title: Role of ribosomal protein RPS2 in controlling let-7a expression in human prostate cancer.
abstract: We discovered that an inverse relationship exists in the expression of ras/c-myc and ribosomal protein RPS2 with pre-let-7a-1/let-7a/let-7f miRNA and prostate tumor cell malignancy. Nonmalignant IBC-10a cells expressed low levels of ras/RPS2 and elevated pre-let-7a-1/let-7a/let-7f miRNA, whereas the reverse occurred in malignant PCa-20a and PC-3ML cells. Stable transfection of IBC-10a cells with pBABE.ras and pBABE.RPS2 induced ras, c-myc, and RPS2 expression, whereas the levels of let-7a/let-7f miRNA dropped to near zero. Conversely, in pBABE.pre-let-7a-1 transfected PCa-20a and PC-3ML clones, let-7a/let-7f increased whereas ras, RPS2, and c-myc dropped greater than 5-fold. Electrophoretic mobility shift assays, antibody "supershift" assays and immunoprecipitation assays revealed that RPS2 specifically binds pre-let-7a-1 to block RNA processing. Immunoflourescent studies and Northern blots confirmed that RPS2 complexes with pre-let-7a-1 (i.e., in episomal structures) to block processing to let-7a/let-7f, indicating RPS2 may prevent let-7a miRNA expression to indirectly promote oncogene expression. Functional studies further showed that the colony-forming ability (CFA) and invasive activities of IBC-10a cells were significantly enhanced in pBABE-ras.IBC-10a and pBABE-RPS2-IBC-10a clones. Conversely, with the "knockdown" of ras and RPS2 in malignant PC-3ML cells (i.e., in pLKO.TRC.shRNA.ras.PC3-ML, pLKO.TRC.shRNA.RPS2.PC-3ML transfected cells), there was both a loss of these functions and a loss of tumorigenesis in SCID mice. Likewise, with the overexpression of let-7a/let-7f in pBABE.pre-let-7a-1.PC-3ML clones (and PCa-20a clones), CFAs, invasive activities in vitro, and tumorigenesis in vivo were significantly reduced. These results show for the first time that RPS2 blocks pre-let-7a-1 processing to enable ras and c-myc expression and the transformation of primary tumor cells.
authors.affiliation: Department of Pathology, Drexel University College of Medicine, MS 435, 15th & Vine Streets, Philadelphia, PA 19102, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.forename: Youji
authors.initials: Y
authors.lastname: Hu
authors.forename: Michael D
authors.initials: MD
authors.lastname: Amatangelo
authors.forename: Mark E
authors.initials: ME
authors.lastname: Stearns
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008297:Male; D051379:Mice; D016513:Mice, SCID; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D009361:Neoplasm Invasiveness; D009368:Neoplasm Transplantation; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism; D014183:Transplantation, Heterologous; D014410:Tumor Stem Cell Assay; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D016271:Proto-Oncogene Proteins c-myc; D012269:Ribosomal Proteins; C487931:mirnlet7 microRNA, human; C030004:ribosomal protein S2; D018631:ras Proteins