25.    Relevance of iNOS expression and nitric oxide production in the maintenance of               cancer stem cells in bladder cancer

Introduction: Bladder cancer (BC) is the second most common tumor of the male urogenital tract, and an important worldwide cause of death. BC is classified as no muscle invasive (NMI) when it is localized from the urothelium to the lamina propria and muscle invasive (MI), when it reaches the detrusor muscle. Increasing evidence has indicated the presence of cancer stem cells (CSCs) in many types of cancers, associated with aggressiveness, chemoresistance and relapse. Nitric oxide (NO) is a free radical produced by enzymes called NO synthases (NOS). The inducible isoform (iNOS) produces high levels of NO in response to inflammatory stimuli. The expression of iNOS in human BC is a poor prognostic factor associated with increased invasion and tumor recurrence. Although there is evidence supporting the role of NO/iNOS in the promotion and progression of BC, the possibility that there are CSCs dependent on endogenous NO generation has not yet been clearly defined. The objective of this study was to evaluate the role of NO in CSC maintenance, modulating NO production, using pharmacological inhibitors (L-NAME and 1400 W) or silencing iNOS expression in a murine BC model. Furthermore, we study the expression of CSC markers and iNOS in a pilot study in human BC samples of patients from the Instituto de Oncología A. H Roffo. Results: The number of CSCs, determined as spheres forming efficiency (SFE), generated by MB49-I (MI murine BC cell line) resulted higher than the NMI MB49 line (p<0.05), in concordance with the higher expression of stemness related genes, Sox-2, Oct-4 and Nanog (qPCR) (p<0.001). iNOS inhibition with L-NAME or 1400 W, diminished SFE in both cell lines (p<0.05 vs MB49; p<0.001 vs MB49-I), and significantly inhibited the expression of pluripontency genes in MB49-I, (p<0.05). Moreover, the reduction of this markers (p<0.0001) and SFE (p<0.001) were more evident in iNOS-silenced cells (MB49-I-shiNOS) vs MB49-I. Regarding patients samples, analyzed by qPCR, we observed that MI tumors presented higher expression of
Sox-2, Oct-4 and Nanog in comparison to NMI tumors. Furthermore, we found in MI tumors, a positive correlation between iNOS expression and the three CSC genes, Sox-2, Oct-4 and Nanog. Conclusion: Our results show the importance of iNOS expression in the maintenance of stem cells in BC. These findings support the inhibition of this enzyme as a target to inhibit the maintenance of cancer stem cells which are responsible for the resistance to chemo and radiotherapy treatments.