Abstract 4998: Molecular profiling reveals heterogeneity of active self-renewal pathways in bladder cancer stem cells

Abstract

Abstract The objective of the current study is to investigate the functional involvement of self-renewal related genes in patient bladder cancer stem cells. First, by fractionating primary patient bladder transitional cell carcinomas (TCCs) into subpopulations with flow cytometry, a unique tumor-initiating subpopulation expressing CD44 was identified in 5 independent TCCs, based on their enriched potential (10-200 folds) to induce xenograft tumors in immunocompromised mice. To extend this finding to a larger sample set, we analyzed a tissue array containing more than 300 bladder TCCs by immunohistochemistry. Approximately 40.4% of TCCs contain CD44+ cells; these cells usually express cytokeratin 5 (CK5) (P<0.0001) but not cytokeratin 20 (P=0.8160). In addition, cellular morphologies of CD44+ tumor cells are often small and round shape. These data suggest that CD44+ tumor cells retain cytokeratin markers and cellular morphologies resembling that of urothelial basal cells. Further, molecular profiling of oncogenic proteins implicated in the self-renewal of stem cells (i.e. Bmi1, Stat3, ß-catenin, Gli1, Oct4 and Nanog) revealed heterogeneity in bladder TCCs. Different subsets of CD44+ bladder TCCs express either one or more of different self-renewal proteins in their active forms (i.e. ~10% with nuclear Bmi1, 30% with nuclear Stat3, 5% with nuclear ß-catenin, 85% with active Gli1 and 0% with active Oct4 nor Nanog). In particular, in situ hybridization demonstrated that Gli1 mRNA was present in over 85% of bladder TCC specimens analyzed. Therefore, we examined the functional significance of the sonic hedgehog pathway in bladder tumor development. In patient bladder xenografts that originally contained active Gli1 in the cancer stem cells, preliminary experiment demonstrated that inhibition of this pathway with cyclopamine resulted in partial reduction of tumor volume (4 out of 6 xenografts). Previous reports demonstrated that genetic mutations in the pathway components (i.e. PTCH or Gli1) leading to constitutive Gli1 activation is rare in bladder TCCs. Preliminary microRNA (miRs) profiling of CD44+ bladder cancer stem cells using a real-time PCR based approach revealed a panel of differentially expressed miRs. Interestingly, in several highly elevated miRs in CD44+ bladder cancer stem cells, the negative regulators of the Gli1 pathways (i.e. PTCH and SUFU) are among the top predicted targets for these miRs. Further experiments are ongoing to validate the possible involvement of miRs in regulating the Gli1 pathways in bladder cancer stem cells. In conclusion, we demonstrated the existence of a functionally unique, CD44+ tumorigenic subpopulation in a subset of bladder TCCs. Diverse “self-renewal” proteins, including Gli1, possibly contribute to the tumorigenic properties of cancer stem cells in different subset of patient bladder TCCs.