Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Oncotarget
2016 Feb 09;76:6948-59. doi: 10.18632/oncotarget.6851.
Show Gene links
Show Anatomy links
IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in JAK2V617F-positive myeloproliferative neoplasms.
de Melo Campos P
,
Machado-Neto JA
,
Eide CA
,
Savage SL
,
Scopim-Ribeiro R
,
da Silva Souza Duarte A
,
Favaro P
,
Lorand-Metze I
,
Costa FF
,
Tognon CE
,
Druker BJ
,
Olalla Saad ST
,
Traina F
.
???displayArticle.abstract???
The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2V617F HEL cells, but not in the JAK2WT U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2V617F-positive but not JAK2WT specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34+ cells from essential thrombocythemia patients compared to healthy donors, and in JAK2V617F MPN patients when compared to JAK2WT. Our data indicate that IRS2 is a binding partner of JAK2V617F in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN.
Figure 1. IRS2 associates with JAK2 in HEL cellsA. Immunoprecipitation (IP) and immunoblotting (IB) with anti-IRS2 and JAK2 antibodies showed a constitutive association between IRS2 and JAK2 in HEL cells harboring the JAK2V617F mutation, but not in JAK2WT cell lines U937, NB4 and HL60. Isotype IgG antibody was used as a negative control of the immunoprecipitation; total cell extracts were used as positive controls for immunoblotting. Blots were cropped to improve the clarity of the figure and retain important bands. B. Confocal analysis of HEL, U937 and NB4 cells displaying JAK2 (green), IRS2 (red) and DAPI (blue) staining; MERGE shows the overlapped images. Colocalization analysis was performed with the “colocalization finder” plug-in of Image J NIH software, and shows merged images of JAK2 and IRS2, with colocalized points in white. The correlation coefficient (r) values are indicated. C. Immunoblotting (IB) with anti-IRS2 and JAK2 antibodies showed high IRS2 and JAK2 expression in HEL, U937, NB4 and HL60 cell lines.
Figure 2. Ruxolitinib decreases IRS2 phosphorylation in JAK2V617F cellsTotal cell extracts of A. JAK2V617F HEL cells or B. JAK2WT U937 cells treated with different doses of ruxolitinib for 6h were submitted to immunoblotting (IB) analysis with anti-IRS2, anti-phosphotyrosine, and antibodies to detect downstream proteins. The asterisk (*) indicates HEL cells used as a positive control along with U937 cells. Membranes were re-probed with the antibody for detection of the respective total and phospho-protein, and developed with the ECLTM Western Blot Analysis System. Blots were cropped to improve the clarity of the figure and retain important bands.
Figure 3. IRS2 silencing decreases STAT5 phosphorylation in HEL (JAK2V617F) cells, but not in U937 (JAK2WT) cellsA. HEL cells or B. U937 cells were transduced with lentivirus-mediated shRNA control (shControl) or IRS2 (shIRS2). IRS2 mRNA and protein expression in shIRS2 cells relative to the shControl cells (upper panel). Western blot analysis for total and phospho-proteins JAK2, STAT3, STAT5, ERK, AKT and P70S6K in total cell extracts of shControl and shIRS2 HEL or U937 cells treated with ruxolitinib (100 or 300nM) or DMSO for 48h (lower panel). The antibodies used for immunoblotting (IB) are indicated; membranes were reprobed with the antibody for detection of the respective total and phospho-protein or actin, and developed with the ECL Western Blot Analysis System. Blots were cropped to improve the clarity of the figure and retain important bands.
Figure 4. IRS2 silencing reduces cell viability and colony formation, and potentiates the effects of ruxolitinib in HEL cellsA. Cell viability was determined by methylthiazole tetrazolium (MTT) assay after 48h of incubation of shIRS2 HEL cells and normalized by the corresponding shControl HEL cells. Results are shown as mean ± SD of eight independent experiments; Mann–Whitney test. B. Colonies containing viable cells were detected by MTT after 8 days of culture of shIRS2 HEL cells and normalized by the corresponding shControl HEL cells. The percentage of colonies relative to controls are shown in the bar graph as mean ± SD of twelve independent experiments; Student's t-test. The assays were performed in the presence of ruxolitinib (100 or 300mM) or DMSO, as indicated. C. U937 cells stably-transduced with lentivirus-mediated shRNA control (shControl) or IRS2 (shIRS2) were submitted to MTT assay or D. colony forming assay, as indicated. All conditions were tested in at least eight independent experiments, and no statistically significant difference was observed for U937 cells.
Figure 5. IRS2 silencing induces apoptosis and has cumulative effects with ruxolitinib in HEL cellsA. Apoptosis was detected by flow cytometry in HEL cells transduced with shControl and shIRS2 using Annexin V+/PI− staining method and a representative dot plot is illustrated. Bar graphs are shown as mean ± SD of eight independent experiments; Student's t-test. The assays were performed in the presence of ruxolitinib (100 and 300mM) or DMSO for 48h, as indicated. B. All conditions were submitted to imunoblotting (IB) analysis for cleaved caspase 3, and a representative experiment is shown. Membranes were reprobed with the antibody for detection of actin, and developed with the ECL Western Blot Analysis System. Blots were cropped to improve the clarity of the figure and retain important bands. C. shControl or shIRS2 U937 cells treated with DMSO only or ruxolitinib (100 or 300nM) for 48h were submitted to Annexin V/PI staining and D. imunoblotting for cleaved caspase 3, as indicated. All conditions were tested in at least eight independent experiments, and no statistically significant difference in apoptosis was observed for U937 cells; no cleaved caspase 3 was observed in any condition. The asterisk (*) indicates ruxolitinib-treated HEL cells used as a positive control for apoptosis along with U937 cells.
Figure 6. Increased IRS2 mRNA levels in CD34+ cells from JAK2V617F-positive myeloproliferative neoplasm patientsA. qPCR analysis of IRS2 mRNA expression in peripheral blood (PB) CD34+ cells from healthy donors and patients with the diagnosis of essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), and stratified by B. JAK2V617F or C. CALR exon 9 mutational status. Horizontal lines indicate medians. The number of subjects and the p values (Mann–Whitney test) are indicated.
Figure 7. IRS2 pharmacological inhibition decreases cell viability in JAK2V617F myeloproliferative neoplasm primary cells and has cumulative effects with ruxolitinibCell viability was determined by methanethiosulfonate (MTS)-based assay (patients #1 to #3, #5 and #6) or by methylthiazole tetrazolium (MTT) (patients #4 and #7) after 72h of incubation of JAK2V617F
A. or JAKWT
B. myeloproliferative neoplasms (MPN) patients' peripheral blood mononuclear cells with different concentrations of NT157 (IRS1/2 inhibitor) or ruxolitinib (JAK1/2 inhibitor) alone or in combination, as indicated. All conditions were tested in triplicate. Data were normalized to the mean percent of the untreated control wells. PMF: primary myelofibrosis; PV: polycythemia vera; ET: essential thrombocythemia.
Anand,
Increased basal intracellular signaling patterns do not correlate with JAK2 genotype in human myeloproliferative neoplasms.
2011, Pubmed
Anand,
Increased basal intracellular signaling patterns do not correlate with JAK2 genotype in human myeloproliferative neoplasms.
2011,
Pubmed
Argetsinger,
Growth hormone, interferon-gamma, and leukemia inhibitory factor utilize insulin receptor substrate-2 in intracellular signaling.
1996,
Pubmed
Baxter,
Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders.
2005,
Pubmed
Carvalheira,
Interaction between leptin and insulin signaling pathways differentially affects JAK-STAT and PI 3-kinase-mediated signaling in rat liver.
2003,
Pubmed
Dearth,
Oncogenic transformation by the signaling adaptor proteins insulin receptor substrate (IRS)-1 and IRS-2.
2007,
Pubmed
Duan,
SH2-B promotes insulin receptor substrate 1 (IRS1)- and IRS2-mediated activation of the phosphatidylinositol 3-kinase pathway in response to leptin.
2004,
Pubmed
Gibson,
Divergent roles for IRS-1 and IRS-2 in breast cancer metastasis.
2007,
Pubmed
Gressot,
Signal transducer and activator of transcription 5b drives malignant progression in a PDGFB-dependent proneural glioma model by suppressing apoptosis.
2015,
Pubmed
Hercus,
The granulocyte-macrophage colony-stimulating factor receptor: linking its structure to cell signaling and its role in disease.
2009,
Pubmed
Hoelbl,
Stat5 is indispensable for the maintenance of bcr/abl-positive leukaemia.
2010,
Pubmed
Ibuki,
The tyrphostin NT157 suppresses insulin receptor substrates and augments therapeutic response of prostate cancer.
2014,
Pubmed
Johnston,
Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.
1995,
Pubmed
Kamishimoto,
Akt activation through the phosphorylation of erythropoietin receptor at tyrosine 479 is required for myeloproliferative disorder-associated JAK2 V617F mutant-induced cellular transformation.
2011,
Pubmed
Levine,
Myeloproliferative disorders.
2008,
Pubmed
Ma,
JAK2/STAT5/Bcl-xL signalling is essential for erythropoietin-mediated protection against apoptosis induced in PC12 cells by the amyloid β-peptide Aβ25-35.
2014,
Pubmed
Machado-Neto,
Knockdown of insulin receptor substrate 1 reduces proliferation and downregulates Akt/mTOR and MAPK pathways in K562 cells.
2011,
Pubmed
Machado-Neto,
Downregulation of IRS2 in myelodysplastic syndrome: a possible role in impaired hematopoietic cell differentiation.
2012,
Pubmed
Miyakawa,
Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes.
2001,
Pubmed
Nagle,
Involvement of insulin receptor substrate 2 in mammary tumor metastasis.
2004,
Pubmed
Patti,
4PS/insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice.
1995,
Pubmed
Ratajczak,
The role of insulin (INS) and insulin-like growth factor-I (IGF-I) in regulating human erythropoiesis. Studies in vitro under serum-free conditions--comparison to other cytokines and growth factors.
1998,
Pubmed
Ren,
Identification of SH2-B as a key regulator of leptin sensitivity, energy balance, and body weight in mice.
2005,
Pubmed
Reuveni,
Therapeutic destruction of insulin receptor substrates for cancer treatment.
2013,
Pubmed
Sathyanarayana,
EPO receptor circuits for primary erythroblast survival.
2008,
Pubmed
Schafranek,
Sustained inhibition of STAT5, but not JAK2, is essential for TKI-induced cell death in chronic myeloid leukemia.
2015,
Pubmed
Socolovsky,
Fetal anemia and apoptosis of red cell progenitors in Stat5a-/-5b-/- mice: a direct role for Stat5 in Bcl-X(L) induction.
1999,
Pubmed
Storz,
Insulin selectively activates STAT5b, but not STAT5a, via a JAK2-independent signalling pathway in Kym-1 rhabdomyosarcoma cells.
1999,
Pubmed
Sun,
Role of IRS-2 in insulin and cytokine signalling.
1995,
Pubmed
Traina,
BCR-ABL binds to IRS-1 and IRS-1 phosphorylation is inhibited by imatinib in K562 cells.
2003,
Pubmed
Velloso,
Cross-talk between the insulin and angiotensin signaling systems.
1996,
Pubmed
Velloso,
The multi-faceted cross-talk between the insulin and angiotensin II signaling systems.
2006,
Pubmed
Verdier,
Erythropoietin induces the tyrosine phosphorylation of insulin receptor substrate-2. An alternate pathway for erythropoietin-induced phosphatidylinositol 3-kinase activation.
1997,
Pubmed
Warsch,
High STAT5 levels mediate imatinib resistance and indicate disease progression in chronic myeloid leukemia.
2011,
Pubmed
White,
The insulin signaling system.
1994,
Pubmed
Yamauchi,
Growth hormone and prolactin stimulate tyrosine phosphorylation of insulin receptor substrate-1, -2, and -3, their association with p85 phosphatidylinositol 3-kinase (PI3-kinase), and concomitantly PI3-kinase activation via JAK2 kinase.
1998,
Pubmed
Yamaura,
STAT signaling in ischemic heart: a role of STAT5A in ischemic preconditioning.
2003,
Pubmed
Yenush,
The IRS-signalling system during insulin and cytokine action.
1997,
Pubmed
Zhang,
Signal transducers and activators of transcription 5 contributes to erythropoietin-mediated neuroprotection against hippocampal neuronal death after transient global cerebral ischemia.
2007,
Pubmed
Zhang,
Withacnistin inhibits recruitment of STAT3 and STAT5 to growth factor and cytokine receptors and induces regression of breast tumours.
2014,
Pubmed
Zumkeller,
The insulin-like growth factor system in normal and malignant hematopoietic cells.
1999,
Pubmed
