T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling.

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.

Macrophages Compensate for Loss of Protein Tyrosine Phosphatase N2 in Dendritic Cells to Protect from Elevated Colitis.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.

Clinical and biological features of PTPN2-deleted adult and pediatric T-cell acute lymphoblastic leukemia.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) is a phosphatase known to be a tumor suppressor gene in T-cell acute lymphoblastic leukemia (T-ALL). Because the full clinicobiologic characteristics of PTPN2 loss remain poorly reported, we aimed to provide a comprehensive analysis of PTPN2 deletions within a cohort of 430 patients, including 216 adults and 214 children treated according to the GRAALL03/05 (#NCT00222027 and #NCT00327678) and the FRALLE2000 protocols, respectively. We used multiplex ligation-dependent probe amplification to identify an 8% incidence of PTPN2 deletion, which was comparable in adult (9%) and pediatric (6%) populations. PTPN2 deletions were significantly associated with an αß lineage and TLX1 deregulation. Analysis of the mutational genotype of adult T-ALL revealed a positive correlation between PTPN2 deletions and gain-of-function alterations in the IL7R/JAK-STAT signaling pathway as well as PHF6 and WT1 mutations. Of note, PTPN2 and PTEN (phosphatase and tensin homolog) deletions were mutually exclusive. Regarding treatment response, PTPN2-deleted T-ALLs were associated with a higher glucocorticoid response and a trend for improved survival in children, but not in adults, with a 5-year cumulative incidence of relapse of 8% for PTPN2-deleted pediatric cases vs 26% (P = .177).

The effects of PTPN2 loss on cell signalling and clinical outcome in relation to breast cancer subtype.

PURPOSE: The protein tyrosine phosphatase PTPN2 dephosphorylates several tyrosine kinases in cancer-related signalling pathways and is thought to be a tumour suppressor. As PTPN2 is not frequently studied in breast cancer, we aimed to explore the role of PTPN2 and the effects of its loss in breast cancer. METHODS: Protein expression and gene copy number of PTPN2 were analysed in a cohort of pre-menopausal breast cancer patients with immunohistochemistry and droplet digital PCR, respectively. PTPN2 was knocked down in three cell lines, representing different breast cancer subtypes, with siRNA transfection. Several proteins related to PTPN2 were analysed with Western blot. RESULTS: Low PTPN2 protein expression was found in 50.2% of the tumours (110/219), gene copy loss in 15.4% (33/214). Low protein expression was associated with a higher relapse rate in patients with Luminal A and HER2-positive tumours, but not triple-negative tumours. In vitro studies further suggested a subtype-specific role of PTPN2. Knockdown of PTPN2 had no effect on the triple-negative cell line, whilst knockdown in MCF7 inhibited phosphorylation of Met and promoted that of Akt. Knockdown in SKBR3 led to increased Met phosphorylation and decreased Erk phosphorylation as well as EGF-mediated STAT3 activation. CONCLUSION: We confirm previous studies showing that the PTPN2 protein is lost in half of the breast cancer cases and gene deletion occurs in 15-18% of the cases. Furthermore, the results suggest that the role of PTPN2 is subtype-related and should be further investigated to assess how this could affect breast cancer prognosis and treatment response.

PTPN2 deficiency along with activation of nuclear Akt predict endocrine resistance in breast cancer.

PURPOSE: The protein tyrosine phosphatase, non-receptor type 2 (PTNP2) regulates receptor tyrosine kinase signalling, preventing downstream activation of intracellular pathways like the PI3K/Akt pathway. The gene encoding the protein is located on chromosome 18p11; the 18p region is commonly deleted in breast cancer. In this study, we aimed to evaluate PTPN2 protein expression in a large breast cancer cohort, its possible associations to PTPN2 gene copy loss, Akt activation, and the potential use as a clinical marker in breast cancer. METHODS: PTPN2 protein expression was analysed by immunohistochemistry in 664 node-negative breast tumours from patients enrolled in a randomised tamoxifen trial. DNA was available for 146 patients, PTPN2 gene copy number was determined by real-time PCR. RESULTS: PTPN2 gene loss was detected in 17.8% of the tumours. Low PTPN2 protein expression was associated with higher levels of nuclear-activated Akt (pAkt-n). Low PTPN2 as well as the combination variable low PTPN2/high pAkt-n could be used as predictive markers of poor tamoxifen response. CONCLUSION: PTPN2 negatively regulates Akt signalling and loss of PTPN2 protein along with increased pAkt-n is a new potential clinical marker of endocrine treatment efficacy, which may allow for further tailored patient therapies.

Obesity Drives STAT-1-Dependent NASH and STAT-3-Dependent HCC.

Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target.

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR-Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

PTPN2-deficiency exacerbates T follicular helper cell and B cell responses and promotes the development of autoimmunity.

Non-coding single nucleotide polymorphisms that repress PTPN2 expression have been linked with the development of type 1 diabetes, rheumatoid arthritis and Crohn's disease. PTPN2 attenuates CD8+ T cell responses to self and prevents overt autoreactivity in the context of T cell homeostasis and antigen cross-presentation. The role of PTPN2 in other immune subsets in the development of autoimmunity remains unclear. Here we show that the inducible deletion of PTPN2 in hematopoietic compartment of adult non-autoimmune prone mice results in systemic inflammation and autoimmunity. PTPN2-deficient mice had increased inflammatory monocytes, B cells and effector T cells in lymphoid and non-lymphoid tissues and exhibited symptoms of dermatitis, glomerulonephritis, pancreatitis and overt liver disease. Autoimmunity was characterised by the formation of germinal centers in the spleen and associated with markedly increased germinal center B cells and T follicular helper (Tfh) cells and circulating anti-nuclear antibodies, inflammatory cytokines and immunoglobulins. CD8+ T cell proliferative responses were enhanced, and interleukin-21-induced STAT-3 signalling in Tfh cells and B cells was increased and accompanied by enhanced B cell proliferation ex vivo. These results indicate that deficiencies in PTPN2 across multiple immune lineages, including naive T cells, Tfh cells and B cells, contribute to the development of autoimmunity.

Loss of protein tyrosine phosphatase, non-receptor type 2 is associated with activation of AKT and tamoxifen resistance in breast cancer.

Breast cancer is a heterogeneous disease and new clinical markers are needed to individualise disease management and therapy further. Alterations in the PI3K/AKT pathway, mainly PIK3CA mutations, have been shown frequently especially in the luminal breast cancer subtypes, suggesting a cross-talk between ER and PI3K/AKT. Aberrant PI3K/AKT signalling has been connected to poor response to anti-oestrogen therapies. In vitro studies have shown protein tyrosine phosphatase, non-receptor type 2 (PTPN2) as a previously unknown negative regulator of the PI3K/AKT pathway. Here, we evaluate possible genomic alterations in the PTPN2 gene and its potential as a new prognostic and treatment predictive marker for endocrine therapy benefit in breast cancer. PTPN2 gene copy number was assessed by real-time PCR in 215 tumour samples from a treatment randomised study consisting of postmenopausal patients diagnosed with stage II breast cancer 1976-1990. Corresponding mRNA expression levels of PTPN2 were evaluated in 86 available samples by the same methodology. Gene copy loss of PTPN2 was detected in 16% (34/215) of the tumours and this was significantly correlated with lower levels of PTPN2 mRNA. PTPN2 gene loss and lower mRNA levels were associated with activation of AKT and a poor prognosis. Furthermore, PTPN2 gene loss was a significant predictive marker of poor benefit from tamoxifen treatment. In conclusion, genomic loss of PTPN2 may be a previously unknown mechanism of PI3K/AKT upregulation in breast cancer. PTPN2 status is a potential new clinical marker of endocrine treatment benefit which could guide further individualised therapies in breast cancer.

Activation of T-cell protein-tyrosine phosphatase suppresses keratinocyte survival and proliferation following UVB irradiation.

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.

Pancreatic T cell protein-tyrosine phosphatase deficiency ameliorates cerulein-induced acute pancreatitis.

BACKGROUND: Acute pancreatitis (AP) is a common clinical problem whose incidence has been progressively increasing in recent years. Onset of the disease is trigged by intra-acinar cell activation of digestive enzyme zymogens that induce autodigestion, release of pro-inflammatory cytokines and acinar cell injury. T-cell protein tyrosine phosphatase (TCPTP) is implicated in inflammatory signaling but its significance in AP remains unclear. RESULTS: In this study we assessed the role of pancreatic TCPTP in cerulein-induced AP. TCPTP expression was increased at the protein and messenger RNA levels in the early phase of AP in mice and rats. To directly determine whether TCPTP may have a causal role in AP we generated mice with pancreatic TCPTP deletion (panc-TCPTP KO) by crossing TCPTP floxed mice with Pdx1-Cre transgenic mice. Amylase and lipase levels were lower in cerulein-treated panc-TCPTP KO mice compared with controls. In addition, pancreatic mRNA and serum concentrations of the inflammatory cytokines TNFα and IL-6 were lower in panc-TCPTP KO mice. At the molecular level, panc-TCPTP KO mice exhibited enhanced cerulein-induced STAT3 Tyr705 phosphorylation accompanied by a decreased cerulein-induced NF-κB inflammatory response, and decreased ER stress and cell death. CONCLUSION: These findings revealed a novel role for pancreatic TCPTP in the progression of cerulein-induced AP.

T-cell protein tyrosine phosphatase attenuates STAT3 and insulin signaling in the liver to regulate gluconeogenesis.

OBJECTIVE: Insulin-induced phosphatidylinositol 3-kinase (PI3K)/Akt signaling and interleukin-6 (IL-6)-instigated JAK/STAT3-signaling pathways in the liver inhibit the expression of gluconeogenic genes to decrease hepatic glucose output. The insulin receptor (IR) and JAK1 tyrosine kinases and STAT3 can serve as direct substrates for the T-cell protein tyrosine phosphatase (TCPTP). Homozygous TCPTP-deficiency results in perinatal lethality prohibiting any informative assessment of TCPTP's role in glucose homeostasis. Here we have used Ptpn2+/- mice to investigate TCPTP's function in glucose homeostasis. RESEARCH DESIGN AND METHODS: We analyzed insulin sensitivity and gluconeogenesis in chow versus high-fat-fed (HFF) Ptpn2+/- and Ptpn2+/+ mice and insulin and IL-6 signaling and gluconeogenic gene expression in Ptpn2+/- and Ptpn2+/+ hepatocytes. RESULTS: HFF Ptpn2+/- mice exhibited lower fasted blood glucose and decreased hepatic glucose output as determined in hyperinsulinemic euglycemic clamps and by the decreased blood glucose levels in pyruvate tolerance tests. The reduced hepatic glucose output coincided with decreased expression of the gluconeogenic genes G6pc and Pck1 and enhanced hepatic STAT3 phosphorylation and PI3K/Akt signaling in the fasted state. Insulin-induced IR-beta-subunit Y1162/Y1163 phosphorylation and PI3K/Akt signaling and IL-6-induced STAT3 phosphorylation were also enhanced in isolated Ptpn2+/- hepatocytes. The increased insulin and IL-6 signaling resulted in enhanced suppression of G6pc and Pck1 mRNA. CONCLUSIONS: Liver TCPTP antagonises both insulin and STAT3 signaling pathways to regulate gluconeogenic gene expression and hepatic glucose output.

Cell cycle-dependent regulation of SFK, JAK1 and STAT3 signalling by the protein tyrosine phosphatase TCPTP.

Janus-activated kinases (JAKs) and Src family kinases (SFKs) and their common substrate signal transducer and activator of transcription (STAT)-3 are frequently hyperactivated in human cancer contributing to the proliferative drive by promoting G(1)/S and G(2)/M progression. Previous studies have established that the protein tyrosine phosphatase TCPTP can dephosphorylate and inactivate the SFK and JAK protein tyrosine kinases (PTKs) to attenuate cytokine signalling in vivo. In this study we determined whether TCPTP regulates SFK and JAK signalling during the cell cycle. We used primary mouse embryonic fibroblasts (MEFs) isolated from TCPTP(-/-) versus +/+ mice, immortalised TCPTP(-/-) MEFs versus those reconstituted with physiological levels of TCPTP and HeLa cells in which TCPTP protein levels had been suppressed by RNA interference, to establish TCPTP as a negative regulator of SFK, JAK1 and STAT3 signalling during the cell cycle. We found that the progression of TCPTP-deficient MEFs after the G(1) restriction point into S-phase was enhanced. We used RNA interference and pharmacological inhibitors to demonstrate that elevated SFK and downstream phosphatidylinositol 3-kinase signalling but not JAK1 or STAT3 signalling were required for the enhanced G(1)/S transition. These results identify TCPTP as a negative regulator of the cell cycle.

DNA replication stalling attenuates tyrosine kinase signaling to suppress S phase progression.

Here we report that T cell protein tyrosine phosphatase (TCPTP)-dependent and -independent pathways attenuate the JAK and Src protein tyrosine kinases (PTKs) and STAT3 phosphorylation to suppress cyclin D1 expression and S phase progression in response to DNA replication stress. Cells that lack TCPTP fail to suppress JAK1, Src, and STAT3, allowing for sustained cyclin D1 levels and progression through S phase despite continued replication stress. Cells that bypass the checkpoint undergo aberrant mitoses with lagging chromosomes that stain for the DNA damage marker gamma H2AX. Therefore, inactivating JAK, Src, and STAT3 signaling pathways in response to DNA replication stress may be essential for the suppression of S phase progression and the maintenance of genomic stability.