Alexandre Iscaife

Comprehensive Study of Gene and microRNA Expression Related to Epithelial-Mesenchymal Transition in Prostate Cancer

Prostate cancer is the most common cancer in men, and most patients have localized disease at the time of diagnosis. However, 4% already present with metastatic disease. Epithelial-mesenchymal transition is a fundamental process in carcinogenesis that has been shown to be involved in prostate cancer progression. The main event in epithelial-mesenchymal transition is the repression of E-cadherin by transcription factors, but the process is also regulated by microRNAs. The aim of this study was to analyze gene and microRNA expression involved in epithelial-mesenchymal transition in localized prostate cancer and metastatic prostate cancer cell lines and correlate with clinicopathological findings. We studied 51 fresh frozen tissue samples from patients with localized prostate cancer (PCa) treated by radical prostatectomy and three metastatic prostate cancer cell lines (LNCaP, DU145, PC3). The expression of 10 genes and 18 miRNAs were assessed by real-time PCR. The patients were divided into groups according to Gleason score, pathological stage, preoperative PSA, biochemical recurrence, and risk group for correlation with clinicopathological findings. The majority of localized PCa cases showed an epithelial phenotype, with overexpression of E-cadherin and underexpression of the mesenchymal markers. MiRNA-200 family members and miRNAs 203, 205, 183, 373, and 21 were overexpressed, while miRNAs 9, 495, 29b, and 1 were underexpressed. Low-expression levels of miRNAs 200b, 30a, and 1 were significantly associated with pathological stage. Lower expression of miR-200b was also associated with a Gleason score ≥8 and shorter biochemical recurrence-free survival. Furthermore, low-expression levels of miR-30a and high-expression levels of Vimentin and Twist1 were observed in the high-risk group. Compared with the primary tumor, the metastatic cell lines showed significantly higher expression levels of miR-183 and Twist1. In summary, miRNAs 200b, 30a, 1, and 183 and the genes Twist1 and Vimentin might play important roles in the progression of prostate cancer and may eventually become important prognostic markers.

Can We Predict Which Patients will Experience Resolution of Detrusor Overactivity after Transurethral Resection of the Prostate

PURPOSE We determined which clinical and urodynamic variables may be related to persistent detrusor overactivity after transurethral resection of the prostate. MATERIALS AND METHODS We studied 46 patients with bladder outlet obstruction due to benign prostatic hyperplasia who were treated with transurethral prostate resection from 2011 to 2012. All patients underwent urodynamic analysis preoperatively and 12 months postoperatively. Clinical and urodynamic variables in the preoperative period were correlated with the resolution of detrusor overactivity postoperatively. RESULTS Patients with detrusor overactivity in the preoperative period were older (65.2 vs 61.1 years, p = 0.041) and had a higher I-PSS (International Prostate Symptom Score) (25.2 vs 19, p = 0.014) and higher maximum flow rate (8.6 vs 6.6 ml per second, p = 0.039). Patients with persistent detrusor overactivity were statistically older than those with resolution (69 vs 63 years, p = 0.043). Detrusor overactivity persisted in 63.6% of patients with maximum cystometric capacity less than 250 ml compared to 20% of those with greater than 250 ml (p = 0.024). When analyzing urodynamic variables together, we found a 66.7% chance of persistent detrusor overactivity in patients with maximum cystometric capacity less than 250 ml and detrusor overactivity amplitude greater than 40 cm H2O (p = 0.041). When these characteristics were associated with early detrusor overactivity, the chance of persistent detrusor overactivity was 83.3% (p = 0.013). CONCLUSIONS Advanced patient age together with low maximum cystometric capacity, and early and high detrusor overactivity amplitude are the most important predictors of persistent detrusor overactivity after relief of bladder outlet obstruction.

Nanoradiopharmaceuticals for breast cancer imaging: development, characterization, and imaging in inducted animals

Monoclonal antibodies as polymeric nanoparticles are quite interesting and endow this new drug category with many advantages, especially by reducing the number of adverse reactions and, in the case of radiopharmaceuticals, also reducing the amount of radiation (dose) administered to the patient. In this study, a nanoradiopharmaceutical was developed using polylactic acid (PLA)/polyvinyl alcohol (PVA)/montmorillonite (MMT)/trastuzumab nanoparticles labeled with technetium-99m (99mTc) for breast cancer imaging. In order to confirm the nanoparticle formation, atomic force microscopy and dynamic light scattering were performed. Cytotoxicity of the nanoparticle and biodistribution with 99mTc in healthy and inducted animals were also measured. The results from atomic force microscopy showed that the nanoparticles were spherical, with a size range of ~200–500 nm. The dynamic light scattering analysis demonstrated that over 90% of the nanoparticles produced had a size of 287 nm with a zeta potential of −14,6 mV. The cytotoxicity results demonstrated that the nanoparticles were capable of reaching breast cancer cells. The biodistribution data demonstrated that the PLA/PVA/MMT/trastuzumab nanoparticles labeled with 99mTc have great renal clearance and also a high uptake by the lesion, as ~45% of the PLA/PVA/MMT/trastuzumab nanoparticles injected were taken up by the lesion. The data support PLA/PVA/MMT/trastuzumab labeled with 99mTc nanoparticles as nanoradiopharmaceuticals for breast cancer imaging.

Loss of TIMP-1 immune expression and tumor recurrence in localized prostate cancer

ABSTRACT Introduction and objective: Overexpression of MMPs has been related to biochemical recurrence after radical prostatectomy. TIMP1 and TIMP2 are controllers of MMPs and the aim of this study is to evaluate the expression levels of MMPs and their regulators using immunohistochemistry in tissue microarray of localized prostate cancer (PC). Materials and Methods: Immune-expression of MMP-9, MMP-2, TIMP1, TIMP-2, MMP-14 and IL8, were analyzed by immunohistochemistry in radical prostatectomy specimens of 40 patients with localized PC who underwent surgery between September 1997 and February 2000. Protein expression was considered as categorical variables, negative or positive. The results of the immune-expression were correlated to Gleason score (GS), pathological stage (TNM), pre-operatory PSA serum levels and biochemical recurrence in a mean follow up period of 92.5 months. Results: The loss of TIMP1 immune-expression was related to biochemical recurrence. When TIMP1 was negative, 56.3% patients recurred versus 22.2% of those whose TIMP1 was positive (p=0.042). MMP-9, MMP-2, IL8 and MMP-14 were positive in the majority of PC. TIMP-2 was negative in all cases. Conclusion: Negative immune-expression of TIMP1 is correlated with biochemical recurrence in patients with PC possibly by failing to control MMP-9, an important MMP related to cancer progression.

The role of microRNAs 371 and 34a in androgen receptor control influencing prostate cancer behavior

BACKGROUND The molecular mechanisms involved in androgen receptor (AR) signaling pathways are not completely understood, and deregulation of microRNAs (miRNAs) expression may play a role in prostate cancer (PC) development and progression. METHODS The expression levels of miRNA and AR were evaluated with quantitative real-time polymerase chain reaction using frozen tissue from the surgical specimens of 83 patients submitted to radical prostatectomy. The expression level of miRNAs was correlated with prognostic factors and biochemical recurrence during a follow-up period of 45 months. In vitro and in vivo experiments were performed to understand the effect of miRNAs over AR in the context of that seen in a PC model. RESULTS MiR-371 underexpression correlated with non-organ-confined (pT3) disease (P = 0.009). In vitro transfection of miR-371 reduced the levels of AR by 22% and 28% in LNCaP and PC3 cell lines, respectively, and in kallikrein 3, it was reduced by 51%. PC was induced in Balb/c mice using PC-3M-luc-C6 cells, and animals were treated with 3 local doses of miR-371. Tumor growth evaluated by in vivo imaging after luciferase injection was slower in animals treated with miR-371. To explore further the possible role of miRNAs in the AR pathway, LNCaP cell line was treated with 5α-dihydrotestosterone and flutamide showing alteration in miRNAs expression, especially miR-34a, which was significantly underexpressed after treatment with high doses of 5α-dihydrotestosterone. CONCLUSION Our data support a role for miRNAs, especially miR-371 and miR-34a, in the complex disarrangement of AR signaling pathway and in the behavior of PC.

Treating metastatic prostate cancer with microRNA-145

Prostate cancer (PCa) is an incurable disease at the metastatic stage. Although there are different options for treatment, the results are limited. MicroRNAs (miRNAs) are a group of small, noncoding, regulatory RNAs with important roles in regulating gene expression. miR-145 is reported to be a key tumor suppressor miRNA (tsmiR) that controls important oncogenes, such as MYC and RAS. In this study, in vitro studies were performed to show the control of MYC and RAS by miR-145. Flow cytometry was used to analyze cell proliferation and apoptosis. The efficacy of miR-145 in treating metastatic PCa was tested in nude mice using a model of bone metastasis promoted by intraventricular injection of PC-3MLuc-C6 cells. Tumor growth was evaluated by an in vivo bioluminescence system. After the full establishment of metastases on day 21, six animals were treated with three intravenous doses of miR-145 (on days 21, 24 and 27), and six were injected with scramble miRNA as controls. Compared to the controls, tumor growth was significantly reduced in animals receiving miR-145, most importantly on day 7 after the third and last dose of miRNA. After discontinuing the treatment, tumor growth resumed, becoming similar to the group of non-treated animals. A decrease in MYC and RAS expression was observed in all cell lines after treatment with miR-145, although statistical significance was achieved only in experiments with LNCaP and PC3 cell lines, with a decrease in 56% (p = 0.012) and 31% (p = 0.013) of RAS expression, respectively. Our results suggest that miR-145 is a potential molecule to be tested for treatment of metastatic, castration-resistant PCa.

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.