Jose Joao Mansure

Inhibition of mammalian target of rapamycin as a therapeutic strategy in the management of bladder cancer

We examined whether mTOR inhibition by RAD001 (Everolimus) could be therapeutically efficacious in the treatment of bladder cancer. RAD001 markedly inhibited proliferation of nine human urothelial carcinoma cell lines in dose- and sensitivity-dependent manners in vitro. FACS analysis showed that treatment with RAD001 for 48h induced a cell cycle arrest in the G0/G1 phase in all cell lines, without eliciting apoptosis. Additionally, RAD001 significantly inhibited the phosphorylation of S6 downstream of mTOR and VEGF production in all cell lines. We also found tumor weights from nude mice bearing human KU-7 subcutaneous xenografts treated with RAD001 were significantly reduced as compared to placebo-treated mice. This tumor growth inhibition was associated with significant decrease in cell proliferation rate and angiogenesis without changes in cell death. In conclusion inhibition of mTOR signaling in bladder cancer models demonstrated remarkable antitumor activity both in vitro and in vivo. This is the first study showing that RAD001 could be exploited as a potential therapeutic strategy in bladder cancer.

Peroxisome proliferator-activated receptor gamma in bladder cancer: a promising therapeutic target.

Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated intracellular transcription factors, members of the nuclear hormone receptor superfamily. The PPAR subfamily consist of three subtypes encoded by distinct genes denoted PPARα, PPARβ/δ, and PPARγ. The peroxisome proliferator-activated receptor γ (PPARγ) is the most extensively studied subtype of the PPARs. Over the last decade, research on PPARγ unveiled its role in important biological processes, including lipid biosynthesis, glucose metabolism, anti-inflammatory response, and atherosclerosis. Recently, PPARγ has been shown to be expressed in many cancers including, lung, prostate, bladder, colon, breast, duodenal, thyroid, and has been demonstrated to potentially play an important role in carcinogenesis. In bladder cancer, PPARγ ligands such as troglitazone and 15d-PGJ2 have shown to inhibit tumor growth. We have recently published the first report to show that a new class of PPARγ agonists, PPARγ-active C-DIMs, which are more potent than the previous generation of drugs, exhibit anti-tumorigenic activity against bladder cancer cells in vitro and bladder tumors in vivo. The following review will discuss the molecular structure of PPARγ, its function, and its role in cancer biology and how it is emerging as a promising therapeutic target in bladder cancer.

Prognostic factors in T1 bladder urothelial carcinoma: the value of recording millimetric depth of invasion, diameter of invasive carcinoma, and muscularis mucosa invasion

pT1 bladder urothelial carcinomas represent a heterogeneous group of tumors with different biologic behaviors, and identifying the subset of tumors that carries a high risk of disease recurrence and progression is therefore important. We evaluated the prognostic significance of substaging 86 cases of pT1 bladder urothelial carcinoma based on different pathologic parameters. The mean tumor depth was 1.1 mm, and the mean diameter of the invasive focus was 2.2 mm. The mean number of tissue fragments with invasion was 4.4. Lymphovascular invasion and concomitant carcinoma in situ were present in 13% and 45% of cases, respectively. Although 56% of patients recurred, 18% experienced disease progression. Multivariate analysis showed a significant association between muscularis mucosa invasion (P = .007), depth of invasion (P = .0001), diameter of invasive focus (P = .014), and progression. Furthermore, depth of invasion more than 3 mm was significantly associated with progression of disease, achieving a sensitivity of 31%, specificity of 99%, and predictive value of 79%. In comparison, the cutoff values for the diameter of invasive carcinoma that correlated best with outcome was 6 mm for progression. Lastly, combining both variables showed a strong prognostic accuracy where it predicted 94% of recurrences. Importantly, all cases with depth of invasion more than 3 mm and diameter more than 6 mm progressed. Lymphovascular invasion or concomitant carcinoma in situ did not correlate with outcome. From the current data, we do recommend reporting muscularis mucosa invasion whenever possible. Alternatively, tumor depth and tumor diameter should be included in the final pathology report in individual cases in which muscularis mucosa invasion cannot be assessed.

Relevance of the mammalian target of rapamycin pathway in the prognosis of patients with high-risk non–muscle invasive bladder cancer☆☆☆

High-risk non-muscle invasive bladder cancer (NMIBC) is associated with higher rates of recurrence and progression. Molecular markers within aberrant signaling pathways in cancer need further evaluation of their role as prognostic indicators and potential future targets for prevention of recurrence. Our objective was to investigate the role of the mammalian target of rapamycin (mTOR) signaling pathway on the stage and outcome of patients with high-risk NMIBC. Tissue microarrays were built from archival bladder tumor specimens (n = 142). Various clinicopathologic variables were collected retrospectively from patients treated with transurethral resection. Immunohistochemical staining was performed for phosphatase and tensin homolog, phosphorylated Akt, phosphorylated mTOR, phosphorylated S6 (p-S6), eukaryotic translation initiation factor 4E-binding protein-1, and p27. Multivariate analysis using Cox regression models addressed recurrence-free survival (RFS), progression-free survival, and worsening-free survival. In multivariate analysis, p-S6 was an independent predictor of shorter RFS (hazard ratio, 3.55; 95% CI, 1.31-9.64). Expression of p27 was inversely correlated with RFS (hazard ratio, 0.27; 95% CI, 0.10-0.74). Low levels of phosphatase and tensin homolog expression were associated with worsening-free survival (P < .03). None of the markers showed correlation with progression-free survival. Our results demonstrate that activation of the mTOR pathway, as assessed by p-S6 and expression of p27, might be used to provide prognostic information, particularly as a predictor of recurrence among patients with high-risk NMIBC.

The Role of HMGB1 in Radioresistance of Bladder Cancer

Although radical cystectomy surgery is the standard-of-care for muscle-invasive bladder cancer, it entails complete removal of the bladder and surrounding organs which leads to substantial loss in the quality-of-life of patients. Radiotherapy, which spares the bladder, would be a more appropriate treatment modality if we can utilize molecular markers to select patients with better response to radiation. In this study, we investigate a protein called high mobility group box protein 1 (HMGB1) as a predictive marker for radiotherapy response in bladder cancer. Our in vitro results indicate a positive correlation between higher levels of HMGB1 protein and resistance to radiation in various cell lines. Upon HMGB1 protein knockdown, highly significant (>1.5-fold) sensitization to radiotherapy was achieved. We saw that loss of HMGB1 was associated with at least two times higher (P < 0.001) DNA damage in cell lines postradiation. Our results also depicted that autophagy was inhibited more than 3-fold (P < 0.001) upon HMGB1 knockdown, implicating its role in autophagy as another cause of bladder cancer radioresistance. Further validation was done in vivo by conducting mouse tumor xenograft experiments, where HMGB1 knockdown tumors showed a significantly better (P < 0.001) response to radiotherapy and decreased autophagy (shown by P62 staining) as compared with controls. The cumulative findings of our in vitro and in vivo studies highlight the significance of HMGB1 as a radiation response marker as well as its utility in radiosensitization of bladder cancer. Mol Cancer Ther; 15(3); 471–9. ©2015 AACR.

A Novel Mechanism of PPAR Gamma Induction via EGFR Signalling Constitutes Rational for Combination Therapy in Bladder Cancer

Background Two signalling molecules that are attractive for targeted therapy are the epidermal growth factor receptor (EGFR) and the peroxisome proliferator-activated receptor gamma (PPARγ). We investigated possible crosstalk between these 2 pathways, particularly in light of the recent evidence implicating PPARγ for anticancer therapy. Principal Findings As evaluated by MTT assays, gefitinib (EGFR inhibitor) and DIM-C (PPARγ agonist) inhibited growth of 9 bladder cancer cell lines in a dose-dependent manner but with variable sensitivity. In addition, combination of gefitinib and DIM-C demonstrated maximal inhibition of cell proliferation compared to each drug alone. These findings were confirmed in vivo, where combination therapy maximally inhibited tumor growth in contrast to each treatment alone when compared to control (p<0.04). Induction of PPARγ expression along with nuclear accumulation was observed in response to increasing concentrations of gefitinib via activation of the transcription factor CCAT/enhancer-binding protein-β (CEBP-β). In these cell lines, DIM-C significantly sensitized bladder cancer cell lines that were resistant to EGFR inhibition in a schedule-specific manner. Conclusion These results suggest that PPARγ agonist DIM-C can be an excellent alternative to bladder tumors resistant to EGFR inhibition and combination efficacy might be achieved in a schedule-specific manner.

Aldehyde dehydrogenase 1 expression in primary and metastatic renal cell carcinoma: An immunohistochemistry study

BackgroundALDH1 has been shown to be a cancer stem cell marker, and its expression correlates with prognosis in a number of malignancies. We aimed to evaluate the expression of ALDH1 in a cohort of primary and metastatic RCC specimens, and to correlate expression with pathological outcomes such as tumor stage and grade, and clinical outcomes such as progression free survival.MethodsThree tissue microarrays were constructed from 244 RCC specimens, taken from 1985 to 2006. Samples were stained using an ALDH1 monoclonal antibody and expression was quantified by degree of staining. Membrane and cytoplasm staining were considered separately. A retrospective chart review enabled correlation with clinical outcomes.ResultsALDH1 expression did not vary significantly based on tumor stage (P = 0.6274) or grade (P = 0.1666). ALDH1 showed significantly more membranous expression in clear cell RCC versus other subtypes (P < 0.0001), as well as in the primary setting compared to metastases (P = 0.0216). In terms of progression free survival, no significant differences were seen based on ALDH1 expression levels. In a subanalysis of clear cell tumors, ALDH1 membranous expression was decreased in tumors of higher stage (P = 0.0233).ConclusionsALDH1 may be useful in characterizing RCC tumors as clear cell subtype. However, unlike in other malignancies, ALDH1 may not be useful in prognosticating renal cancers. The clinical significance of decreased ALDH1 expression in the high stage and metastatic setting remains to be determined in further investigations.

Enhancer of Zeste Homolog 2 Expression Is Associated With Metastasis and Adverse Clinical Outcome in Clear Cell Renal Cell Carcinoma: A Comparative Study and Review of the Literature

CONTEXT Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase mediating chromatin condensation and epigenetic modulation, is overexpressed in various human carcinomas and is associated with adverse clinicopathologic characteristics and biologic behavior. The expression of EZH2 in renal cell carcinomas (RCCs) has not been fully characterized yet. OBJECTIVE To evaluate the prognostic role of EZH2 in RCC by analyzing the immunohistochemical staining pattern of the marker in relation to pathologic features and clinical outcome. DESIGN We correlated the immunolabeling of EZH2 with multiple clinicopathologic features, including Fuhrman nuclear grade, pathologic stage, metastatic status, and clinical outcome in 223 clear cell RCCs (CRCCs) and 21 papillary RCCs, by using tissue microarrays of primary and metastatic cases. RESULTS Most CRCCs (75%) showed positive EZH2 staining, with most primary tumors showing focal staining in comparison to nonfocal staining in metastatic cases. In primary tumors, EZH2 expression was associated with higher nuclear grade and lower pathologic stage. Metastatic tumors showed a higher number of positive cases (81% versus 67%) and a more diffuse and more intense pattern of staining than primary CRCCs. For the 22 locally advanced primary tumors (T3/4) and 43 metastatic RCCs, patients who experienced RCC-related deaths significantly overexpressed the marker in comparison to patients who did not experience RCC-related mortality. CONCLUSIONS By showing that EZH2 expression is associated with increased metastatic potential and a worse clinical outcome, this study suggests that EZH2 can serve as a prognostic biomarker for RCC, thus confirming it as a key molecule driving oncogenesis and metastasis.

Combining mTOR Inhibition with Radiation Improves Antitumor Activity in Bladder Cancer Cells In Vitro and In Vivo: A Novel Strategy for Treatment

Purpose Radiation therapy for invasive bladder cancer allows for organ preservation but toxicity and local control remain problematic. As such, improving efficacy of treatment requires radiosensitization of tumor cells. The aim of study is to investigate if the mammalian Target of Rapamycin (mTOR), a downstream kinase of the phosphatidylinositol 3-kinase (PI3K)/AKT survival pathway, may be a target for radiation sensitization. Experimental Design Clonogenic assays were performed using 6 bladder cancer cell lines (UM-UC3, UM-UC5, UM-UC6, KU7, 253J-BV, and 253-JP) in order to examine the effects of ionizing radiation (IR) alone and in combination with RAD001, an mTOR inhibitor. Cell cycle analysis was performed using flow cytometry. In vivo, athymic mice were subcutaneously injected with 2 bladder cancer cell lines. Treatment response with RAD001 (1.5 mg/kg, daily), fractionated IR (total 9Gy = 3Gy×3), and combination of RAD001 and IR was followed over 4 weeks. Tumor weight was measured at experimental endpoint. Results Clonogenic assays revealed that in all bladder cell lines tested, an additive effect was observed in the combined treatment when compared to either treatment alone. Our data indicates that this effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined. Furthermore, our data show that this arrest is primarily regulated by changes in levels of cyclin D1, p27 and p21 following treatments. In vivo, a significant decrease in tumor weight was observed in the combined treatment compared to either treatment alone or control. Conclusions Altering cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and is a promising therapeutic modality for bladder cancer.

Desulfovibrio vulgaris Hildenborough HydE and HydG interact with the HydA subunit of the [FeFe] hydrogenase

HydE, HydF, and HydG participate in the synthesis of the complex di-iron center of [FeFe] hydrogenases. The hydE, hydF, hydG, hydA, and hydB genes of Desulfovibriovulgaris Hildenborough were cloned and His-tag pull-down assays were used to study the potential interaction between HydE, HydF, and HydG with the HydA and HydB protein subunits of the D.vulgaris [FeFe] hydrogenase. Interaction of HydE and HydG with HydA was demonstrated. HydF did not interact with HydA, and none of the accessory proteins appeared to interact with HydB. This suggests that specific protein–protein interactions may be required during [FeFe] cluster synthesis and/or insertion.