Advertisement
Molecular Cell Biology

Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia

Samantha Le Sommer, Nicola Morrice, Martina Pesaresi, Dawn Thompson, Mark A. Vickers, Graeme I. Murray, Nimesh Mody, Benjamin G. Neel, Kendra K. Bence, Heather M. Wilson and Mirela Delibegović
DOI: 10.1158/0008-5472.CAN-17-0946 Published January 2018

Abstract

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B−/− mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia.

Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75–87. ©2017 AACR.

Introduction

Protein tyrosine phosphatase 1B (PTP1B) is a nonreceptor tyrosine-phosphatase that plays critical roles in a number of signaling cascades, notably insulin signaling, where it directly dephosphorylates the insulin receptor (1, 2). Consequently, it has been identified as an attractive drug target for the treatment of type 2 diabetes mellitus (T2DM) and obesity. PTP1B inhibitors are currently in phase II clinical trials for T2DM treatment, as both PTP1B inhibition and genetic ablation improve glucose homeostasis and promote weight loss (3–5). New substrates for the enzyme continue to be identified, but recent evidence has shown it plays a critical role in the regulation of inflammation, cell proliferation, differentiation and invasion, supporting its use as a therapeutic target for inflammatory and autoimmune diseases and cancer (6).

Whilst global PTP1B knockout (PTP1B−/−) mice exhibited resistance to diet-induced obesity and insulin resistance (1, 2), they also showed a higher rate of death when challenged with high-dose lipopolysaccharide (LPS) injections (7), and an enhanced response to irradiation, when challenged with LPS and d-galactosamine (8). These studies suggest a critical role for PTP1B in regulation of immune responses.

Mice lacking PTP1B in the myeloid cell lineage specifically (LysM-PTP1B−/−) also exhibited improved glucose homeostasis and protection against high-fat-diet–induced inflammation and lipopolysaccharide (LPS) induced endotoxemia, via an IL10–signal transducer and activation of transcription 3 (STAT3) dependent mechanism (9). This was further confirmed by a transcriptomic study, which illustrated macrophage PTP1B as a key regulator of IL10 signaling via STAT3 (10).

We recently provided the first evidence for a major regulatory role of PTP1B in dendritic cells (DC); PTP1B-deficient DC exhibited hyperphosphorylation of STAT3, even under basal conditions. DC-PTP1B inhibition resulted in decreased migration, increased IL10 secretion and a decreased ability to prime adaptive immune responses (11).

It has been shown previously that PTP1B plays a key role in the pathogenesis of tumor development. Overexpression of PTP1B can be a driving factor in the pathogenesis of breast cancer (6, 12), whereas deficiency has been shown to increase the invasiveness of prostate cancer (13). STAT3 overexpression and/or overactivation also plays a critical role in multiple tumorigenic processes (14). Deletion of chromosome 20q(del(20q)) is a common chromosomal abnormality associated with myeloid neoplasms and the del(20q) involving PTPN1 deletion was observed in 8% of secondary acute myeloid leukemia cases and 17% of myeloproliferative neoplasms. Given the poorly defined and often opposing roles that PTP1B and STAT3 play in the etiology of inflammation and cancers, we sought to characterize the long-term implications of PTP1B deficiency and associated STAT3 hyperphosphorylation in myeloid cells that drive the pathogenesis of both conditions.

We report here that the absence of myeloid PTP1B (LysM-PTP1B−/−) in mice results in a shortened lifespan due to the late development of acute leukemia. By contrast, heterozygous deficiency (LysM-PTP1B+/−) does not lead to development of leukemia, suggestive that a complete deficiency of PTP1B is required for the phenotype. This recapitulates the shortened lifespan we observe in global PTP1B−/− mice.

Materials and Methods

Animal studies

All animal procedures were approved by the UK Home Office under The Animals (Scientific Procedures) Act 1986 (PPL60/3951). PTP1B−/− and wild-type PTP1B+/+ littermates were maintained for lifespan studies at Beth Israel Deaconess Medical Center, Boston, MA (2). PTP1Bfl/fl mice (15) and mice expressing Cre under the LysM promoter (LysM-PTP1B−/−; ref. 16) were maintained for lifespan studies at the Medical Research Facility, Aberdeen, United Kingdom. Mice were housed and maintained at 22- to 24°C on a 12-hour light–dark cycle, with free access to food and water on standard chow diet and aged to 24 months (104 weeks).

Bone marrow–derived macrophage preparation and treatment

Bone marrow–derived macrophage (BMDM) were prepared from mononuclear phagocyte precursors flushed from femurs and tibias as previously described (9). BMDM were stimulated with either 100 ng/mL LPS (InvivoGen) or 20 ng/mL IL10 (Peprotech). In selected experiments, mature BMDM were pretreated with 5 μmol/L of ruxolitinib (Selleck) before stimulation with LPS.

Adhesion assays

Adhesion assays were performed as previously described (17). In brief, 96-well plates were precoated with 50 μg/mL collagen. BMDM (5 × 105 per well) were seeded and left to adhere for 30 minutes. Nonadherent cells were washed off, adherent cells stained with crystal violet, washed, and lysed. OD595 value was measured in a plate reader OpsysMR (DYNEX Technologies).

Antigen presentation assays

We used an OVA-peptide system, which assesses the presentation of the SIINFEKL OVA-peptide on MHC class I to the T-cell reporter cell line B3Z. Mature BMDM were seeded in triplicate in 96-well plates (5 × 104 cells per well). Cells were left to adhere and incubated with the SIINFEKL peptide for 6 hours; washed with PBS, fixed with 0.05% glutaraldehyde, and quenched with 0.2 mol/L glycine. Cells were washed, and incubated with 5 × 104 cells from the CD8 T-cell hybridoma cell line B3Z for 16 hours. Stimulation of the B3Z hybridoma (18) was measured by a luminescent β-galactosidase assay (Clontech).

ELISA

Serum and media supernatant, TNFα and IL10, concentrations were determined using ELISA (R&D Systems).

Differential blood counts

Blood smears were prepared and fixed in methanol and then stained with eosin and methylene blue. Slides were mounted using aqueous media and visualized using light microscopy (Carl Zeiss) and AxioVision 4.8 digital image processing software (Carl Zeiss). To count reticulocytes, an oil 100× objective was used and 1,000 cells counted.

Flow cytometry

Single-cell suspensions were prepared from spleens and red blood cells lysed for 1 minute with lysis buffer (Sigma Aldrich). Fc receptor binding sites were blocked with rat anti-mouse CD16/CD32 (BD Cat# 553141, 0.5 mg/reaction) and cells labeled with: PE Rat Anti-Mouse CD45R/B220 (BD Pharmingen); Alexa Fluor 700 Rat Anti-Mouse F4/80 (Biolegend); PE-CF594 Rat Anti-Mouse CD11b (BD Horizon); V450 Rat Anti-Mouse CD8a (BD Horizon); Alexa Fluor 488 Hamster Anti-Mouse CD3e (BD Pharmingen); PerCP-Cy5.5 Rat Anti-Mouse CD4 (BD Pharmingen); PE Rat Anti-Mouse CD25 (BD Pharmingen). Data were acquired on a BD Fortessa and analyzed using FlowJo.

Hematocrit

Fresh blood was drawn into two heparinized capillary tubes. Samples were spun in a microhematocrit centrifuge, packed cell volume was measured, and percentages were calculated.

White blood cell count

Whole blood was diluted in 5 volumes of red blood cell (RBC) lysing buffer (Sigma Aldrich). White blood cells (WBC) were counted using an automated Becton Coulter Z2 cell counter.

Hematoxylin and eosin staining

Tissues were fixed in 4% formaldehyde, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Histological analysis on H&E-stained sections was performed by an experienced histopathologist (M. Vickers).

Immunoblotting

Tissues and cells were homogenized in radioimmunoprecipitation-assay buffer containing sodium-orthovanadate and protease inhibitors (9). Proteins were separated by SDS-PAGE (4%–12%) and transferred to nitrocellulose. Immunoblotting was performed using antibodies from Cell Signaling Technology, unless otherwise stated: p-STAT3 Y705, p-STAT3 S727, STAT3, p-P38 T108/Y182, P38, p-ERK T202/204, ERK, GAPDH, pSTAT5 Y694, STAT5, and PTP1B (Millipore). Immunoblots were visualized using enhanced chemilluminescence and quantified using Bio-1D densitometry scanning software (PeqLab).

Immunohistochemistry

Immunohistochemistry was performed as previously described (9). Briefly, tissues were fixed in formalin, embedded in paraffin, sectioned, dewaxed in Histoclear, rehydrated, subjected to antigen retrieval (10 mmol/L trisodium citrate HCl, pH6, 0.05% (v/v) Tween), quenched and blocked (TBS-Tween, 0.05% (v/v) goat serum). Primary antibody was incubated at optimal concentration, followed by secondary HRP-conjugated antibody. DAB substrate was used for detection with a hematoxylin nuclear counterstain. Sections were visualized by light microscopy (Carl Zeiss Microscopy) and AxioVision 4.8 digital image processing software (Carl Zeiss Microscopy).

Gene expression analysis

Tissues were homogenized in TRIzol reagent (Sigma-Aldrich) and cDNA synthesis carried out from 1 μg of RNA using a Tetro cDNA Synthesis Kit (Bioline). Quantitative PCR was performed using a Light Cycler 480 (Roche), and gene expression of f4/80, cd11b, cd68, arginase, il4, il6, il10, stat3, stat5, bcl-2, bcl-6, bcl-x, Fas, Hgf, Myc, Nfe212,Hdec4, and Fos was determined in relation to the most stable housekeeping gene (either ywhaz or nono). Primer sequences are available in Supplementary data.

Chromatin immunoprecipitation-seq analysis

Detailed description can be found in Supplementary Methods. BMDM extracted from PTP1Bfl/fl and LysM-PTP1B−/− mice aged 6 months were treated ± IL10 20 ng/mL (Peprotech) for 4 hours (n = 4/group). Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology) according to the manufacturer's instructions, with chromatin sheered to 200 bp and 10 μL of STAT3 Mouse mAb #9139 per IP (Cell Signaling Technology).

Microarray bioinformatics analysis

Detailed description can be found in Supplementary Methods. PTP1Bfl/fl and LysM-PTP1B−/− mice (n = 5 and 4, respectively) were injected with 0.5 mg/kg LPS i.p. for 3 hours. Microarray was performed using Gene 2.0ST array (Affymetrix), according to the manufacturer's instructions.

Data analysis

Data are expressed as mean ± SEM unless otherwise stated. Statistical analyses were conducted using one-way ANOVAs with Turkey post hoc testing, two-way ANOVAs with Bonferroni post hoc testing, two-tailed Student t tests, or Kaplan–Meier analyses, as appropriate, using GraphPad Prism 5 software.

Results

Global and myeloid cell–specific PTP1B deficiency in mice shortens their lifespan

To determine the effects of global PTP1B deficiency on lifespan, mice deficient in PTP1B globally (PTP1B−/−) and wild-type controls (PTP1B+/+) male (n = 40 vs. n = 22, respectively) and female (n = 23 vs. n = 17, respectively) littermates were left undisturbed until the time of death (Supplementary Fig. S1A and S1B) at Beth Israel Deaconess Medical Center animal facility. Global PTP1B−/− mice exhibited significantly decreased survival times, with a median survival time of 64.6 weeks in female, and 76.4 weeks in male, global PTP1B−/− mice (P < 0.0001; Supplementary Tables S1–S3). At the conclusion of the study at 104 weeks, the female wild-type PTP1B+/+ mice had reached a median survival time of 90 weeks, whilst the male PTP1B+/+ mice remained healthy. Global PTP1B−/− mice exhibited splenomegaly (Supplementary Fig. S1C–S1E), associated with excessive hematopoiesis in the spleen and liver, neutrophil invasion, and liver necrosis.

To determine if the shortened lifespan observed in global PTP1B−/− mice is due to myeloid-PTP1B deficiency, given the pivotal role of PTP1B in regulating functions of myeloid cells (9), we conducted at the Medical Research Facility, University of Aberdeen, an aging study to 104 weeks of age, comparing mice that have PTP1B deleted specifically in myeloid cells (LysM-PTP1B−/−), mice heterozygous for myeloid PTP1B (LysM-PTP1B+/−) and control littermate PTP1Bfl/fl mice expressing wild-type level of PTP1B (n = 12/genotype).

LysM-PTP1B−/− mice exhibited a dramatically decreased median survival time and a shortened lifespan in comparison with PTP1Bfl/fl mice and LysM-PTP1B+/− mice (median survival 76 weeks), as demonstrated by Kaplan–Meier analysis of survival (Fig. 1A). LysM-PTP1B−/− mice exhibited spontaneous development of extranodal tumors at approximately 40 weeks (±6.9 weeks SEM), accompanied by swollen abdomen due to splenomegaly. LysM-PTP1B+/− lifespans were shorter than PTP1Bfl/fl littermates, although the difference was not statistically significant by a log-rank (Mantel–Cox) test (P = 0.056; Supplementary Tables S1–S3).

Figure 1.
Figure 1.

Myeloid-specific PTP1B deletion leads to decreased lifespan and development of an acute myeloid leukemia. A, Survival curve of LysM-PTP1B−/− mice (white diamonds), LysM-PTP1B+/− mice (gray diamonds), and PTP1Bfl/fl control mice (black diamonds) in weeks (n = 12/genotype). LysM-PTP1B−/− mice have a significantly shortened lifespan in comparison with LysM-PTP1B+/− and PTP1Bfl/fl control mice (P < 0.001). B, Number of bone marrow cells harvested from LysM-PTP1B−/− mice (gray) and PTP1Bfl/fl mice (black) over time in weeks (P < 0.001, at all time points; n = 4/time point). C, Percentage of blast cells in peripheral blood over time in weeks relative to total WBCs (P < 0.001; n = 12/genotype). D, Differential blood counts from PTP1B+/+ and LysM-PTP1B−/− mice at time of death; cell subsets are expressed as a percentage of WBCs; neutrophils were significantly decreased in LysM-PTP1B−/− mice (P < 0.05), and there was a significant increase in the blast cell population in LysM-PTP1B−/− mice in comparison with PTP1B+/+ mice (P < 0.001; n = 12/genotype). E, Hematocrits of PTP1Bfl/fl mice (black) were reduced in comparison with LysM-PTP1B−/− mice (gray; P < 0.001; n = 8/genotype). F, Total WBC counts were increased in PTP1Bfl/fl mice (black) in comparison with LysM-PTP1B−/− (gray) mice (P < 0.01) from 10 μL of whole blood (n = 8/group). G, Percentage of immature red blood cells (reticulocytes) relative to total red blood cell count; LysM-PTP1B−/− mice had an increase in reticulocytosis (P < 0.001). Representative image of blood slide is also shown with black arrows indicating reticulocytes. H, Representative (magnification, ×40) myeloperoxidase IHC staining of tumor-burdened liver and lung tissue.

To confirm that the phenotype was dependent on the extent of myeloid-PTP1B deficiency, PTP1B expression in BMDMs was determined. LysM-PTP1B−/− mice that spontaneously developed tumors had a complete BMDM PTP1B-deficiency, whereas LysM-PTP1B+/− mice displayed decreased PTP1B expression (50%–80% decrease compared with PTP1Bfl/fl controls) and did not develop tumors (Supplementary Fig. S2A and S2B; n = 12/genotype).

LysM-PTP1B−/− mice had increased total numbers of bone marrow cells between 4 and 12 months of age (Fig. 1B), but these numbers declined sharply after 12 months of age compared with that of PTP1Bfl/fl mice.

LysM-PTP1B−/− mice develop acute myeloid-like leukemia phenotype

The decline in total numbers of bone marrow cells with age could be indicative of the release of immature cells from the bone marrow; therefore, the characteristics of circulating cells were assessed. LysM-PTP1B−/− mice exhibited an increase in leukocytosis (Fig. 1C–E), with an increasing proportion of blast cells and 50% decrease in the percentage of neutrophils (Fig. 1D) at the time of death. Most strikingly, this was accompanied by the appearance of a morphologically distinct blast cell population (Supplementary Fig. S2C and S2D) in LysM-PTP1B−/− mice, which had irregular nuclei with some folding, and a high nuclear:cytoplasm ratio with poor granulation. This population was absent in PTP1Bfl/fl mice (Fig. 1D) and comprised <1% of circulating WBC in LysM-PTP1B+/− mice (Supplementary Fig. S2E and S2F).

The development of this blast cell population was time dependent, with a 5-fold increase from 9 months to 18 months of age (Fig. 1C). Further assessment of blood parameters confirmed a decrease in hematocrit, suggesting anemia in LysM-PTP1B−/− mice (Fig. 1E), along with an increase in total WBC counts (Fig. 1F) in the circulation, and an increase in the percentage of reticulocytes (immature red blood cells; Fig. 1G).

Liver and lung tumors in LysM-PTP1B−/− mice all stained positively for the myeloid-specific enzyme myeloperoxidase, verifying their myeloid lineage, and thereby suggesting the development of tumors to be similar to previously reported myeloproliferative disorders, with mutations in PTP1B (Fig. 1H; refs. 19, 20).

Tumor development in LysM-PTP1B−/− mice is characterized by changes in monocytic-macrophage cell populations in the spleen and liver

LysM-PTP1B−/− mice that developed a leukemia-like blood phenotype, exhibited swollen abdomens due to splenomegaly and hepatomegaly, with visible development of extranodal tumors (Fig. 2A); next, we sought to determine the nature of these tumors. Large tumors between 8 and 10 mm in diameter developed in multiple locations outside of the organs, including around the small intestine, limb joints, and neck (Supplementary Fig. S3A–S3C).

Figure 2.
Figure 2.

LysM-PTP1B−/− phenotype is characterized by tumor formation in multiple tissues and severe disruption to splenic architecture. A, Spleen-to-body-weight ratio at 72 weeks (18 months) is significantly increased in LysM-PTP1B−/− mice (gray) when compared with those of PTP1Bfl/fl (black) control mice (P < 0.0001; n = 6/genotype). Representative post mortem photograph of LysM-PTP1B−/− mouse with splenomegaly is shown. B, Histological analysis of tumor-burdened tissues using H&E staining. Representative slides from LysM-PTP1B−/− liver, lungs, and solid tumors are shown (n = 12/genotype) at ×40 magnification. C, Histology from PTP1Bfl/fl and LysM-PTP1B−/− mice; representative images from both genotypes are displayed at ×40 magnification. From top to bottom: H&E (n = 12/genotype), F4/80 (n = 6/genotype), and Ki67 (n = 6/genotype). D, Representative liver F4/80 IHC staining at ×40 magnification is shown from left to right; PTP1Bfl/fl control mice, LysM-PTP1B+/−, and LysM-PTP1B−/− (n = 6/genotype). Gene expression was investigated using qPCR, f4/80 (E), cd11b (F), and cd68 (G) in liver tissue of PTP1Bfl/fl (black), LysM-PTP1B+/− (gray), and LysM-PTP1B−/− (white) mice (n = 12/genotype). *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Top gene ontology terms for genes (involved in biological processes) significantly upregulated or downregulated from hepatic gene expression microarray of Ptp1b fl/fl mice (n = 5) or LysM-Ptp1b−/− mice (n = 4), ages 6 months, injected with LPS for 3 hours.

H&E staining of extramedullary tumors revealed clusters of densely packed cells (Fig. 2B). A large number of tumor cells displayed prominent nucleoli, abnormal mitotic figures and highly positive Ki67 staining, suggesting that these cells were synthesizing large amounts of protein, were in mitosis and actively proliferating (21, 22). Moreover, LysM-PTP1B−/− mice exhibited a major disruption in the normal architecture of the follicular structure of the spleen compared with PTP1Bfl/fl mice, as determined by H&E and F4/80 macrophage staining (Fig. 2C). Flow cytometry analysis of splenocytes isolated from 4- and 12-month-old mice, demonstrated that in the spleen, there was a 2-fold increase in markers of both dendritic cells (F4/80+ CD11c+) and macrophages (F4/80+ CD11b+) at 12 months of age (Table 1).

View this table:
Table 1.

Cellular composition of PTP1Bfl/fl and LysM-PTP1B−/− mouse spleens at 4 and 12 months of age

Hepatic F4/80 staining indicated an increase in a macrophage-type population in LysM-PTP1B−/− livers, which were not contained solely within the tumor mass, but distributed throughout the organ (Fig. 2D). LysM-PTP1B−/− mice also demonstrated an increase in hepatic gene expression of the monocytic-macrophage markers, F4/80, CD11b, and CD68 compared with PTP1Bfl/fl mice (Fig. 2E–G), in agreement with hepatic F4/80 staining, suggestive of an increase in monocytic-macrophage cells with tumor development in the liver.

To identify global hepatic gene expression changes associated with tumor development, but prior to secondary changes allied with full tumor onset, microarray analysis was performed in livers from presymptomatic (6-month-old) mice, injected with low-dose LPS (0.5 mg/kg) for 3 hours to mimic an inflammatory environment (Fig. 2H). In total, 1989 genes were significantly upregulated and 1,490 genes were significantly downregulated in LysM PTP1B−/− mice. Principal component analysis and hierarchical clustering of differentially expressed genes showed a unique profile of hepatic gene expression between LysM-PTP1B−/− and PTP1Bfl/fl mice (Supplementary Fig. S4A and S4B). Pathway analysis of differentially expressed genes determined a significant upregulation of genes involved in lipid metabolism and downregulation of genes involved in immune system processes/responses (Fig. 2H; Supplementary Fig. S4C and S4D). Many of these lipid metabolism genes were ∼1.5-fold upregulated and are known targets of peroxisome proliferator–activated receptor α (PPARα) and sterol regulatory-element-binding protein (SREBP; Supplementary Table S4; ref. 23). Upregulation of mTOR and amino acid sensing activators of mTOR, lamtor 4 and 5, provide a further link to these pathways because aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in cancer, by specifically activating bioenergetic and anabolic cellular processes (24). In addition, Sirt3 and Sirt5 were upregulated and both of these protein deacetylase sirtuins have been reported to positively regulate mitochondrial fatty acid oxidation (25). These results suggest a hallmark of cancer, i.e., metabolic reprogramming in the form of increased lipid metabolism is associated with tumor development in livers of LysM-PTP1B−/− mice.

Myeloid PTP1B deficiency alters expression of splenic and hepatic tissue cytokines

Given the changes in cell populations in splenic and hepatic tissue from LysM-PTP1B−/− mice with tumors, we investigated whether the expression levels of immunosuppressive genes were altered. Interleukin-4 (IL4), an anti-inflammatory cytokine (26), was found to be significantly upregulated in the liver of LysM-PTP1B−/− mice as compared with PTP1Bfl/fl mice (Fig. 3A). The lack of an intermediate phenotype in terms of IL4 expression in LysM-PTP1B+/− is likely due to a combination of changes in cell population between genotypes and tissues, coupled with a decrease in IL6, which has been demonstrated to enhance macrophage polarization toward the M1 end of the spectrum and attenuate arginase and IL4 production.

Figure 3.
Figure 3.

Dysregulation of cytokines in LysM-PTP1B−/− mice is accompanied by changes in STAT3 expression. Gene expression was assessed by qPCR in the liver: il4 (A); arginase (B); il6 (C); il10 (D), and spleen: il4 (E); arginase (F); il6 (G); il10 (H) of PTP1Bfl/fl (black), LysM-PTP1B+/− (gray), and LysM-PTP1B−/− (white) mice (n = 12/genotype). I, Plasma IL10 levels were investigated by ELISA, showing a significantly increased plasma IL10 in LysM-PTP1B−/− mice (P < 0.001) and LysM-PTP1B+/− (P < 0.05) in comparison with PTP1Bfl/fl control mice (n = 12/genotype). J, Protein levels of STAT3 in liver and spleen tissues, as analyzed by immunoblotting; representative STAT3 blot is shown using GAPDH loading control (n = 10/genotype). K and L, Quantification of STAT3 in liver (K) and spleen (L) relative to GAPDH (n = 10/genotype; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Arginase I, an enzyme highly intertwined with IL4-mediated macrophage activation, postulated to contribute toward cancers evasion of the immune system (27), was also significantly upregulated in the liver of LysM-PTP1B−/− mice (Fig. 3B). IL6, a cytokine more associated with inflammatory responses, but also known to play a role in regulation of the tumor microenvironment (28), was significantly upregulated in the liver of LysM-PTP1B−/− mice, without significant alterations in LysM-PTP1B+/− (Fig. 3C).

High circulating levels of IL10 are associated with immunosuppression and have been shown to encourage tumor growth, by suppressing APC-mediated immune responses (29). We previously reported that LysM-PTP1B−/− mice exhibited increased expression in BMDM- and serum-IL10 (9). LysM-PTP1B−/− mice also had markedly increased expression of IL10 in the liver (15-fold compared with PTP1Bfl/fl controls; Fig. 3D).

In the spleen, there were no differences in IL4 and Arginase I expression between genotypes (Fig. 3E and F), whilst IL6 was significantly upregulated in LysM-PTP1B−/− mice (Fig. 3G) and IL10 was upregulated 60-fold in LysM-PTP1B−/− mice in comparison with the controls (Fig. 3H).

As expected, LysM-PTP1B−/− mice had increased levels of serum IL10 (Fig. 3I). LysM-PTP1B+/− mice had intermediate levels of expression in spleen and liver and circulating levels of IL10 compared with PTP1Bfl/fl controls (Fig. 3I). Finally, both STAT3 and STAT5 expression in hepatic and splenic tissues were also increased in LysM-PTP1B−/− mice (Fig. 3J–L, and Supplementary Fig. S5A–S5D).

Dysregulation of cytokines in LysM-PTP1B−/− mice is accompanied by upregulation of STAT3 and STAT5

Constitutive activation of multiple STAT family members has been determined to be major pro-oncogenic drivers (30), and tyrosine-phosphorylated STATs are major substrates for PTP1B (9, 10, 31). We have demonstrated previously that deletion of PTP1B can increase both STAT3 tyrosine phosphorylation and STAT3 expression levels in BMDM (9) and dendritic cells (11), and we also demonstrate that total levels of STAT5 protein and mRNA increase in LysM-PTP1B−/− livers and spleens (Supplementary Fig. S5A–S5F).

To determine the level of STAT3 activity through DNA binding, ChIP-seq analysis was performed in BMDM from PTP1Bfl/fl and LysM-PTP1B−/− mice, under basal and IL10 stimulated conditions. There were relatively few STAT3 binding peaks in control mice of both genotypes (<200), but IL10 treatment resulted in a >10-fold increase in STAT3 binding in total and in regions associated with genes (Fig. 4A). Motif analysis revealed the increase in STAT3 binding occurred at the canonical STAT3 binding-motif (TTCnnnGAA, e.g., TTCCnGGAA; Fig. 4B) and also TTCC and GGAA half-sites (Supplementary Fig. S6). STAT3 binding peaks at selected genes were visualized in the UCSC genome browser and revealed binding near STAT3 and Diap1 genes in BMDMs from all four treatment groups; binding at the Bcl6 gene specifically in IL10-treated mice (from both genotypes) and specific binding at several known targets of STAT3 was observed only in LysM-PTP1B−/− mice treated with IL10 (e.g., Stat6, Klf4, Epas1, and Lims1; Fig. 4C; Supplementary Fig. S6). These results strongly suggest that increased DNA binding activity of STAT3 activity in LysM-PTP1B−/− BMDM, combined with increased levels of circulating IL10 cytokine, may contribute to the development of the acute leukemia phenotype.

Figure 4.
Figure 4.

STAT3 ChIP-seq in BMDM from Ptp1bfl/fl and LysM-PTP1B−/− mice. A, Number of STAT3 peaks (clear) and associated genes (black bars) as determined by ChIP-seq in BMDM treated with IL10 (4 hours) from Ptp1b fl/fl and LysM-Ptp1b−/− mice (n = 4 mice per group). B, Occurrences of known classic STAT3 motifs in ChIP peaks from each group. C, STAT3 binding peaks at selected genes were visualized in the UCSC genome browser. Chromosomal locations are shown at the top of each panel and gene positions are indicated at the bottom. Diap1, death-associated inhibitor of apoptosis 1; Bcl6, B-cell lymphoma-6; Klf4, Kruppel-like factor 4; Epas1, endothelial PAS domain protein 1, also known as HIF-2α; Lims1, LIM and senescent cell antigen-like domains 1, also known as PINCH.

LysM-PTP1B−/− mice display upregulation of Bcl-2 and Bcl-XL

STAT3 and STAT5 can directly regulate the gene expression levels of mitochondrial proteins that play a key role in promoting or inhibiting apoptosis (28). Splenic STAT3 was upregulated in LysM-PTP1B+/− and further increased in LysM-PTP1B−/− mice (Fig. 5A). B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma extra-large (Bcl-XL), two strongly antiapoptotic proteins downstream of STAT3, were also induced in LysM-PTP1B+/− splenic tissue compared with PTP1Bfl/fl and were further increased in LysM-PTP1B−/− mice (Fig. 5B and C). Oncogenic transcription factor, B-cell lymphoma 6 (Bcl-6), which can be regulated by STAT5, was significantly increased in the splenic tissue of LysM-PTP1B+/− mice, but was completely suppressed in LysM-PTP1B−/− mice (Fig. 5D).

Figure 5.
Figure 5.

LysM-PTP1B−/− mice have increased expression of the Bcl-2 family antiapoptotic proteins Bcl-2 and Bcl-XL. Gene expression analysis of spleen stat3 (A), bcl-2 (B), bcl-xl (C), bcl-6 (D) and liver, stat3 (E), bcl-2 (F), bcl-xl (G) and bcl-6 (H; n = 12/genotype). *, P < 0.05; **, P < 0.01; ***, P < 0.001. I, Proposed model for the role of PTP1B in the development of leukemia. Deletion of PTP1B in myeloid cells results in increased proliferation and survival in the bone marrow, possibly via CSF-1–STAT3 signaling axis, leading to increase in bone marrow cells. Immature blast cells in the bone marrow are released prematurely into the circulation, where they proliferate and seed into tissues such as the liver and lung to disrupt normal homeostasis. Infiltration and expansion is accompanied by changes in JAK-STAT signaling and changes in tissue cytokine profile, leading to establishment of a tumor microenvironment within the tissue.

In the liver, STAT3 expression was significantly upregulated in LysM-PTP1B−/− mice only (Fig. 5E). Similarly, both Bcl-2 and Bcl-XL expression were upregulated in hepatic tumor-burdened tissue of LysM-PTP1B−/− mice (Fig. 5F and G). The expression of these proteins was unaltered in LysM-PTP1B+/− mice compared with PTP1Bfl/fl. In addition, Bcl-6 was increased in the hepatic tissue of LysM-PTP1B+/− mice, but completely suppressed in LysM-PTP1B−/− mice (potentially due to differing cell composition between mouse genotypes; Fig. 5H). The altered balance of these mitochondrial proteins in LysM-PTP1B−/− mice would strongly promote cell survival over cell death and thus suggests a mechanism for the myeloproliferative disorder and development of tumors in these mice.

Overall, our data show for the first time that myeloid PTP1B is required to inhibit the overactivation of JAK–STAT signaling and the development of myeloid leukemia. Moreover, high expression levels of STAT3, Bcl-2, and Bcl-XL (with chronic loss of myeloid PTP1B) may be due to infiltration and expansion of leukemic blast cells that have infiltrated the hepatic tissue and developed into solid extramedullary masses (chloromas), rather than primary defects within the hepatic cells themselves (Fig. 5I).

Ruxolitinib treatment inhibits JAK/STAT signaling in LysM–PTP1B−/− BMDM and reverses the functional phenotype observed

In order to investigate if changes in signaling and gene expression in LysM-PTP1B−/− cells were driven by overactivation of the JAK/STAT pathway, BMDM from presymptomatic, 6-month-old mice were treated with 5 μmol/L ruxolitinib, a Janus kinase (JAK1/2) inhibitor. Ruxolitinib works by binding to the active site and thereby preventing binding of JAKs to their substrates, thus decreasing activity of the JAK–STAT pathway. The phosphorylation status of JAK2, a reported substrate for PTP1B in vitro (32), did not change between PTP1Bfl/fl controls and LysM-PTP1B−/− BMDM stimulated with LPS, with or without ruxolitinib treatment (Fig. 6A and B). Treatment with ruxolitinib significantly inhibited both basal and LPS-induced STAT3 phosphorylation at its activation site Y705 (P < 0.001), but not the S727 site (P > 0.5; Fig. 6C–E). STAT3 Y705 was hyperphosphorylated in LysM-PTP1B−/− mice (Fig. 6C and D) and is required for the dimerization of STAT3 and subsequent shuttling to the nucleus, where it can exert its transcriptional effects. Transcriptional activity of STAT3 is via the S727 site, which was not inhibited by ruxolitinib treatment, despite being basally hyperphosphorylated in LysM-PTP1B−/− BMDM (Fig. 6C and E). However, it is important to note that without phosphorylation at Y705, STAT3 is unable to dimerize and translocate, thus diminishing its transcriptional effects. STAT5 phosphorylation at Y694 was similar between LysM-PTP1B−/− and PTP1Bfl/fl BMDM, both basally and in response to LPS, and was completely blocked upon ruxolitinib treatment (Fig. 6C and F).

Figure 6.
Figure 6.

Treating LysM PTP1B−/− BMDM with ruxolitinib restores phenotype. A, Representative blots from immunoprecipitation of p-Tyr, and immunoblotting for JAK2 in PTP1Bfl/fl and LysM PTP1B−/− BMDM. B, Quantification of p-Tyr JAK2 IP immunoblots relative to GAPDH input control (n = 3/genotype; mean ± SEM). C, Immunoblots for pSTAT3 Y705, pSTAT3 S727, STAT3, pSTAT5 Y694, STAT5, p-mTOR S2448, mTOR, pERK1/2 T202/T204, ERK, GAPDH, p-p38 T180/T182, and p38. Quantification by rolling densitometry scanning of pSTAT3 Y705 (D), pSTAT3 S727 (E), pSTAT5 Y694 (F), p-mTOR S2448 (G), pERK1/2 T202/Y204 (H), p-p38 T180/Y182 (I; n = 3/genotype for all targets; mean ± SEM). J and K, TNFα ELISA (J) and IL10 ELISA (K) of culture media from ruxolitinib-treated PTP1Bfl/fl and LysM PTP1B−/− BMDM (n = 3/genotype in duplicate; mean ± SEM). L, β-galactosidase assay with B3Z cells shows that LysM PTP1B−/− BMDM are not able to activate a T reporter cell line to the same extent as control BMDM; this is reversed by ruxolitinib treatment (n = 3 in triplicates; mean ± SEM). M, LysM PTP1B−/− BMDM are more adhesive to collagen substrate than PTP1Bfl/fl BMDM at basal level and following LPS treatment; this is reversed by ruxolitinib treatment (100 ng/mL, 24 hours; n = 3/genotype; mean ± SEM; significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001).

In canonical JAK-STAT signaling, activated JAK additionally leads to the induction of downstream mTOR and ERK signaling; hence, we assessed the phosphorylation status of mTOR S2448. mTOR S2448 was significantly decreased in LysM-PTP1B−/− BMDM versus PTP1Bfl/fl (P < 0.05; Fig. 6C and G), and phosphorylation was significantly blocked by ruxolitinib treatment in both genotypes (P < 0.0101; Fig. 6C and G). The mitogen activated protein kinase, ERK2/1, is also activated following JAK phosphorylation. While PTP1Bfl/fl BMDM treated with LPS did not respond at T202/Y204, LysM-PTP1B−/− mice display a response curve with phosphorylation peaking at 60 minutes following stimulation (Fig. 6C and H). In both genotypes, ERK signaling is diminished with ruxolitinib treatment (P < 0.001; Fig. 6C and H). MAPK p38 phosphorylation at T180/Y182 is increased significantly in LysM-PTP1B−/− mice compared with PTP1Bfl/fl (P < 0.01; Fig. 6C and I). While ruxolitinib treatment decreased p38 signaling significantly in both genotypes (P < 0.05), it was not completely suppressed (Fig. 6C and I).

It has been robustly demonstrated that p38 induction can be required for IL10 production in macrophages (33); hence, we assayed culture media for TNFα and IL10 production following treatment with ruxolitinib (Fig. 6J and K). TNFα secretion was inhibited upon PTP1B deficiency in LPS-stimulated BMDM, and this was reversed upon ruxolitinib treatment (Fig. 6J). Conversely, IL10 production was significantly reduced by blockade of the JAK–STAT signaling pathway in LysM-PTP1B−/− BMDM (P < 0.001; Fig. 6K), thus demonstrating that dysregulation of STAT3 signaling due to myeloid-PTP1B deficiency is the driving factor in the enhancement of IL10 production in the LysM-PTP1B−/− model.

It was important to determine whether inhibiting STAT3 activity could reverse and restore the functional defects observed in LysM PTP1B−/− cells. We previously demonstrated that DCs in LysM-PTP1B−/− mice have altered functionality, with a decreased ability to prime antigen-specific T-cell responses and increased adherence in vivo in response to LPS stimulation (11). We now demonstrate that LysM-PTP1B−/− BMDM also display this phenotype, with a significant decrease in LysM-PTP1B−/− BMDM ability to activate the B3Z reporter T-cell model (P < 0.001; Fig. 6L). However, treatment with ruxolitinib decreases activation of B3Z T cells in both PTP1Bfl/fl and LysM-PTP1B−/− BMDM, indicating that suppression of the JAK–STAT pathway downregulates functions of both genotypes to equal levels (Fig. 6L). In addition, DCs from LysM-PTP1B−/− mice exhibited an increase in adhesion to collagen (11). We demonstrate that this phenotype is present in LysM-PTP1B−/− BMDM and that treatment with ruxolitinib restores the phenotype of unstimulated LysM-PTP1B−/− BMDM to that of control unstimulated PTP1Bfl/fl BMDM (Fig. 6M). These data demonstrate that inhibition of the JAK–STAT pathway leads to inability of BMDM to increase adhesion to collagen substrate following LPS stimulation and suggest that changes in the functionality of LysM-PTP1B−/− BMDM are JAK/STAT-driven.

Discussion

Our study demonstrates that long-term complete myeloid PTP1B deficiency, as achieved in LysM-PTP1B−/− mice, decreases their lifespan, which recapitulates the phenotype observed with global PTP1B deletion. This phenotype is not observed with neuronal, muscle, liver, or adipose-specific PTP1B knockout models. PTP1B has been implicated in the development of many cancers, functioning either as a tumor promoter (34) or suppressor (35) in a tissue-dependent manner. Our findings elucidate PTP1B function in myeloid cells and provide new evidence that PTP1B acts as a tumor suppressor in myeloid lineage cells. We present data that LysM-PTP1B−/− mice exhibit greater than normal numbers of bone marrow cells when <12 months of age, which declines with increasing age, concurrently with an increase in blast cells in peripheral blood, normally diagnostic of an acute leukemia.

PTP1B has been suggested to play a role in both, colony stimulating factor 1 (CSF1; ref. 7) signaling, and fms-like tyrosine kinase (FLT-3) signaling (36), which can regulate proliferation and mobilization of myeloid cells from the bone marrow (37). Both these molecules have been reported to signal through STAT3 (37, 38), and thus activation of these pathways should be a focus of future studies.

Histological analysis of LysM-PTP1B−/− tissues revealed infiltration of leukemic cells into hepatic and lung tissues, with tumor development. The tumors contained cells with a high nuclear: cytoplasmic ratio, a common feature of malignant cells (39). As the cell becomes malignant, this can affect packing of the nuclear material, often giving rise to large clumped heterochromatin and hypochromatic nuclei with wrinkled nuclear membranes (39), both of which are evident in the LysM-PTP1B−/− tumors.

LysM-PTP1B−/− mice exhibit an increase in IL10 expression in BMDMs (9). We now report that in aged mice, the increase in IL10 expression is systemic and found both in serum and at mRNA level in multiple tissues. With respect to inflammatory metabolic disorders, such as T2DM, heightened IL10 production may be advantageous, because T2DM is associated with inflammation-mediated PTP1B upregulation in multiple tissues. However, long-term exposure has been suggested to contribute toward tumor development, by suppressing natural tumor immunity (29).

LysM-PTP1B−/− myeloid cells exhibit hyperphosphorylation of STAT3 under normal homeostatic conditions (9), in both BMDM and dendritic cells, from 6-month-old mice, suggesting that changes in STAT3 occur prior to development of leukemia. STAT5 activation has been suggested to play a role in myeloproliferative neoplasms (19). Importantly, STAT5 is not hyperphophorylated basally or upon LPS stimulation in LysM-PTP1B−/−BMDM. STAT3 is at the center of multiple signaling cascades, and both, overexpression and constitutively active STAT3, are associated with multiple cancers, due to its antiapoptotic and proliferative effects (28, 40–43). Given that LysM-PTP1B+/− mice exhibit an increase in STAT3 mRNA and protein levels in the spleen and liver without the development of tumors, it is likely that that even some PTP1B mediated STAT3 de-phosphorylation in the LysM-PTP1B−/+ mice is enough to confer protection. Furthermore, we demonstrate that STAT3 inhibition in BMDM from presymptomatic LysM-PTP1B−/− mice, that exhibit hyperphosphorylation of STAT3 Y705, is blocked with ruxolitinib treatment. This blockade of STAT3 phosphorylation suppresses enhanced IL10 secretion and restores the functional phenotype of LysM-PTP1B−/− BMDMs.

STAT3 exerts many of its prosurvival and proliferative effects via downstream effects in the B-cell lymphoma family of proteins, including changing Bcl-6 and Bcl-2/Bcl-XL (pro- and antiapoptotic, respectively) ratios, which can alter the survival of a cell (31). The ratio of Bcl-6 to Bcl-2/Bcl-XL can be used as a prognostic marker for cancers and is correlated with poor survival (44). We demonstrate that this ratio is skewed in favor of the antiapoptotic Bcl-2 and Bcl-XL in LysM-PTP1B−/− mice, thus favoring cell survival. It has been established that enhanced survival due to increased Bcl-2 (45) is STAT3 dependent, and high levels of Bcl-2 can confer resistance to chemotherapy in myeloid leukemias. Bcl-XL also protects cells against a number of cytotoxic insults, including induction of cytokine resistance (46), and promotes cell survival (47), and again high expression can confer a multidrug resistant phenotype (48). It has been suggested that this is due to Bcl-XL–mediated inhibition of p53. Importantly, studies have revealed that Bcl-6−/− macrophages display a hyperproliferative phenotype and demonstrate spontaneous IL6 production, which may, in part, explain the high levels of IL6 seen in LysM-PTP1B−/− mice (49). Our data demonstrate a direct imbalance, with the prosurvival Bcl-2 and Bcl-XL increased and their partner Bcl-6 suppressed, tilting the balance toward survival and proliferation. It is also important to note that PTP1B deletion in hematopoetic compartments in mice, using Mx1-Cre promoter, also results in enlargement of spleen size and induction of extramedullary hematopoiesis, resulting in expansion of myeloid lineage cells and increased phosphorylation of STAT5, Akt, and ERK in bone marrow. This contributed toward the development of a myeloproliferative neoplasm/myelofibrosis-like phenotype in these mice, although no tumors were observed presumably because of the younger age studied (19). This further contributes to the body of evidence demonstrating the importance of PTP1B regulation of the JAK/STAT pathway and importance of this enzyme as a tumor suppressor.

We propose that complete deficiency of PTP1B in myeloid cells results in the development of leukemia through multiple mechanisms (Fig. 5I). An increase in bone marrow cellularity in young mice, coupled with increased survival and proliferation due to STAT3 mediated effects (potentially involving FLT3), increases the chance of secondary mutations. Meanwhile, the development of a systemic immunosuppressive environment through high IL10 levels and inability of antigen-presenting cells to prime immune responses may lead to a lack of immune surveillance. This would be further exacerbated through the IL10–STAT3 signaling axis, thus establishing a positive feedback loop to further drive unregulated proliferation and survival through imbalance of Bcl-6 and Bcl-2/Bcl-XL. In summary, our data demonstrate a tumor-suppressive role for myeloid PTP1B and provide further evidence that PTP1B is a critical gene in the pathogenesis of del(20q) myeloid malignancies (19).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Authors' Contributions

Conception and design: S. Le Sommer, N. Mody, B.G. Neel, H.M. Wilson, M. Delibegović

Development of methodology: S. Le Sommer, H.M. Wilson, M. Delibegović

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): S. Le Sommer, N. Morrice, M. Pesaresi, D. Thompson, K.K. Bence, M. Delibegović

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Le Sommer, N. Morrice, M. Pesaresi, B.G. Neel, H.M. Wilson, M. Delibegović

Writing, review, and/or revision of the manuscript: S. Le Sommer, N. Morrice, M.A. Vickers, N. Mody, H.M. Wilson, M. Delibegović

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D. Thompson, M. Delibegović

Study supervision: N. Mody, H.M. Wilson, M. Delibegović

Acknowledgments

The authors thank the University of Aberdeen Histology and Microscopy core facility, as well as the National Health Services (NHS) Grampian Pathology services for processing of histology slides. The authors also thank the Centre for Genome Enabled Biology and Medicine at the University of Aberdeen where microarray and ChIP sequencing experiments were processed. Flow cytometry analyses were performed at the University of Aberdeen Ian Fraser Flow Cytometry Centre. This work was performed with the funds from the Wellcome Trust ISSF grant to M. Delibegovic and BHF project grant to M. Delibegovic (PG/11/8/28703). S. Le Sommer is a recipient of the University of Aberdeen Institute of Medical Sciences PhD studentship.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

  • Received April 4, 2017.
  • Revision received June 29, 2017.
  • Accepted October 25, 2017.

References

  1. 1.↵
    1. Elchebly M,
    2. Payette P,
    3. Michaliszyn E,
    4. Cromlish W,
    5. Collins S,
    6. Loy AL,
    7. et al.
    Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene. Science 1999;283:1544.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    1. Klaman LD,
    2. Boss O,
    3. Peroni OD,
    4. Kim JK,
    5. Martino JL,
    6. Zabolotny JM,
    7. et al.
    Increased energy expenditure, decreased adiposity, and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice. Mol Cell Biol 2000;20:5479–89.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    1. Agouni A,
    2. Mody N,
    3. Owen C,
    4. Czopek A,
    5. Zimmer D,
    6. Bentires-Alj M,
    7. et al.
    Liver-specific deletion of protein tyrosine phosphatase (PTP) 1B improves obesity- and pharmacologically-induced endoplasmic reticulum stress. Biochem J 2011;438:369–78.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    1. Delibegovic M,
    2. Bence KK,
    3. Mody N,
    4. Hong E,
    5. Ko HJ,
    6. Kim JK,
    7. et al.
    Improved glucose homeostasis in mice with muscle-specific deletion of protein-tyrosine phosphatase 1B. Mol Cell Biol 2007;27:7727–34.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    1. Owen C,
    2. Czopek A,
    3. Agouni A,
    4. Grant L,
    5. Judson R,
    6. Lees EK,
    7. et al.
    Adipocyte-specific protein tyrosine phosphatase 1B deletion increases lipogenesis, adipocyte cell size and is a minor regulator of glucose homeostasis. PLoS One 2012;7:e32700.
    OpenUrlCrossRefPubMed
  6. 6.↵
    1. Balavenkatraman KK,
    2. Aceto N,
    3. Britschgi A,
    4. Mueller U,
    5. Bence KK,
    6. Neel BG,
    7. et al.
    Epithelial protein-tyrosine phosphatase 1B (PTP1B) contributes to the induction of mammary tumors by HER2/Neu but is not essential for tumor maintenance. MCR 2011;9:1377–84.
    OpenUrl
  7. 7.↵
    1. Heinonen KM,
    2. Dubé N,
    3. Bourdeau A,
    4. Lapp WS,
    5. Tremblay ML
    . Protein tyrosine phosphatase 1B negatively regulates macrophage development through CSF-1 signaling. Proc Natl Acad Sci U S A 2006;103:2776–81.
    OpenUrlAbstract/FREE Full Text
  8. 8.↵
    1. Traves PG,
    2. Pardo V,
    3. Pimentel-Santillana M,
    4. Gonzalez-Rodriguez A,
    5. Mojena M,
    6. Rico D,
    7. et al.
    Pivotal role of protein tyrosine phosphatase 1B (PTP1B) in the macrophage response to pro-inflammatory and anti-inflammatory challenge. Cell Death Dis 2014;5:e1125.
    OpenUrlCrossRefPubMed
  9. 9.↵
    1. Grant L,
    2. Shearer KD,
    3. Czopek A,
    4. Lees EK,
    5. Owen C,
    6. Agouni A,
    7. et al.
    Myeloid-cell protein tyrosine phosphatase-1B deficiency in mice protects against high-fat diet and lipopolysaccharide-induced inflammation, hyperinsulinemia, and endotoxemia through an IL-10 STAT3-dependent mechanism. Diabetes 2014;63:456.
    OpenUrlAbstract/FREE Full Text
  10. 10.↵
    1. Pike KA,
    2. Hutchins AP,
    3. Vinette V,
    4. Théberge J,
    5. Sabbagh L,
    6. Tremblay ML,
    7. et al.
    Protein tyrosine phosphatase 1B is a regulator of the interleukin-10–induced transcriptional program in macrophages. Sci Signal 2014;7:ra43.
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    1. Martin-Granados C,
    2. Prescott AR,
    3. Le Sommer S,
    4. Klaska IP,
    5. Yu T,
    6. Muckersie E,
    7. et al.
    A key role for PTP1B in dendritic cell maturation, migration, and T cell activation. J Mol Cell Biol 2015;7:517–28.
    OpenUrlCrossRefPubMed
  12. 12.↵
    1. Soysal S,
    2. Obermann EC,
    3. Gao F,
    4. Oertli D,
    5. Gillanders WE,
    6. Viehl CT,
    7. et al.
    PTP1B expression is an independent positive prognostic factor in human breast cancer. Breast Cancer Res Treat 2012;137:637–44.
    OpenUrl
  13. 13.↵
    1. Lessard L,
    2. Labbé DP,
    3. Deblois G,
    4. Bégin LR,
    5. Hardy S,
    6. Mes-Masson A,
    7. et al.
    PTP1B is an androgen receptor-regulated phosphatase associated with tumor-promoting functions in prostate cancer. Cancer Res 2012;72:1529–37.
    OpenUrlAbstract/FREE Full Text
  14. 14.↵
    1. Yu H,
    2. Lee H,
    3. Herrmann A,
    4. Buettner R,
    5. Jove R
    . Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat Rev Cancer 2014;14:736–46.
    OpenUrlCrossRefPubMed
  15. 15.↵
    1. Bence KK,
    2. Delibegovic M,
    3. Xue B,
    4. Gorgun CZ,
    5. Hotamisligil GS,
    6. Neel BG,
    7. et al.
    Neuronal PTP1B regulates body weight, adiposity and leptin action. Nat Med 2006;12:917–24.
    OpenUrlCrossRefPubMed
  16. 16.↵
    1. Clausen BE,
    2. Burkhardt C,
    3. Reith W,
    4. Renkawitz R,
    5. Förster I
    . Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res 1999;8:265–77.
    OpenUrlCrossRefPubMed
  17. 17.↵
    1. Spurrell DR,
    2. Luckashenak NA,
    3. Minney DC,
    4. Chaplin A,
    5. Penninger JM,
    6. Liwski RS,
    7. et al.
    Vav1 regulates the migration and adhesion of dendritic cells. J Immunol 2009;183:310.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    1. Karttunen J,
    2. Shastri N
    . Measurement of ligand-induced activation in single viable T cells using the lacZ reporter gene. Proc Natl Acad Sci U S A 1991;88:3972–6.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    1. Jobe F,
    2. Patel B,
    3. Kuzmanovic T,
    4. Makishima H,
    5. Yang Y,
    6. Przychodzen B,
    7. et al.
    Deletion of Ptpn1 induces myeloproliferative neoplasm. Leukemia 2017;31:1229–34.
    OpenUrl
  20. 20.↵
    1. Mei W,
    2. Wang K,
    3. Huang J,
    4. Zheng X
    . Cell transformation by PTP1B truncated mutants found in human colon and thyroid tumors. PLoS One 2016;11:e0166538.
    OpenUrl
  21. 21.↵
    1. Derenzini M,
    2. Treré D,
    3. Pession A,
    4. Montanaro L,
    5. Sirri V,
    6. Ochs RL
    . Nucleolar function and size in cancer cells. Am J Pathol 1998;152:1291–7.
    OpenUrlPubMed
  22. 22.↵
    1. Scholzen T,
    2. Gerdes J
    . The Ki-67 protein: From the known and the unknown. J Cell Physiol 2000;182:311–22.
    OpenUrlCrossRefPubMed
  23. 23.↵
    1. Kersten S
    . Integrated physiology and systems biology of PPARα. Mol Metab 2014;3:354–71.
    OpenUrlCrossRefPubMed
  24. 24.↵
    1. Düvel K,
    2. Yecies JL,
    3. Menon S,
    4. Raman P,
    5. Lipovsky AI,
    6. Souza AL,
    7. et al.
    Activation of a metabolic gene regulatory network downstream of mTOR complex 1. Mol Cell 2010;39:171–83.
    OpenUrlCrossRefPubMed
  25. 25.↵
    1. Rardin MJ,
    2. He W,
    3. Nishida Y,
    4. Newman JC,
    5. Carrico C,
    6. Danielson SR,
    7. et al.
    SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks. Cell Metab 2013;18:920–33.
    OpenUrlCrossRefPubMed
  26. 26.↵
    1. Hosoyama T,
    2. Aslam MI,
    3. Abraham J,
    4. Prajapati SI,
    5. Nishijo K,
    6. Michalek JE,
    7. et al.
    IL-4R drives dedifferentiation, mitogenesis and metastasis in rhabdomyosarcoma. Clin Cancer Res 2011;17:2757–66.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    1. Rodriguez PC,
    2. Quiceno DG,
    3. Zabaleta J,
    4. Ortiz B,
    5. Zea AH,
    6. Piazuelo MB,
    7. et al.
    Arginase I production in the tumor microenvironment by mature myeloid cells inhibits T-cell receptor expression and antigen-specific T-cell responses. Clin Cancer Res 2004;64:5839–49.
    OpenUrl
  28. 28.↵
    1. Bromberg J,
    2. Wang TC
    . Inflammation and cancer: IL-6 and STAT3 complete the link. Cancer Cell 2009;15:79–80.
    OpenUrlCrossRefPubMed
  29. 29.↵
    1. Sharma S,
    2. Stolina M,
    3. Lin Y,
    4. Gardner B,
    5. Miller PW,
    6. Kronenberg M,
    7. et al.
    T cell-derived IL-10 promotes lung cancer growth by suppressing both T cell and APC function. J Immunol 1999;163:5020–8.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    1. Yu H,
    2. Pardoll D,
    3. Jove R
    . STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer 2009;9:798–809.
    OpenUrlCrossRefPubMed
  31. 31.↵
    1. Zhang X,
    2. Guo A,
    3. Yu J,
    4. Possemato A,
    5. Chen Y,
    6. Zheng W,
    7. et al.
    Identification of STAT3 as a substrate of receptor protein tyrosine phosphatase T. Proc Natl Acad Sci U S A 2006;104:4060–4.
    OpenUrl
  32. 32.↵
    1. Myers MP,
    2. Andersen JN,
    3. Cheng A,
    4. Tremblay ML,
    5. Horvath CM,
    6. Parisien JP,
    7. et al.
    TYK2 and JAK2 are substrates of protein-tyrosine phosphatase 1B. J Biol Chem 2001;276:47771.
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    1. Symons A,
    2. Beinke S,
    3. Ley SC
    . MAP kinase kinase kinases and innate immunity. Trends Immunol 2006;27:40–8.
    OpenUrlCrossRefPubMed
  34. 34.↵
    1. Wiener J,
    2. Kernsm B,
    3. Harvey E,
    4. Conaway M,
    5. Iglehart J,
    6. Berchuck A,
    7. et al.
    Overexpression of the protein tyrosine phosphatase PTP1B in human breast cancer: association with p185c-erbB-2 protein expression. J Natl Cancer Inst 1994;86:372–8.
    OpenUrlCrossRefPubMed
  35. 35.↵
    1. Labbé DP,
    2. Uetani N,
    3. Vinette V,
    4. Lessard L,
    5. Aubry I,
    6. Migon E,
    7. et al.
    PTP1B deficiency enables the ability of a high-fat diet to drive the invasive character of PTEN-deficient prostate cancers. Cancer Res 2016;76:3130–5.
    OpenUrlAbstract/FREE Full Text
  36. 36.↵
    1. Schmidt-Arras D,
    2. Böhmer S,
    3. Koch S,
    4. Müller JP,
    5. Blei L,
    6. Cornils H,
    7. et al.
    Anchoring of FLT3 in the endoplasmic reticulum alters signaling quality. Blood 2009;113:3568–76.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    1. Fukuda S,
    2. Broxmeyer HE,
    3. Pelus LM
    . Flt3 ligand and the Flt3 receptor regulate hematopoietic cell migration by modulating the SDF-1α(CXCL12)/CXCR4 axis. Blood 2005;105:3117–26.
    OpenUrlAbstract/FREE Full Text
  38. 38.↵
    1. Novak U,
    2. Harpur A,
    3. Paradiso L,
    4. Kanagasundaram V,
    5. Jaworowski A,
    6. Wilks A,
    7. et al.
    Colony-stimulating factor 1-induced STAT1 and STAT3 activation is accompanied by phosphorylation of Tyk2 in macrophages and Tyk2 and JAK1 in fibroblasts. Blood 1995;86:2948–56.
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    1. Zink D,
    2. Fischer AH,
    3. Nickerson JA
    . Nuclear structure in cancer cells. Nat Rev Cancer 2004;4:677–87.
    OpenUrlCrossRefPubMed
  40. 40.↵
    1. Bhattacharya S,
    2. Ray R,
    3. Johnson L
    . STAT3-mediated transcription of Bcl-2, Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells. Biochem J 2005;392:335–44.
    OpenUrlAbstract/FREE Full Text
  41. 41.↵
    1. Catlett-Falcone R,
    2. Landowski TH,
    3. Oshiro MM,
    4. Turkson J,
    5. Levitzki A,
    6. Savino R,
    7. et al.
    Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity 1999;10:105–15.
    OpenUrlCrossRefPubMed
  42. 42.↵
    1. Kujawski M,
    2. Kortylewski M,
    3. Lee H,
    4. Herrmann A,
    5. Kay H,
    6. Yu H
    . Stat3 mediates myeloid cell dependent tumor angiogenesis in mice. J Clin Invest 2008;118:3367–77.
    OpenUrlCrossRefPubMed
  43. 43.↵
    1. Shain KH,
    2. Yarde DN,
    3. Meads MB,
    4. Huang M,
    5. Jove R,
    6. Hazlehurst LA,
    7. et al.
    β1 integrin adhesion enhances IL-6–mediated STAT3 signaling in myeloma cells: implications for microenvironment influence on tumor survival and proliferation. Cancer Res 2009;69:1009–15.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    1. Campos L,
    2. Rouault J,
    3. Sabido O,
    4. Oriol P,
    5. Roubi N,
    6. Vasselon C,
    7. et al.
    High expression of bcl-2 protein in acute myeloid leukemia cells is associated with poor response to chemotherapy. Blood 1993;81:3091–6.
    OpenUrlAbstract/FREE Full Text
  45. 45.↵
    1. Bhattacharya S,
    2. Ray RM,
    3. Johnson LR
    . STAT3-mediated transcription of Bcl-2, Mcl-1 and c-IAP2 prevents apoptosis in polyamine-depleted cells. Biochem J 2005;392:335–44.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    1. Huang DC,
    2. Cory S,
    3. Strasser A
    . Bcl-2, Bcl-xL and adenovirus protein E1B19kD are functionally equivalent in their ability to inhibit cell death. Oncogene 1997;14:405–14.
    OpenUrlCrossRefPubMed
  47. 47.↵
    1. Motoyama N,
    2. Wang F,
    3. Roth K,
    4. Sawa H,
    5. Nakayama K,
    6. Nakayama K,
    7. et al.
    Massive cell death of immature hematopoietic cells and neurons in Bcl-x-deficient mice. Science 1995;267:1506–10.
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    1. Minn A,
    2. Rudin C,
    3. Boise L,
    4. Thompson C
    . Expression of bcl-xL can confer a multidrug resistance phenotype. Blood 1995;86:1903–10.
    OpenUrlAbstract/FREE Full Text
  49. 49.↵
    1. Yu RY,
    2. Wang X,
    3. Pixley FJ,
    4. Yu JJ,
    5. Dent AL,
    6. Broxmeyer HE,
    7. et al.
    BCL-6 negatively regulates macrophage proliferation by suppressing autocrine IL-6 production. Blood 2005;105:1777.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top
View this article with LENS

Open full page PDF
Article Alerts
Email Article
Citation Tools
Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia
Samantha Le Sommer, Nicola Morrice, Martina Pesaresi, Dawn Thompson, Mark A. Vickers, Graeme I. Murray, Nimesh Mody, Benjamin G. Neel, Kendra K. Bence, Heather M. Wilson and Mirela Delibegović
Cancer Res January 1 2018 (78) (1) 75-87; DOI: 10.1158/0008-5472.CAN-17-0946
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement