Silencing of Dicer1 temporally separates pro- and anti-apoptotic signaling and confers susceptibility to chemotherapy in p53 mutated cells.
miRNAs are critically implicated in the initiation process of and progression through cancerogenesis. The mechanisms, however, by which miRNAs interfere with the signalosomes of human cancer cells, are still obscure. We utilized the p53-mutated human keratinocyte cell line HACAT to investigate the biological significance and extent to which miRNAs regulate proliferation, cell growth, and apoptosis in transformed phenotypes. Silencing of the miRNA-processing enzyme Dicer1 resulted in cell cycle arrest at the G1/S border, along with restoration of CDK inhibitor p21(CIP)expression. Employing a cell cycle-wide phospho-proteomic approach, we detected neglectable changes in abundance and schedule of overall and cell cycle periodic protein expression despite cell cycle arrest of Dicer1-depleted cells. Instead, we found substantially delayed post-translational modifications of some, but not all, signaling nodes. Phospho-site-specific analyses revealed that pro-apoptotic information elicited by Myc, β-catenin, and other mitotic pathways early in G1 are absorbed and balanced by anti-apoptotic signaling from AKT and NFκB in Dicer1-competent cells. The absence of regulatory miRNAs, however, led to a substantial delay of anti-apoptotic signaling, leaving pro-apoptotic stress unbalanced in Dicer1-deprived cells. We here show that this temporal separation of pro- and anti-apoptotic signaling induced by inhibition of Dicer1 is synergistic and synthetic lethal to low-dose 5-FU chemotherapy in p53-mutated HACAT cells. The findings reported here contribute to the understanding of the complex interactions of miRNAs with the signalosom of transformed phenotypes and may help to design novel strategies to fight cancer.
Department of Internal Medicine II; Julius-Maximilians University; Wuerzburg, Germany.
Tatyana S
TS
Nekova
Physiological Chemistry I; Biocenter; Julius-Maximilians University; Wuerzburg, Germany.
Susanne
S
Kneitz
Department of Internal Medicine II; Julius-Maximilians University; Wuerzburg, Germany.
Hermann
H
Einsele
Department of Internal Medicine II; Julius-Maximilians University; Wuerzburg, Germany.
Gernot
G
Stuhler
D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005472:Fluorouracil / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D009154:Mutation; D010641:Phenotype; D034622:RNA Interference; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D000964:Antimetabolites, Antineoplastic; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D005472:Fluorouracil
5-FU; Dicer; HACAT; apoptosis; cell cycle; miRNA; p21; p53; signaling pathway