) complex is vital for telomere maintenance and participates in several steps of DNA metabolism, such as repair and replication, essential functions for malignant cells. Despite this, little is known about these genes in cancer biology. Here, using bioinformatics tools, we performed a study in 33 cancer types and over 10,000 TCGA samples analyzing the role of the CST complex in cancer. We obtained the somatic landscape and gene expression patterns of each of the subunits of the complex studied. Furthermore, we show that CST is important for genetic stability and nucleic acid metabolism in cancer. We identify possible interactors, transcription factors, and microRNAs associated with CST and two drugs that may disrupt their pathways. In addition, we show that CST gene expression is associated with cancer survival and recurrence in several tumor types. Finally, we show negative and positive correlations between immune checkpoint genes and CST in different types of cancer. With this work, we corroborate the importance of these genes in cancer biology and open perspectives for their use in other works in the field.

Can we use Ki67 expression to predict prostate cancer aggressiveness? / Podemos usar a expressão de Ki67 para prever a agressividade do câncer de próstata?

ABSTRACT Introduction: specialists have an urge for biomarkers that can discriminate indolent prostate cancer from aggressive tumors. Ki67 is a proliferation marker, and its expression is associated with the aggressiveness of several cancers. Objective: analyze the expression of Ki67 in prostate cancer samples correlating with the aggressiveness of the disease. Methods: Ki67 mRNA levels were determined utilizing data from a TCGA cohort (Tumor(n)=492 and control(n)=52). The protein expression was determined on 94 biopsies from patients by immunohistochemical assay. Results: in mRNA, the Ki67 upregulation is associated with cancer tissue (p<0.0001) and worst disease-free survival (p=0.035). The protein upregulation is associated with increase of the ISUP score (p<0.0001), cancer stage (p=0.05), biochemical recurrence (p=0.0006) and metastasis (p<0.0001). We also show a positive correlation between Ki67 expression and ISUP score (r=0.5112, p<0.0001) and disease risk stratification (r=0.3388, p=0.0009). Ki67 expression is a factor independently associated with biochemical recurrence (p=0.002) and metastasis (p<0.0001). Finally, the patients with high Ki67expression shows better survival regarding biochemical recurrence (p=0.008) and metastasis (p=0.056). Patients with high Ki67 expression are 2.62 times more likely to develop biochemical recurrence (p=0.036). Conclusion: Ki67 upregulation is associated with prostate cancer aggressiveness.

RESUMO Introdução: especialistas precisam biomarcadores que podem discriminar o câncer de próstata indolente de tumores agressivos. Ki67 é um marcador de proliferação, e sua expressão está associada à agressividade de vários tumores. Objetivo: analisar a expressão do Ki67 em amostras de câncer de próstata correlacionando com a agressividade da doença. Métodos: os níveis de mRNA de Ki67 foram determinados utilizando dados de uma coorte de TCGA (Tumor(n)=492 e controle(n)=52). A expressão da proteína foi determinada em 94 biópsias de pacientes por ensaio imuno-histoquímica. Resultados: no mRNA, a superexpressão Ki67 está associada ao tecido canceroso (p<0,0001) e à pior sobrevida livre de doença (p=0,035). A superexpressão proteica está associada ao aumento do escore ISUP (p<0,0001), estágio de câncer (p=0,05), recorrência bioquímica (p=0,0006) e metástase (p<0,0001). Também mostramos uma correlação positiva entre a expressão Ki67 e o escore ISUP (r=0,5112, p<0,0001) e a estratificação de risco de doença (r=0,3388, p=0,0009). A expressão Ki67 é um fator independentemente associado à recorrência bioquímica (p=0,002) e metástase (p<0,0001). Finalmente, os pacientes com alta expressão de Ki67 expression mostram melhor sobrevivência em relação à recorrência bioquímica (p=0,008) e metástase (p=0,056). Os pacientes com alta expressão de Ki67 são 2,62 vezes mais propensos a desenvolver recorrência bioquímica (p=0,036). Conclusão: a superexpressão Ki67 está associada à agressividade do câncer de próstata.

Robotically assisted laparoscopic radical prostatectomy induces lower tissue trauma than radical retropubic prostatectomy.

To compare tissue trauma between Retropubic Radical Prostatectomy and Robotically Assisted Laparoscopic Radical Prostatectomy by inflammatory mediators. Serum samples from 40 patients submitted to RALP and 20 patients submitted to RRP were withdrawn at four different time points. The cytokines IL-4, IL-8, IL-6, IL-1B, IL-10 and TNF-α were detected using ELISA/Multiplex assays and xMAP-Luminex®. With both techniques, IL-10 and IL-6 were higher in T4 than in T1-T3 (p = 0.001). IL-10 and IL-6 were higher in T4 in open surgery than in robotic surgery (p = 0.000 and p = 0.001, respectively). Compared with both groups, IL-6 and IL-10 were higher in T4 in open surgery than in robotic surgery. Thus, we can postulate that RALP causes less tissue trauma than classical RRP, as indicated by the more limited increase in inflammatory mediators such as IL-6 and IL-10.

Digital application developed to evaluate functional results following robot-assisted radical prostatectomy: App for prostate cancer.

INTRODUCTION: Mobile applications ("apps") developed for smartphones and tablets are increasingly used in healthcare, allowing remote patient support or promoting self-health care. Prostate cancer (PC) screening allows for early-stage PC diagnosis, resulting in high rates of curative procedures such as radical prostatectomy. The main complications following surgery are urinary incontinence and erectile dysfunction. However, the exact numbers related to these morbidities are often missing due to brief interviews during consultations in the medical office. Therefore, the aim of this study was to create an app to determine whether response rates to pre- and post-surgical PC questionnaires will increase. METHODS: The app was built using the IONIC framework system and provided to patients through a prospective randomized study. We included 100 patients divided into two groups: 1. first group used the app (n = 50); and 2. second group responded via validated printed questionnaires (control group) (n = 50). All patients received discharge counseling to respond to the questionnaires 1, 3, 6 and 12 months after the procedure. The app group received verbal guidance on how monitoring would occur, received an SMS containing a username and password providing access to the system and received reminder alerts to respond to the questionnaires. RESULTS: The new app is called UroHealth and is available for download in the Apple App Store or at www.urohealth.com.br. When we evaluated the response rates, we found that 42.9% of the patients answered the preoperative questionnaire in the app group, while 16% responded in the control group (p = 0.003). By the end of the follow-up, we found that 24.5% of the patients answered the questionnaire in the app group, while 4% responded in the control group (p = 0.003). CONCLUSION: This app enabled almost 6 times more patients to answer long-term follow-up questions after surgical procedures, providing high-quality information regarding morbidity related to treatment. Although our initial results indicate that this app may become a useful tool in obtaining more frequent and realistic answers, thus helping to improve surgical techniques, other ways of reaching the patient should be tested to achieve higher response rates.

Urinary biomarkers of inflammation and tissue remodeling may predict bladder dysfunction in patients with benign prostatic hyperplasia.

PURPOSE: To evaluate the expression of urinary biomarkers of inflammation and tissue remodeling in patients with BPH undergoing surgery and evaluate the association of biomarkers with postoperative urodynamic outcomes MATERIALS AND METHODS: We analyzed urine samples from 71 patients treated with TURP from 2011 to 2017. Urinary levels of epidermal growth factor (EGF), matrix-metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), nerve growth factor (NGF) and monocyte-chemoattractant protein-1 (MCP-1) (by commercial ELISA kit) were measured, adjusted by urinary creatinine (Cr) and analyzed according to patients clinical and urodynamic characteristics (baseline and 12-month postoperative urodynamic) RESULTS: MMP-1/Cr levels were significantly higher among subjects with higher detrusor pressure on preoprative urodynamic. MCP-1/Cr levels were significantly higher amongs subjects with preoperative DO. Preoperative levels of NGF/Cr (0.13 vs 0.08, p = 0.005) and MMP-1/Cr (0.11 vs 0.04, p = 0.021) were predictors of persistent DO 12 months after surgery. The following factors were shown to be useful for predicting the persistence of DO in the postoperative period: NGF/Cr, with an AUC of 0.77 (95% CI 0.62-0.92) (p = 0.006), and MMP-1/Cr, with an AUC of 0.72 (95% CI 0.56-0.88) (p = 0.022). CONCLUSIONS: MMP-1/Cr was associated with higher detrusor pressure and MCP-1/CR with DO. NGF/Cr and MMP-1/Cr were shown to be predictors of persistent postoperative DO.

Polymorphism in the PBX1 gene is related to cystinuria in Brazilian families.

The aim of our study was to determine regions of loss of heterozygosity, copy number variation analysis, and single nucleotide polymorphisms (SNPs) in Brazilian patients with cystinuria. A linkage study was performed using DNA samples from six patients with cystinuria and six healthy individuals. Genotyping was done with the Genome-Wide Human SNP 6.0 arrays (Affymetrix, Inc., Santa Clara, CA, USA). For validation, SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. The homozygote polymorphic genotype of SNP rs17383719 in the gene PBX1 was more frequent (P = 0.015) in cystinuric patients. The presence of the polymorphic allele for this SNP increased the chance of cystinuria by 3.0-fold (P = 0.036). Pre-B-cell leukaemia transcription factor 1 (PBX1) was overexpressed 3.3-fold in patients with cystinuria. However, when we compared the gene expression findings with the genotyping, patients with a polymorphic homozygote genotype had underexpression of PBX1, while patients with a heterozygote or wild-type homozygote genotype had overexpression of PBX1. There is a 3-fold increase in the risk of the development of cystinuria among individuals with this particular SNP in the PBX1 gene. We postulate that the presence of this SNP alters the expression of PBX1, thus affecting the renal absorption of cystine and other amino acids, predisposing to nephrolithiasis.

MicroRNA-23b and microRNA-27b plus flutamide treatment enhances apoptosis rate and decreases CCNG1 expression in a castration-resistant prostate cancer cell line.

The acquisition of a castration-resistant prostate cancer phenotype by prostate cancer cells is the alteration that has the worst prognosis for patients. The aim of this study was to evaluate the role of the microRNAs-23b/-27b as well as the possible CCNG1 target gene in tissue samples from patients with localized prostate cancer that progressed to castration-resistant prostate cancer and in a castration-resistant prostate cancer cell line (PC-3). The microRNAs and target gene expression levels of the surgical specimens were analyzed by quantitative real-time polymerase chain reaction. The prostate cancer cell line, PC-3, was transfected with pre-miR-23b, pre-miR-27b, and their respective controls using Lipofectamine RNAiMAX and exposed or not to flutamide. After transfections, expression levels of both the microRNAs and the gene, CCNG1, were analyzed by quantitative real-time polymerase chain reaction. The apoptosis and cell cycle assays were performed on the mini MUSE cytometer. MicroRNAs-23b/-27b were underexpressed in surgical specimens of prostate cancer; however, their target gene, CCNG1, was overexpressed in 69% of the cases. After transfection with the microRNAs-23b/-27b and flutamide, we observed a reduction in gene expression compared with cells that were treated only with microRNAs or only with flutamide. In the apoptosis assay, we demonstrated cell sensitization following transfection with microRNAs-23b/-27b and potentiation when co-administered with flutamide. The number of cells in apoptosis was almost three times higher with the simultaneous treatments (miR + flutamide) compared with the control (p < 0.05). In the cell cycle assay, only flutamide treatment showed better results; a higher number of cells were found in the G0-G1 phase, and a lower percentage of cells completed the final phase of the cycle (p < 0.05). We conclude that microRNAs-23b/-27b are downexpressed in prostate cancer, and their target gene, CCNG1, is overexpressed. We postulated that microRNAs-23b/-27b sensitize the PC-3 cell line and that after the addition of flutamide in the apoptosis assay, we would observe synergism in the treatments between miR and flutamide. In the cell cycle assay, the use of flutamide was sufficient to decrease the number of cells in mitosis. Therefore, we postulate that microRNAs, along with other drugs, may become very useful therapeutic tools in the treatment of castration-resistant prostate cancer.

miR-618: possible control over TIMP-1 and its expression in localized prostate cancer.

BACKGROUND: The imbalance between the action of the tissue inhibitors of matrix metalloproteinases (TIMPs) and the matrix metalloproteinases (MMPs) is one component of metastasis physiology. TIMP-1 overrides MMP-9 activity in cancer and might be regulated by miR-618. The aims of this study were to clarify whether TIMP-1 expression is modified by miR-618 and to clarify the effect of miR-618 expression on the invasion of prostate cancer cells. We also studied miR-618 expression in surgical specimens of patients with localized prostate cancer submitted to open radical prostatectomy. METHODS: After transfection of miR-618 or its antagonist in DU145 cells, qRT-PCR for TIMP-1/MMP-9 and both ELISA and zymography for MMP-9 were performed. Total miRNA was extracted from surgical specimens of PCa, and miR-618 expression was examined for correlations with Gleason score, pathological status and biochemical recurrence. RESULTS: DU145 cells transfected with miR-618 had a 76% reduction in TIMP-1 expression relative to control cells (p = 0.003). miR-618 inhibition reduced MMP-9 expression by 31% (p = 0.032) and MMP-9 absorbance evaluated with ELISA assay (p = 0.06).Zymography suggested higher MMP-9 activity in DU145 cells transfected with miR-618 than those transfected with miR-618 inhibitor, but the difference was not significant (p = 0.55). However, miR-618 expression was lower in surgical specimens of patients with Gleason score > 7 (p = 0.08) and more advanced disease (p = 0.07). CONCLUSIONS: In vitro, miR-618 overexpression decreases TIMP-1 and miR-618 inhibition decreases MMP-9, suggesting that miR-618 might be an oncomiR. However, the analysis of clinical samples of localized prostate cancer revealed an inconsistent pattern, as increased miR-618 expression was associated with lower Gleason score and pathological status. Further studies are needed to address whether miR-618 is a context-dependent miRNA.

miR-29b enhances prostate cancer cell invasion independently of MMP-2 expression.

BACKGROUND: The ability to metastasize is one of the most important characteristics of neoplastic cells. An imbalance between the action of some matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs drives the invasion process. Some studies have suggested that MMP-2 is involved in metastasis, while other studies have reported that collagen production by cancer cells might also contribute to motility. However, decreased expression of microRNA-29b (miR-29b), which may control MMP-2 and collagen gene expression, has been shown in prostate cancer (PCa). The objectives of the present study were to clarify whether MMP-2 as well as collagens I and III (encoded by COL1A1 and COL3A1, respectively) are controlled by miR-29b and to determine whether metastasis is altered by this relationship. METHODS: PCa DU145 and PC-3 cells were transfected with 100 µL of OPTI-MEM I containing 100 nmol of miR-29b (or its inhibitor) along with 1.5 µL of lipofectamine. Positive and negative controls were prepared using the same protocol. MMP-2, COL1A1 and COL3A1 messenger RNA (mRNA) levels were evaluated via real-time polymerase chain reaction (qRT-PCR). For qRT-PCR, 6 × 104 cells were used. Invasion studies were conducted with Matrigel assays, which simulate invasion of the extracellular matrix by neoplastic cells. After transfection of 3 × 104 cells, invasion was allowed to proceed for 48 h. Invasive cells were counted under an optical microscope. Each experiment was performed in triplicate. RESULTS: MMP-2 mRNA was not expressed in DU145 cells after transfection with miR-29b. After transfection of cells with the miR-29b inhibitor, COL1A1 (p = 0.02) and COL3A1 (p = 0.06) mRNA expression was increased in DU145 cells, and a large number of transfected DU145 and PC3 cells invaded the Matrigel membrane. CONCLUSIONS: In vitro studies showed that reducing the amount of miR-29b may lead to higher PCa cell invasion via a process that is independent of MMP-2. Collagen expression, controlled by miR-29b, may facilitate this motility process. Thus, the present study suggests that collagen production plays an active role in metastasis control and restoration of miR-29b levels may decrease metastasis. Altogether, these findings support further exploration of drug therapy targeting this aspect of the metastasis circuit.

The role of urinary KIM-1, NGAL, CA19-9 and ß2-microglobulin in the assessment of ureteropelvic junction obstruction in adults.

PURPOSE: The objective of this study is to evaluate the diagnostic properties of urinary biomarkers in adults with ureteropelvic junction obstruction: KIM-1, NGAL, CA19-9, and ß2-microglobulin. We also assessed urinary biomarker concentrations following pyeloplasty. MATERIAL AND METHODS: We prospectively studied adults from December 2013 to February 2015. We included 47 patients with a mean age of 38.6 ± 12.7 years. Each patient provided four samples of voided urine for biomarker measurement, one at pre-operative consultation and the others at 1, 3, and 6 months of post-operative follow-up. The control group consisted of 40 healthy individuals with no hydronephrosis on ultrasound evaluation. RESULTS: KIM-1 had an area under the curve of 0.79 (95% CI 0.70-0.89), NGAL 0.71 (95% CI 0.61-0.83), CA19-9 0.70 (95% CI 0.60-0.81), and ß2-microgloblin 0.61 (95% CI 0.50-0.73). KIM-1 was the most sensitive marker with a cut-off of 170.4 pg/mg creatinine (sensitivity 91.4%, specificity 59.1%), whereas CA19-9 was the most specific with a cut-off of 51.3 U/mg creatinine (sensitivity 48.9%, specificity 88.0%). Urinary concentrations of biomarkers decreased after pyeloplasty. CONCLUSIONS: The evaluation of urinary biomarkers is useful in adults undergoing pyeloplasty. KIM-1, NGAL, and CA19-9 were elevated and significantly decreased after surgery.

Gene expression profile of renal cell carcinomas after neoadjuvant treatment with sunitinib: new pathways revealed.

BACKGROUND: In renal cell carcinoma (RCC) of the clear cell type, inactivity of the VHL gene induces overexpression of HIF1 α and its targets, the tyrosine kinase receptors, promoting RCC development and progression. The discovery of tyrosine kinase inhibitors (TKIs) changed the treatment of these tumors. Other molecular pathways involved in the TKI mechanisms of action have not been described in the literature. The aim of our study was to elucidate alternative mechanisms of action of sunitinib in tumor tissue after neoadjuvant treatment of RCC. METHODS: The gene expression profile was accessed using microarray (Affymetrix Human Genome U133 Plus 2.0 platform) and frozen RCC tissues collected from 5 patients with locally advanced non-metastatic tumors who underwent nephrectomy after being treated with 2 cycles of neoadjuvant sunitinib. The results were compared with matched controls comprising 6 patients with no neoadjuvant intervention. RESULTS: There was underexpression of the majority of genes after sunitinib treatment. The lower expression levels of IGFBP1, CCL20, CXCL6 and FGB were confirmed by qRT-PCR in all cases. The downregulation of gene expression leads us to search for methylation as a mechanism of action of the TKI. IGFBP1 was shown to be methylated by methylation-sensitive high-resolution melting technique. CONCLUSIONS: The ultimate genetic effects of sunitinib may explain its actions as an antitumor drug that apparently suppresses the expression of important genes related to cell survival, adhesion, invasion and immunomodulation. The methylation of gene promoters was shown to be part of the mechanism of action of this class of drugs.

Role of Genetic Polymorphisms in the Development and Prognosis of Sporadic and Familial Prostate Cancer.

BACKGROUNDS: Our aim was to evaluate the role of 20 genetic polymorphisms in the development and prognosis of sporadic and familial PC. A case-control study of 185 patients who underwent radical prostatectomy from 1997 to 2011. These patients were divided into two groups based on their family history. Gleason grade, PSA value and pathological TNM 2002 stage were used as prognostic factors. Blood samples from 70 men without PC were used as controls. The SNPs were genotyped using a TaqMan® SNP Genotyping Assay Kit. RESULTS: Considering susceptibility, the polymorphic allele in the SNP rs2660753 on chromosome 3 was significantly more prevalent in controls (p = 0.01). For familial clustering, the polymorphic homozygote genotype of the SNP rs7931342 was five times more frequent in patients with familial PC compared to sporadic PC (p = 0.01). Regarding the SNP 1447295, the polymorphic homozygote genotype was more prevalent in patients with organ-confined PC (p = 0.05), and most importantly, the polymorphic allele occurred more frequently in patients without biochemical recurrence (p = 0.01). Kaplan-Meier analysis showed a median biochemical recurrence free survival of 124.2 compared to 85.6 months for patients with the wild-type allele (p = 0.007). CONCLUSION: Our findings provide the evidence for the association of 20 recently highlighted SNPs and their susceptibility, familial clustering, staging, Gleason score and biochemical recurrence of PC. We believe that the association between these SNPs and PC may contribute to the development of alternative tools that can facilitate the early detection and prognosis of this disease.

Expression profile of standard and variants forms of CD44 related to prostate cancer behavior.

CD44 is a transmembrane glycoprotein and is regarded as a potential marker in various tumors. The aim of our study was to analyze the expression of the standard form of CD44 (CD44s) and its isoforms in localized prostate cancer (PCa), and to correlate these data with the classical prognostic factors and biochemical recurrence.Ninety-four surgical specimens were analyzed in this study. The expression levels of CD44s and all its 9 variants were analyzed by quantitative real time PCR (qRT-PCR). The control group consisted of 14 specimens from patients with benign prostatic hyperplasia. We correlated all the expression profiles with biochemical recurrence, as defined by a PSA >0.4 ng/mL in a mean follow-up period of 53.3 months. In PCa, CD44s was underexpressed and all the other isoforms were overexpressed. The mean expression level of most variants was higher in patients who had not recurred, and a higher expression of CD44v2 independently correlated with a better recurrence-free survival rate (p=0.045). This variant was also underexpressed in metastatic PCa cell lines. There was no correlation between the expression levels of any of the CD44 isoforms and the classical prognostic factors.We here demonstrated that PCa cases are characterized by a change in the expression of CD44, with a loss of CD44s and an overexpression of all the other CD44 variants. However, during cancer progression we found a loss of expression of all CD44 variants, and a correlation between higher expression of CD44v2 and a better recurrence-free survival rate. The understanding of the CD44 expression patterns in PCa could contribute to its use as a new prognostic marker.