The Characterization and Role of RREB1 in Human Bladder Cancer

RREB1 is a C 2 H 2 zinc-finger protein that has previously been associated with malignancy but little is known about its expression, composition of splice variants, or role in cancer phenotypes. Total RREB1 expression decreased in human bladder cancer tumors compared to paired normal tissue. Rigorous comparison of commercial RREB1 antibodies allowed for the identification of RREB1 splice variants in the cytoplasm and nucleus on western blot. Five RREB1 splice variants were identified and designated: RREB1α, RREB1β, RREB1γ, RREB1δ, RREB1ε.. Immunohistochemistry of RREB1 in a bladder cancer tissue microarray demonstrated that RREB1 expression positively correlates with the squamous cell histology. Transient depletion of RREB1 in bladder cancer cell lines (UMUC3 and KU7) decreased cell growth in vitro and similar results were seen in prostate cancer cell lines (LNCAP and PC3). siRNA duplexes that target RREB1 isoforms showed RREB1α and RREB1β, but not RREB1β alone, were necessary for in vitro UMUC3 cell growth and in vivo subcutaneous tumor growth. RREB1 depletion was found to decrease the expression of tumor associated protein CD24. Cloning of the CD24 promoter revealed a hypoxia response element, which led to the discovery of hypoxia induced CD24 RNA expression and promoter activity. RREB1 depletion decreased the expression of two additional hypoxia responsive genes (VEGFA and IGFBP3) without affecting the mRNA stability. Loss of RREB1 was found to reduce HIF1A protein levels and decrease its occupancy of the CD24 promoter. Depletion of HIF1A caused a similar degree of cell growth inhibition as seen with II RREB1 depletion. Thus, we propose the necessity of RREB1 in urologic malignancies at least partially acts by the stabilization of HIF1A protein and maintenance of downstream gene expression.

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