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Involvement of matrix metalloproteinases (MMPs) in the pathogenesis of urothelial carcinoma elects them to be sensitive marker for clinical and prognostic implications. MMPs regulate tumor growth and invasion by inducing epithelial-to-mesenchymal transition (EMT) which is characterized by the complex reprogramming of epithelial cells and ultimately bring about major changes in the structural organization of bladder urothelium. The present study has been undertaken to evaluate the clinical relevance of MMPs in two distinct types of bladder cancer disease. Expression analysis of MMPs namely MMP-2, MMP-7, MMP-9 and EMT markers including epithelial marker, E-cadherin; mesenchymal markers, N-cadherin and Vimentin; and EMT-activating transcriptional factors (EMT-ATFs), Snail, Slug, Twist and Zeb was done in 64 cases of bladder tumor tissues [{Non-muscle invasive bladder cancer (NMIBC): 35 cases} and {Muscle invasive bladder cancer (MIBC): 29 cases}] by real-time quantitative polymerase chain reaction (RT-qPCR). Immunohistochemistry (IHC) staining was done in matched bladder tumor tissues to evaluate the protein expression and localization of E-cadherin, N-cadherin, Vimentin, Snail, and Slug. Our data showed overexpression of MMP-2, MMP-7 and MMP-9 at transcriptome level in 32.8%, 25% and 37.5% bladder tumor cases respectively. These tumor tissues were examined for higher expression of mesenchymal markers (N-cadherin and Vimentin) at mRNA and protein level and exhibited statistical association with tumor stage and tumor grade (p = 0.02, p = 0.04, Mann-Whitney test). Significant statistical correlation in tumor tissues with overexpressed MMPs has also been observed between gain of transcriptional factors and weak expression of E-cadherin with tumor stage, grade, gender, presence of hematuria and smoking history of the patients. Gene expression patterns of EMT markers in bladder tumors with overexpressed MMPs and their significant association with clinical profile validate the important role of MMPs in the pathogenesis of urothelial carcinoma of bladder (UCB). Increased expression of specific MMPs may affect several downstream EMT programs and thus may improve its diagnostic and prognostic utility in clinical setting.

BACKGROUND Aberrant activation of phosphorylated form of glycogen synthase kinase-3β [pS9GSK-3β (Serine 9 phosphorylation)] is known to trigger Wnt/β-catenin signal cascade but its clinicohistopathological implications in bladder carcinogenesis remain unknown. AIM To investigate the diagnostic and prognostic relevance of expressions of pS9GSK-3β, β-catenin and its target genes in the pathobiology of bladder cancer. METHODS Bladder tumor tissues from ninety patients were analyzed for quantitative expression and cellular localization of pS9GSK-3β by immunohistochemical (IHC) staining. Real time-quantitative polymerase chain reaction and IHC were done to check the expression of β-catenin, Cyclin D1, Snail and Slug at transcriptome and protein level respectively. Clinicohistopathological variables were obtained from histology reports, follow up and OPD visits of patients. Expressions of the markers were statistically correlated with these variables to determine their significance in clinical setting. Results were analysed using SPSS 20.0 software. RESULTS Aberrant (low or no membranous/high nuclear/high cytoplasmic) expression of pS9GSK-3β was noted in 51% patients and found to be significantly associated with tumor stage and tumor grade ( P = 0.01 and 0.04; Mann Whitney U test). Thirty one percent tumors exhibited aberrant co-expression of pS9GSK-3β and β–catenin proteins and showed strong statistical association with tumor stage, tumor type, smoking/tobacco chewing status ( P = 0.01, 0.02 and 0.04, Mann-Whitney U test) and shorter overall survival probabilities of patients ( P = 0.02; Kaplan Meier test). Nuclear immunostaining of Cyclin D1 in tumors with altered pS9GSK-3β/β–catenin showed relevance with tumor stage, grade and type. CONCLUSION β–catenin and pS9GSK-3β proteins are identified as markers of diagnostic/prognostic significance in disease pathogenesis. Observed histopathological association of Cyclin D1 identifies it as marker of potential relevance in tumors with altered pS9GSK-3β/β-catenin.

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In the present study, quantitative expression of epithelial-to-mesenchymal transition (EMT)-associated markers was investigated immunohistochemically and their diagnostic and prognostic significance was evaluated in patients diagnosed with non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Immunohistochemical (IHC) staining was performed in a cohort of 65 formalin-fixed and paraffin-embedded human urinary bladder tumor tissues. Tissues' sections were obtained from archives of the Department of Pathology at SGPGIMS, India. Epithelial marker (Ecadherin), mesenchymal markers (N-cadherin and vimentin), and EMT-activating transcription factors (ATFs) (Snail and Slug) were examined for their cellular localization (membranous/cytoplasmic/nuclear) and quantitative expressions in terms of IHC score. Expression of the aforesaid markers was statistically correlated with various clinicohistopathological variables. These variables were obtained from histopathology reports and subsequent follow-up and OPD visits of patients. Impact of these markers was assessed on recurrence-free survival (RFS) in NMIBC cases and progression-free survival (PFS) in MIBC cases. The data was analyzed using SPSS 20.0 software. Focal loss of membranous E-cadherin showed statistical relevance with hematuria and tumor grade (p < 0.001, p = 0.005: independent sample t test) in MIBC patients. Novel membranous expression of N-cadherin exhibited relevance with hematuria (p = 0.011, one sample t test) in NMIBC, while membranous expression of vimentin showed correlation with tumor grade and age (p < 0.001, p < 0.001, one sample t test) in MIBC cases. Nuclear immunopositivity of Snail showed statistical association with tumor grade in both NMIBC and MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test) and with hematuria in MIBC cases (p = 0.007, independent sample t test). Additionally, nuclear immunopositivity of Slug showed statistical association with tumor type in NMIBC and tumor grade in MIBC cases (p < 0.001, Moses non-parametric test; p < 0.001, one sample t test). Kaplan-Meier along with logrank statistics examined association between EMT profile and RFS in NMIBC cases (p < 0.001). Significant association of EMT biomarkers with clinicohistopathological outcomes may aid urologists to correlate its dynamic functions in urothelial tumorigenesis.