title: Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells.
abstract: BCR-ABL kinase activates downstream signaling pathways, including the PI3K-Akt/mTOR and the MAPK pathway. IRS1 has been previously described as constitutively phosphorylated and associated with BCR-ABL in K562 cells, suggesting that IRS1 has role in the BCR-ABL signaling pathways. In this study, we analyzed the effect of IRS1 silencing, by shRNA-lentiviral delivery, in K562 cells, a CML cell line that presents the BCR-ABL. IRS1 silencing decreased cell proliferation and colony formation in K562 cells, which correlates with the delay of these cells at the G0/G1 phase and a decrease in the S phase of the cell cycle. Furthermore, IRS1 silencing in K562 cells resulted in a decrease of Akt, P70S6K and ERK1/2 phosphorylation. Nevertheless, apoptosis was unaffected by IRS1 knockdown and no alterations were found in the phosphorylation of BAD and in the expression of BCL2 and BAX. BCR-ABL and CRKL phosphorylation levels remained unaffected upon IRS1 silencing, and no synergistic effect was observed with imatinib treatment and IRS1 knockdown, indicating that IRS1 is downstream from BCR-ABL. In conclusion, we demonstrated that inhibition of IRS1 is capable of inducing the downregulation of Akt/mTOR and MAPK pathways and further decreasing proliferation, and clonogenicity and induces to cell cycle delay at G0/G1 phase in BCR-ABL cells.
authors.affiliation: Hematology and Hemotherapy Center-University of Campinas/Hemocentro-Unicamp, Instituto Nacional de Ciência e Tecnologia do Sangue, Campinas, São Paulo, Brazil.
authors.forename: João Agostinho
authors.initials: JA
authors.lastname: Machado-Neto
authors.forename: Patricia
authors.initials: P
authors.lastname: Favaro
authors.forename: Mariana
authors.initials: M
authors.lastname: Lazarini
authors.forename: Fernando Ferreira
authors.initials: FF
authors.lastname: Costa
authors.forename: Sara T
authors.initials: ST
authors.lastname: Olalla Saad
authors.forename: Fabiola
authors.initials: F
authors.lastname: Traina
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D017209:Apoptosis; D001483:Base Sequence; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle; D049109:Cell Proliferation; D003114:Colony-Forming Units Assay; D017931:DNA Primers / Q000235:genetics; D015536:Down-Regulation; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020014:K562 Cells; D020935:MAP Kinase Signaling System; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C082869:CRKL protein; D017931:DNA Primers; C526292:IRS1 protein, human; D055504:Insulin Receptor Substrate Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C546842:MTOR protein, human; D016044:Fusion Proteins, bcr-abl; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C505410:Casp3 protein, rat; D053148:Caspase 3