Nayara Viana

Increased expression of MMP-9 and IL-8 are correlated with poor prognosis of Bladder Cancer

BackgroundExtracellular matrix homeostasis is strictly maintained by a coordinated balance between the expression of metalloproteinases (MMPs) and their inhibitors. The purpose of this study was to investigate whether the expression of MMP-9, MMP-2 and its specific inhibitors, are expressed in a reproducible, specific pattern and if the profiles are related to prognosis in Bladder Cancer (BC).MethodsMMP-9, MMP-2 and its specific inhibitors expression levels were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in fresh-frozen malignant tissue collected from 40 patients with BC submitted to transurethral resection of bladder. The control group consisted of normal bladder tissue from five patients who had undergone retropubic prostatectomy to treat benign prostatic hyperplasia.ResultsMMP-9 was overexpressed in 59.0 % of patients, and MMP-2, TIMP-1, TIMP-2, MMP-14, RECK and IL-8 was underexpressed in most of the patients. Regarding prognostic parameters we observed that high-grade tumors exhibited significantly higher levels of MMP-9 and IL-8 (p = 0.012, p = 0.003). Invasive tumors (pT1-pT2) had higher expression levels of MMP-9 than superficial tumors (pTa) (p = 0.026). The same was noted for IL-8 that was more expressed by invasive tumors (p = 0.015, p = 0.048). Most importantly tumor recurrence was related with higher levels of both MMP-9 (p = 0.003) and IL-8 (p = 0.005).ConclusionWe have demonstrated that the overexpression of MMP-9 and higher expression of IL-8 are related to unfavorable prognostic factors of urothelial bladder cancer and tumor recurrence and may be useful in the follow up of the patients.

Stage, Grade and Behavior of Bladder Urothelial Carcinoma Defined by the MicroRNA Expression Profile

PURPOSE We identified miRNA expression profiles in urothelial carcinoma that are associated with grade, stage, and recurrence-free and disease specific survival. MATERIALS AND METHODS The expression of 14 miRNAs was evaluated by quantitative reverse transcriptase-polymerase chain reaction in surgical specimens from 30 patients with low grade, noninvasive (pTa) and 30 with high grade, invasive (pT2-3) urothelial carcinoma. Controls were normal bladder tissue from 5 patients who underwent surgical treatment for benign prostatic hyperplasia. Endogenous controls were RNU-43 and RNU-48. miRNA profiles were compared and Kaplan-Meier curves were constructed to analyze disease-free and disease specific survival. RESULTS miR-100 was under expressed in 100% of low grade pTa specimens (p <0.001) and miR-10a was over expressed in 73.3% (p <0.001). miR-21 and miR-205 were over expressed in high grade pT2-3 disease (p = 0.02 and <0.001, respectively). The other miRNAs were present at levels similar to those of normal bladder tissue or under expressed in each tumor group. miR-21 over expression (greater than 1.08) was related to shorter disease-free survival in patients with low grade pTa urothelial carcinoma. Higher miR-10a levels (greater than 2.30) were associated with shorter disease-free and disease specific survival in patients with high grade pT2-3 urothelial carcinoma. CONCLUSIONS Four miRNAs were differentially expressed in the 2 urothelial carcinoma groups. miR-100 and miR-10a showed under expression and over expression, respectively, in low grade pTa tumors. miR-21 and miR-205 were over expressed in pT2-3 disease. In addition, miR-10a and miR-21 over expression was associated with shorter disease-free and disease specific survival. miRNAs could be incorporated into the urothelial carcinoma molecular pathway. These miRNAs could also serve as new diagnostic or prognostic markers and new target drugs.

Perineural invasion detection in prostate biopsy is related to recurrence-free survival in patients submitted to radical prostatectomy.

OBJECTIVE Perineural invasion (PNI) is detected in almost 20% of prostate biopsies and has been related to worse prognostic factors in radical prostatectomy (RP) specimens and lower disease-free survival rates. The aim of this study was to evaluate the importance of PNI during periods of extended prostate biopsies and to determine the value of this preoperative parameter as a predictor of pathologic findings in surgical specimens and in biochemical recurrence. MATERIALS AND METHODS Between 2001 and 2009, 599 prostate biopsies and their respective RP specimens were examined in our laboratory. The RP specimens were always examined completely. The mean age of the patients was 61 years, and the mean PSA was 6.4 ng/mL. The mean and median number of biopsy cores obtained was 14.4 and 14, respectively. PNI was identified in 105 biopsies (17.5%). We studied the ability of PNI in prostate biopsies to determine the tumor stage in surgical specimens and the relationship of PNI with biochemical recurrence during a mean follow-up time of 51.4 months. RESULTS The presence of PNI in prostate biopsies was observed in older patients (63 vs. 61 years old, P = 0.008). All of the prognostic factors determined for the RP specimens were significantly worse in patients with PNI compared with those without PNI. PNI was strongly associated with a higher pathologic stage (87% specificity, 40% sensitivity, odds ratio 4.8). Stage pT3 prostatic cancer was determined in 46 (43.8%) of 105 patients with PNI on biopsy compared to 69 (14%) of 494 patients without PNI (P = 0.01). Fifty-six (19.6%) patients had a biochemical recurrence, and PNI correlated significantly with PSA recurrence. A Kaplan-Meier analysis revealed a significant difference in recurrence-free survival between patients with and without PNI (45% vs. 53%, respectively, P = 0.021, log-rank test = 0.19). CONCLUSION PNI is an important morphologic preoperative predictor of the pathologic stage as well as biochemical recurrence and must always be mentioned when adenocarcinoma is diagnosed on prostate biopsies.

Tgf-β1 expression as a biomarker of poor prognosis in prostate cancer

OBJECTIVE: To evaluate the correlation between transforming growth factor beta (TGF-β1) expression and prognosis in prostate cancer. PATIENTS AND METHODS: TGF-β1 expression levels were analyzed using the quantitative real-time polymerase chain reaction to amplify RNA that had been isolated from fresh-frozen malignant and benign tissue specimens collected from 89 patients who had clinically localized prostate cancer and had been treated with radical prostatectomy. The control group consisted of 11 patients with benign prostate hyperplasia. The expression levels of TGF-β1 were compared between the groups in terms of Gleason scores, pathological staging, and prostate-specific antigen serum levels. RESULTS: In the majority of the tumor samples, TGF-β1 was underexpressed 67.0% of PCa patients. The same expression pattern was identified in benign tissues of patients with prostate cancer. Although most cases exhibited underexpression of TGF-β1, a higher expression level was found in patients with Gleason scores ≥7 when compared to patients with Gleason scores <7 (p = 0.002). Among the 26 cases of TGF-β1 overexpression, 92.3% had poor prognostic features. CONCLUSIONS: TGF-β1 was underexpressed in prostate cancers; however, higher expression was observed in tumors with higher Gleason scores, which suggests that TGF-β1 expression may be a useful prognostic marker for prostate cancer. Further studies of clinical specimens are needed to clarify the role of TGF-β1 in prostate carcinogenesis.

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.

MicroRNA 100: a context dependent miRNA in prostate cancer

OBJECTIVE: MicroRNAs are noncoding RNA molecules involved in the development and progression of tumors. We have found that miRNA-100 is underexpressed in metastatic prostate cancer compared to localized disease. Conversely higher levels of miR-100 are related to biochemical recurrence after surgery. This suggests that miR-100 may be a context-dependent miRNA, acting as oncogene or tumor suppressor miRNA. Our aim is to demonstrate the role of miR-100 in the control of predicted target genes in prostate cancer cell lines. METHODS: Cell lines DU145 and PC3 were transfected with miR-100, antimiR-100 and after 24 h and 48 h of exposure, qRT-PCR and western blot were performed for mTOR, FGFR3, THAP2, SMARCA5 and BAZ2A. RESULTS: There was reduction in mTOR (p = 0.025), THAP2 (p = 0.038), SMARCA5 (p = 0.001) and BAZ2A (p = 0.006) mRNA expression in DU145 cells after exposure to miR-100. In PC3 cells, mTOR expression was decreased by miR-100 (p = 0.01). There was a reduction in the expression levels of proteins encoded by studied genes, ranging from 34% to 69%. CONCLUSIONS: We demonstrate that miR-100 is a context-dependent miRNA controlling BAZ2, mTOR, FGFR3, SMARCA5 and THAP2 that might be involved in PC progression. The elucidation of the roles of miRNAs in tumors is important because they can be used as therapeutic targets in the future.

Controlling RECK miR21 Promotes Tumor Cell Invasion and Is Related to Biochemical Recurrence in Prostate Cancer

The search for biomarkers to characterize prostate cancer aggressiveness has been the objective for the majority of researchers involved with the most prevalent tumor in men. MiRNAs are important for the control of many cellular functions and their deregulation is involved with tumor development and progression. To find miRNAs differentially expressed in prostate cancer and their relation to prognostic factors and biochemical recurrence we studied 53 surgical specimens from men who underwent radical prostatectomy, through a microarray analysis using the microarray platform (GeneChip® miRNA Array - Affymetrix) with more than 46,000 probes and 847 mature human miRNAs and transcripts. We defined different as an expression level greater or less than 1.1 with p<0.05. The validation study using qRT-PCR had confirmed miR21 as overexpressed in tumor that have recurred with a risk of 2.5. Transfection of miR-21 using lipid based assay in DU145 cell line, showed decrease in expression of RECK resulting in increase in expression of MMP9. Invasion assay with Matrigel showed increase in tumor cell invasion after miR-21 transfection. We conclude that miR-21 overexpression is related to increased biochemical recurrence after surgical treatment of prostate cancer. And the negative control of RECK results in overexpression of MMP9 promotes increasing tumor cell invasion supporting miR-21 as an oncomiR related to aggressiveness in prostate cancer.

The role of micro RNAs let7c, 100 and 218 expression and their target RAS, C-MYC, BUB1, RB, SMARCA5, LAMB3 and Ki-67 in prostate cancer

OBJECTIVE: The aim of this study is to verify the expression of proteins that are controlled by miR-let7c, 100 and 218 using immunohistochemistry in tissue microarray representative of localized and metastasized the lymph nodes and bone prostate cancer. METHODS: To verify the expression of proteins that are controlled by miR-let7c (C-MYC, BUB1, RAS) 100 (SMARCA5, RB) and 218 (LAMB3) and cell proliferation (Ki-67) we used immunohistochemistry and computerized image system ImageJ MacBiophotonics in three tissue microarrays representative of localized prostate cancer and lymph node and bone metastases. miRNA expression was evaluated by qRT-PCR using 60 paraffin blocks to construct the tissue microarray representative of localized disease. RESULTS: RAS expression was increased in localized prostate cancer and bone metastases compared to the lymph nodes (p = 0.017). RB showed an increase in expression from localized prostate cancer to lymph node and bone metastasis (p = 0.036). LAMB3 was highly expressed in localized and lymph node metastases (p<0.001). Cell proliferation evaluated by Ki-67 showed an increase from localized prostate cancer to metastases (p<0.001). We did not found any relationship between C-MYC (p = 0.253), BUB1 (p = 0.649) and SMARCA5 (p = 0.315) protein expression with prognosis or tumor behavior. CONCLUSION: We found that the expression of RAS, RB, LAMB3 and Ki-67 changed in the different stages of prostate cancer. Furthermore, we confirmed the overexpression of the miRNAs let7c, 100 and 218 in localized prostate cancer but failed to show the control of protein expression by the putative controller miRNAs using immunohistochemistry.

The involvement of miR-100 in bladder urothelial carcinogenesis changing the expression levels of mRNA and proteins of genes related to cell proliferation, survival, apoptosis and chromosomal stability

IntroductionMicroRNAs (miRNA) are small non-coding RNAs that play an important role in the control of gene expression by inhibiting protein translation or promoting messenger RNA degradation. Today, miRNAs have been shown to be involved in various physiological and pathological cellular processes, including cancer, where they can act as oncogenes or tumor suppressor genes. Recently, lowered expression of miR-100, resulting in upregulation of FGFR3, has been correlated with low-grade, non-invasive bladder urothelial cancer, as an alternative oncogenesis pathway to the typical FGFR3 gene mutation. Our aim is to analyze the role of miR-100 in bladder cancer cell lines in controlling the expression of some of its possible target genes, including FGFR3 and its relationship with proliferation, apoptosis and DNA ploidy.MethodsThe bladder cancer cell lines RT4 and T24 were transfected with pre-miR 100, anti-miR 100 and their respective controls using a lipid-based formulation. After transfection mRNA and protein levels of its supposed target genes THAP2, BAZ2A, mTOR, SMARCA5 and FGFR3 were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and western blotting. Cell proliferation, apoptosis and DNA ploidy were analyzed by flow cytometry. For statistical analysis, a t-test was applied, p < 0.05 was considered significant.ResultsAfter miR-100 transfection, there was a significant reduction in the mRNA of mTOR (p = 0.006), SMARCA5 (p = 0.007) and BAZ2A (p = 0.029) in RT4, mTOR (p = 0.023) and SMARCA5 (p = 0.015) in T24. There was a reduction in the expression of all proteins, variable from 22.5% to 57.1% in both cell lines. In T24 miR-100 promoted an increase in cell proliferation and anti-miR 100 promoted apoptosis characterizing miR-100 as an oncomiR in this cell line representative of a high-grade urothelial carcinoma.ConclusionmiR-100 transfection reduces expression of BAZ2A, mTOR and SMARCA5 mRNA and protein in BC cell lines. miR-100 would be classified as an oncomiR in T24 cells representative of high grade urothelial carcinoma promoting increase in cell proliferation and reduction in apoptosis. The knowledge of miRNA role in tumors will allow their use as tumor markers and targets for new therapies.

Immune expression of E-cadherin and α, β and γ-Catenin adhesion molecules and prognosis for upper urinary tract urothelial carcinomas

INTRODUCTION Cell adhesion molecules (CAM) are required for maintaining a normal epithelial phenotype, and abnormalities in CAM expression have been related to cancer progression, including bladder urothelial carcinomas. There is only one study that correlates E-cadherin and Α-, Β- and y-catenin expression with prognosis of upper tract urothelial carcinomas. Our aim is to study the pattern of immune expression of these CAMs in urothelial carcinomas from the renal pelvis and ureter in patients who have been treated surgically. Our goal is to correlate these expression levels and characteristics with well-known prognostic parameters for disease-free survival. MATERIALS AND METHODS We evaluated specimens from 20 patients with urothelial carcinomas of the renal pelvis and ureter who were treated with nephroureterectomy or ureterectomy between June 1997 and January 2007. CAM expression was evaluated by immunohistochemistry in a tissue microarray and correlated with histopathological characteristics and patient outcomes after a mean follow-up of 55 months. RESULTS We observed a relationship between E-cadherin expression and disease recurrence. Disease recurrence occurred in 87.5% of patients with strong E-cadherin expression. Only 50.0% of patients with moderate expression and 0% of patients with weak or no expression of E-cadherin had disease recurrence (p = 0.014). There was also a difference in disease-free survival. Patients with strong E-cadherin expression had a mean disease-free survival rate of 49.1 months, compared to 83.9 months for patients with moderate expression (p = 0.011). Additionally, an absence of Α-catenin expression was associated with tumors that were larger than 3 cm (p = 0.003). CONCLUSIONS We demonstrated for the first time that immune expression of E-cadherin is related to tumor recurrence and disease-free survival rates, and the absence of Α-catenin expression is related to tumor size in upper tract urothelial carcinomas.