FLT3/ITD Mutation Signaling Includes Suppression of SHP-1
2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês
10.1074/jbc.m411974200
ISSN1083-351X
AutoresPeili Chen, Mark J. Levis, Patrick A. Brown, Kyu-Tae Kim, Jeffrey Allebach, Donald Small,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoMutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by ∼3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations. Mutations in the FLT3 gene are the most common genetic alteration found in AML patients. FLT3 internal tandem duplication (ITD) mutations result in constitutive activation of FLT3 tyrosine kinase activity. The consequences of this activation are an increase in total phosphotyrosine content, persistent downstream signaling, and ultimately transformation of hematopoietic cells to factor-independent growth. The Src homology (SH)2 domain-containing protein-tyrosine phosphatase (SHP)-1 is involved in the down-regulation of a broad range of growth factor and cytokine-driven signaling cascades. Loss-of-function or deficiency of SHP-1 activity results in a hyperproliferative response of myelomonocytic cell populations to growth factor stimulation. In this study, we examined the possible role of SHP-1 in regulating FLT3 signaling. We found that transformation of TF-1 cells with FLT3/ITD mutations suppressed the activity of SHP-1 by ∼3-fold. Suppression was caused by decreased SHP-1 protein expression, as analyzed at both the protein and RNA levels. In contrast, protein levels of SHP-2, a phosphatase that plays a stimulatory role in signaling through a variety of receptors, did not change significantly in FLT3 mutant cells. Suppressed SHP-1 protein levels in TF-1/ITD cells were partially overcome after cells were exposed to CEP-701, a selective FLT3 inhibitor. SHP-1 protein levels also increased in naturally occurring FLT3/ITD expressing AML cell lines and in primary FLT3/ITD AML samples after CEP-701 treatment. Furthermore, a small but reproducible growth/survival advantage was observed in both TF-1 and TF-1/ITD cells when SHP-1 expression was knocked down by RNAi. Taken together, these data provide the first evidence that suppression of SHP-1 by FLT3/ITD signaling may be another mechanism contributing to the transformation by FLT3/ITD mutations. Deregulation of signaling through protein kinases has been identified as one of the most important mechanisms in human cancers (1Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3072) Google Scholar, 2Porter A.C. Vaillancourt R.R. Oncogene. 1998; 17: 1343-1352Crossref PubMed Scopus (277) Google Scholar). Activating mutations of tyrosine kinases cause hyperphosphorylation of downstream targets and transformation of cells. c-Kit and Bcr-Abl are examples of kinases mutated in mastocytoma and gastrointestinal stromal tumors (GISTs) (3Boissan M. Feger F. Guillosson J.J. Arock M. J. Leukoc. Biol. 2000; 67: 135-148Crossref PubMed Scopus (107) Google Scholar, 4Hirota S. Isozaki K. Moriyama Y. Hasimoto K. Nishida T. Ishigura S. Kawano K. Hanada M. Kurata A. Takeda M. Muhammad T.G. Matsuzawa Y. Kanakura Y. Shinomura Y. Kitamura Y. Science. 1998; 279: 577-580Crossref PubMed Scopus (3758) Google Scholar, 5Tsujimura T. Furitsu T. Morimoto M. Isozaki K. Nomura S. 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FLT3 1The abbreviations used are: FLT3, FMS-like tyrosine kinase 3; ITD, internal tandem duplication; AML, acute myeloid leukemia; JMML, juvenile myelomonocytic leukemia; IFN, interferon; IL, interleukin; Epo, erythropoietin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SH2, Src homology domain 2; SHIP, SH2 domain-containing inositol 5-phosphatase; SHP, SH2 domain-containing protein-tyrosine phosphatase; PTP, protein-tyrosine phosphatase; GFP, green fluorescent protein; p-NPP, p-nitrophenyl phosphate; MAPK, mitogen-activated protein kinase; RT, reverse transcription.1The abbreviations used are: FLT3, FMS-like tyrosine kinase 3; ITD, internal tandem duplication; AML, acute myeloid leukemia; JMML, juvenile myelomonocytic leukemia; IFN, interferon; IL, interleukin; Epo, erythropoietin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SH2, Src homology domain 2; SHIP, SH2 domain-containing inositol 5-phosphatase; SHP, SH2 domain-containing protein-tyrosine phosphatase; PTP, protein-tyrosine phosphatase; GFP, green fluorescent protein; p-NPP, p-nitrophenyl phosphate; MAPK, mitogen-activated protein kinase; RT, reverse transcription. (FMS-like tyrosine kinase 3, Flk2, Stk-1), a member of the type III receptor-tyrosine kinase (RTK) family, is normally expressed in hematopoietic stem/progenitor cells (HSCs) (7Small D. Levenstein M. Kim E. Carow C. Amin S. Rockwell P. Witte L. Burrow C. Ratajczak M.Z. Gewirtz A.M. Civin C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 459-463Crossref PubMed Scopus (372) Google Scholar). Wild-type FLT3 is activated following binding of the cognate ligand (FL) and plays a role in differentiation, proliferation, and survival of HSCs and dendritic cells (8Lyman S.D. Curr. Opin. Hematol. 1998; 5: 192-196Crossref PubMed Scopus (44) Google Scholar). FLT3 is also expressed in ∼90% of cases of acute myeloid leukemia (AML) and almost 100% of cases of B-cell lineage acute lymphoid leukemia (ALL) (9Carow C.E. Levenstein M. Kaufmann S.H. Chen J. Amin S. Rockwell P. Witte L. Borowitz M.J. Civin C.I. Small D. Blood. 1996; 87: 1089-1096Crossref PubMed Google Scholar, 10Rosnet O. Buhring H.J. 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A. 1996; 93: 14665-14669Crossref PubMed Scopus (142) Google Scholar). These observations together provide strong support that SHP-1 functions as a tumor suppressor during myelopoiesis. This prompted us to investigate whether SHP-1 is involved in cell transformation and leukemogenesis mediated by FLT3/ITD signaling. In this study, we report for the first time that SHP-1 activity is suppressed by FLT3/ITD mutations and that this suppression is associated with a cell growth and a survival advantage. Reagents—Monoclonal mouse anti-phosphotyrosine antibody 4G10 and recombinant protein A-agarose beads were purchased from Upstate Biotechnology (Lake Placid, NY), monoclonal antibody to SHP-1 and SHP-2 from BD Pharmingen (San Diego, CA), and polyclonal antibodies to FLT3, SHP-1, SHP-2, and actin from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibodies and the enhanced chemiluminescence (ECL) detection system were from Amersham Biosciences. Recombinant human GM-CSF and FL were obtained from Pepro Tech (Rocky Hill, NJ). CEP-701 was kindly provided by Cephalon Inc. (West Chester, PA). RNeasy total RNA purification and QuantiTect SYBR Green RT-PCR kits were obtained from Qiagen (Valencia, CA). Dicer small interfering RNA (siRNA) generation kit was obtained from Gene Therapy Systems, Inc. (San Diego, CA). Cell Culture, Transfection, and Treatment—Primary AML samples were collected through an institutional review board-approved protocol. Frozen aliquots of AML samples were thawed and incubated for 12 h followed by centrifugation over Ficoll to eliminate cells dying from the freeze-thaw process. All cells were grown at 37 °C in a humidified incubator with 5% CO2. MV4-11 cells were maintained in Iscoves modified Dulbecco medium (IMDM) (Invitrogen) with 20% heat-inactivated fetal bovine serum (Gemini BioProducts, Woodland, CA). All other cells (including primary AML samples) were maintained in RPMI 1640 medium (Invitrogen) with 10% fetal bovine serum. Medium was supplemented with penicillin/streptomycin, and 2 ng/ml GM-CSF for TF-1 cells (American Type Culture Collection (ATCC), Manassas, VA). TF-1 cells were stably transfected with pCI-neo vectors (Promega, Madison, WI) containing full-length cDNA coding for either a wild-type FLT3 or a FLT3/ITD by electroporation, as described previously (21Tse K.F. Mukherjee G. Small D. Leukemia. 2000; 14: 1766-1776Crossref PubMed Scopus (182) Google Scholar). Transfected cells were selected in 1 mg/ml G418 (Invitrogen) for 2 weeks and subcloned by limiting dilution. Cells were maintained in log-phase growth for experiments. Pervanadate was freshly prepared by mixing equal volumes of 0.1 m H2O2 and 0.1 m sodium orthovanadate and incubated for 10–20 min before use. When CEP-701 (stored as a 4 mm stock solution in dimethyl sulfoxide (Me2SO) at –20 °C) was used to treat cells, the corresponding control samples also contained identical amounts of Me2SO (<0.25%). MTT Proliferation Assay—Cells were aliquoted into 96-well plates at an initial density of 1–5 × 104/ml in duplicate or triplicate. Plates were incubated at 37 °C, 5% CO2 for the indicated period of time. The MTT assays (Roche Applied Science) were performed according to the manufacturer's instructions. Plates were read at 562 nm in a microplate reader (Molecular Devices, Sunnyvale, CA). Immunoprecipitation and Immunoblotting—Cells were washed with ice-cold phosphate-buffered saline and lysed on ice for 30 min in lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, 100 mm NaF, 10% glycerol, 10 mm EDTA) containing protease and phosphatase inhibitors (2 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 50 μg/ml antipain, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 1 μg/ml pepstatin). To immunoprecipitate specific proteins, clarified whole cell lysates were incubated with corresponding antibodies followed by binding to protein A-agarose beads overnight at 4 °C. Immunoprecipitates were resolved by SDS-PAGE and transferred onto immobilon membranes (Millipore, Bedford, MA). Immunoblotting assays were performed with the designated antibodies and immunoreactive bands were visualized using chemiluminescence (ECL, Amersham Biosciences). For reprobing, blots were stripped with a buffer containing 50 mm Tris-HCl, pH 6.8, 2% SDS, and 0.1 m 2-mercaptoethanol. Quantitation of immunoblots by densitometry was performed with NIH Image 1.62. p-Nitrophenyl Phosphate (p-NPP) Hydrolysis Assay—SHP-1 and SHP-2 were immunoprecipitated and washed three times with lysis buffer, three times with lysis buffer without phosphatase inhibitors, and twice with p-NPP assay buffer (50 mm Tris-HCl, pH 7.4, 5 mm dithiothreitol). Phosphatase catalytic activity was assayed at 37 °C for 15 min in 200 μl of p-NPP buffer containing 20 mmp-NPP. Optical density was determined at 405 nm in a microplate reader. RNA Purification and Northern Blot Analysis—Total RNA was extracted from 1 × 107 cells using RNeasy total RNA purification kits. The RNA samples (15 μg/lane) were separated on 1% formaldehyde-denaturing agarose gels and transferred to nylon membranes (PerkinElmer Life Sciences). Full-length SHP-1 cDNA labeled with [32P]dCTP by random primer labeling (Stratagene) was used as a probe. Hybridization was performed in 5× SSC (1× SSC = 0.15 m NaCl, 0.15 m sodium citrate), 50% deionized formamide, 5× Denhardt's solution, 1% SDS, 10% dextran sulfate (Mr 500,000), 0.1 mg/ml salmon sperm DNA at 42 °C for 16–24 h. The membrane was washed under monitoring and then exposed to XAR film (Kodak, Rochester, NY) at –80 °C. SHP-1 RNA Interference—SHP-1 siRNA was prepared with Dicer siRNA generation kit according to the manufacturer's instructions. Briefly, T7 promoters were added to both ends of selected SHP-1 sequence (5′-GTCAGGGTGGGGGATCAGGTG... GCCAAGGCTGGCTTCTGGGAG-3′) by means of PCR. The resultant DNA templates were used for an in vitro transcription reaction with T7 RNA polymerase to generate double strand (ds) RNA, which was digested into small interference RNA fragments with recombinant Dicer enzyme. GFP siRNA was prepared simultaneously from gWIZ/GFP control plasmid as a control. The SHP-1 siRNA transfected TF-1 cells were incubated overnight and aliquoted for MTT proliferation assays or for determination of SHP-1 mRNA levels and protein levels by quantitative real time RT-PCR and immunoblotting assays at intervals of 24 h. Quantitative Real-time RT-PCR—Total RNAs were purified as above. One-step real-time reverse transcription-polymerase chain reaction (qPCR) was performed according to the manufacturer's instructions (Qiagen) on an iCycler iQ Real-time PCR System (Bio-Rad). Primers used in the reactions were as follow: SHP-1 FP, 5′-ATGCAGAGACCCTGCTCAAG-3′, SHP-1 RP, 5′-ACCAGCTCTGTCAGAGTCG-3′; GAPDH FP, 5′-CCTCAACGACCACTTTGTCA-3′, GAPDH RP, 5′-GGTGGTCCAGGGGTCTTACT-3′. Serially diluted β-actin cDNA and primers were included as a standard curve. FLT3/ITD Induces Factor-independent Growth and Increases in Phosphotyrosine Levels in TF-1 Cells—TF-1 is a Philadelphia chromosome-negative (Ph–), CD34+, FLT3-negative human GM-CSF-dependent myeloid leukemia-derived cell line (58Kitamura T. Tange T. Terasawa T. Chiba S. Kuwaki T. Miyagawa K. Piao Y.-F. Miyazono K. Urabe A. Takaku F. J. Cell. Physiol. 1989; 140: 323-334Crossref PubMed Scopus (710) Google Scholar). It is capable of differentiating upon treatment with differentiation agents, with δ-aminolevulinic acid inducing erythroid differentiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) inducing macrophage differentiation. In this study, TF-1 cells were transfected by electroporation with pCI-neo vectors expressing either wild-type FLT3 or FLT3/ITD. After selection of stable transfectants in G418 + GM-CSF for 2 weeks, the ability of the cells to survive in the absence of GM-CSF was determined by trypan blue exclusion. Wild-type FLT3-expressing TF-1 cells (TF-1/FLT3) still required GM-CSF to maintain normal growth. In contrast, FLT3/ITD-expressing TF-1 cells (TF-1/ITD) grew in the absence of GM-CSF (data not shown). The same results have been observed when BaF3 and 32D cells are transfected with wild-type FLT3 or FLT3/ITDs (29Levis M. Allebach J. Tse K.F. Zheng R. Baldwin B.R. Smith B.D. Jones-Bolin S. Ruggeri B. Dionne C. Small D. Blood. 2002; 99: 3885-3891Crossref PubMed Scopus (418) Google Scholar, 59Zheng R. Friedman A.D. Small D. Blood. 2002; 100: 4154-4161Crossref PubMed Scopus (73) Google Scholar). The phosphotyrosine levels of cell extracts from TF-1, TF-1/FLT3, and TF-1/ITD cells were examined by immunoblotting with anti-phosphotyrosine antibody, 4G10. TF-1/ITD cells were notable for significantly increased levels of phosphotyrosine proteins when compared with TF-1 and TF-1/FLT3 cells (Fig. 1, compare lane 5 to lanes 1 and 3). In addition, after a brief treatment with pervanadate, a potent membrane-permeable PTP inhibitor, the phosphotyrosine level in TF-1/ITD cells was further dramatically increased (Fig. 1, lane 6). This contrasts with the minimal to moderate increases observed in TF-1 and TF-1/FLT3 cells after pervanadate treatment, respectively. Total phosphotyrosine protein levels are the result of the balance between protein-tyrosine kinase and phosphatase activities. The increased k
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