Title: The Role of microRNAs in Bladder Urothelium Development and Tumorigenesis
Description: There are two morphologically distinct cell types in the normal urothelium: umbrella cells and basal/intermediate cells. Immunohistochemical studies from our group suggest that there may be more than one urothelial progenitor. Bladder cancer is the fifth most common cancer in the United States and the second most prevalent genitourinary malignancy. Urothelial carcinoma accounts for 90% of bladder cancers. Based on clinical and histological studies, urothelial carcinomas are thought to develop through two independent pathways and are classified into two main phenotypic variants: low-grade tumors (usually papillary and "superficial" with high recurrence), and high-grade tumors (usually flat carcinoma <italic>in situ</italic> lesions that are often associated with and progress to muscle invasion). MicroRNAs (miRNAs) are single-stranded non-coding RNA molecules, approximately 21-23 nucleotides in length, that regulate gene expression. Since their discovery in 1993, they have emerged as major mediators of cellular functions and tissue homeostasis.

Importantly, distortion of their normal function is commonly observed in human malignancies, suggesting that they act as a new class of tumor suppressors and oncogenes. Despite the strong links reported between miRNAs and the pathogenesis of numerous human cancers, there are few studies centering on their characterization in normal urothelium and there is little consensus on which miRNAs contribute to urothelial tumor initiation and progression. Through a series of studies, we profiled the expression of miRNAs in distinct compartments of the normal bladder, including umbrella and basal-intermediate urothelial cells, as well as the muscularis propria; and bladder carcinoma <italic>in situ</italic> (CIS) lesions. We discovered and validated the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. This study represents the first molecular characterization of miRNA expression in the normal urothelium. Strikingly, we found that miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells.

Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Once more, this analysis revealed that CIS lesions shared a common phenotype with umbrella cells through the expression of umbrella-specific markers. Mechanistic studies were performed in parallel to further delineate the potential role of two critical miRNAs involved in cell invasion that were previously unassociated with urothelial carcinomas: miR-198 and miR-126. Overexpression of miR-198 increased cell invasion in non-invasive bladder cancer cells, an effect that was magnified with concurrent down-regulation of the miR-200 family. In contrast, elevated levels of miR-126 suppressed cell invasion in invasive bladder cancer cells, possibly through regulation of gene expression of the matrix metalloproteinase ADAM9. Correspondingly, knock-down studies of ADAM9 in invasive bladder cancer cells also inhibited cell invasion. We further demonstrated preferential expression of ADAM9 in muscle-invasive bladder tumors compared to non-muscle invasive tumors, and that ADAM9 expression significantly correlated with a poor prognosis in patients with urothelial carcinoma.

Our studies represent a comprehensive and accurate description of the different miRNAs expressed in distinct urothelial cellular compartments and CIS tumors. This study is also the first to provide evidence of the possible origin of CIS lesions from umbrella cells. Additionally, important translational results of our studies support the use of miR-198, miR-126, and ADAM9 as clinical biomarkers of disease progression, and provide a rationale for the therapeutic inhibition of ADAM9 in aggressive urothelial carcinomas. Overall, the findings reported here indicate that several miRNAs are differentially regulated in urothelium development and tumorigenesis, and may form a basis for clinical development of new biomarkers for urothelial carcinoma.