Protein Tyrosine Phosphatase H1 Research Articles

Prognostic Significance of Metastatic Lymph Nodes Ratio (MLNR) Combined with Protein-Tyrosine Phosphatase H1 (PTPH1) Expression in Operable Breast Invasive Ductal Carcinoma.

PurposeThe metastatic lymph node ratio (MLNR) is one of the most important factors in prognostic analysis of breast cancer. The objective of this study was to determine if MLNR combined with protein-tyrosine phosphatase H1 (PTPH1) pathological expression can be used to predict the prognosis of patients with breast invasive ductal carcinoma (IDC) better than one factor only.Patients and MethodsA total of 136 patients with invasive ductal carcinoma (IDC) of breast who underwent modified radical mastectomy and were treated with chemotherapy after operation at Qilu Hospital of Shandong University from December 2008 to October 2011 were included. PTPH1 expression was evaluated by immunohistochemistry in surgical specimens retrospectively collected from patients with histologically proven invasive ductal breast cancer. Kaplan–Meier survival analysis and Cox regression analysis were performed to assess the prognostic significance of PTPH1 expression. A prognostic factor for disease-free survival (DFS) was identified by univariate and multivariate analyses. ROC analysis was used to evaluate the performance of single factors and combined feature.ResultsOne hundred and thirty-six patients were included in the analysis. By cut-point survival analysis, MLNR cut-off was designed as 0.2. On multivariate analysis, a MLNR>0.2 was associated with a worse DFS (HR=2.581, 95% CI=1.303–5.113, P=0.007). PTPH1 overexpression is correlated with a better DFS (HR=0.391, 95% CI=0.162–0.945, P=0.037). In addition, MLNR and PTPH1 combined feature had better performance in predicting clinical outcomes after surgery long before recurrence had occurred (Area under the curve=0.795 [95% CI=0.694–0.896], P<0.001).ConclusionThese findings indicate that both PTPH1 and MLNR are accurate independent prognostic parameters in patients with IDC of the breast. Better information on IDC prognosis could be obtained from the combined feature.

The Protein Tyrosine Phosphatase H1 PTPH1 Supports Proliferation of Keratinocytes and is a Target of the Human Papillomavirus Type 8 E6 Oncogene.

Human papillomaviruses (HPV) replicate their DNA in the suprabasal layer of the infected mucosa or skin. In order to create a suitable environment for vegetative viral DNA replication HPV delay differentiation and sustain keratinocyte proliferation that can lead to hyperplasia. The mechanism underlying cell growth stimulation is not well characterized. Here, we show that the E6 oncoprotein of the βHPV type 8 (HPV8), which infects the cutaneous skin and is associated with skin cancer in Epidermodysplasia verruciformis patients and immunosuppressed organ transplant recipients, binds to the protein tyrosine phosphatase H1 (PTPH1), which resulted in increased protein expression and phosphatase activity of PTPH1. Suppression of PTPH1 in immortalized keratinocytes reduced cell proliferation as well as the level of epidermal growth factor receptor (EGFR). Furthermore, we report that HPV8E6 expressing keratinocytes have increased level of active, GTP-bound Ras. This effect was independent of PTPH1. Therefore, HPV8E6-mediated targeting of PTPH1 might result in higher level of EGFR and enhanced keratinocyte proliferation. The HPV8E6-mediated stimulation of Ras may be an additional step to induce cell growth. Our results provide novel insights into the mechanism how βHPVE6 proteins support proliferation of infected keratinocytes, thus creating an environment with increased risk of development of skin cancer particularly upon UV-induced DNA mutations.

NOTCH3 inactivation increases triple negative breast cancer sensitivity to gefitinib by promoting EGFR tyrosine dephosphorylation and its intracellular arrest

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.

The K-Ras effector p38γ MAPK confers intrinsic resistance to tyrosine kinase inhibitors by stimulating EGFR transcription and EGFR dephosphorylation

Mutations in K-Ras and epidermal growth factor receptor (EGFR) are mutually exclusive, but it is not known how K-Ras activation inactivates EGFR, leading to resistance of cancer cells to anti-EGFR therapy. Here, we report that the K-Ras effector p38γ MAPK confers intrinsic resistance to small molecular tyrosine kinase inhibitors (TKIs) by concurrently stimulating EGFR gene transcription and protein dephosphorylation. We found that p38γ increases EGFR transcription by c-Jun-mediated promoter binding and stimulates EGFR dephosphorylation via activation of protein-tyrosine phosphatase H1 (PTPH1). Silencing the p38γ/c-Jun/PTPH1 signaling network increased sensitivities to TKIs in K-Ras mutant cells in which EGFR knockdown inhibited growth. Similar results were obtained with the p38γ-specific pharmacological inhibitor pirfenidone. These results indicate that in K-Ras mutant cancers, EGFR activity is regulated by the p38γ/c-Jun/PTPH1 signaling network, whose disruption may be a novel strategy to restore the sensitivity to TKIs.

Tyrosine dephosphorylation enhances the therapeutic target activity of epidermal growth factor receptor (EGFR) by disrupting its interaction with estrogen receptor (ER).

Protein-protein interactions can increase or decrease its therapeutic target activity and the determining factors involved, however, are largely unknown. Here, we report that tyrosine-dephosphorylation of epidermal growth factor receptor (EGFR) increases its therapeutic target activity by disrupting its interaction with estrogen receptor (ER). Protein tyrosine phosphatase H1 (PTPH1) dephosphorylates the tyrosine kinase EGFR, disrupts its interaction with the nuclear receptor ER, and increases breast cancer sensitivity to small molecule tyrosine kinase inhibitors (TKIs). These effects require PTPH1 catalytic activity and its interaction with EGFR, suggesting that the phosphatase may increase the sensitivity by dephosphorylating EGFR leading to its dissociation with ER. Consistent with this notion, a nuclear-localization defective ER has a higher EGFR-binding activity and confers the resistance to TKI-induced growth inhibition. Additional analysis show that PTPH1 stabilizes EGFR, stimulates the membranous EGFR accumulation, and enhances the growth-inhibitory activity of a combination therapy of TKIs with an anti-estrogen. Since EGFR and ER both are substrates for PTPH1 in vitro and in intact cells, these results indicate that an inhibitory EGFR-ER protein complex can be switched off through a competitive enzyme-substrate binding. Our results would have important implications for the treatment of breast cancer with targeted therapeutics.

Protein-Tyrosine Phosphatase H1 Increases Breast Cancer Sensitivity to Antiestrogens by Dephosphorylating Estrogen Receptor at Tyr537

Estrogen receptor α (ERα or ER) is the only target of breast cancer therapy using antiestrogens. However, about 50% of ER-expressing breast cancer is intrinsically refractory to the antihormone therapy and strategies to improve the therapeutic response are urgently needed. Dynamic ER phosphorylation and dephosphorylation play an important role in ER activity and antihormone response. Although more than 10 kinases participate in phosphorylating ER protein, phosphatases involved remain mostly unidentified. Here, we tested the hypothesis that the protein-tyrosine phosphatase H1 (PTPH1) may regulate ER tyrosine phosphorylation and thereby impact breast cancer antihormone sensitivity. Our results showed that PTPH1 dephosphorylates ER at Tyr537 in vitro and in breast cancer cells. Moreover, PTPH1 stimulates ER nuclear accumulation and increases breast cancer sensitivity to tamoxifen (TAM) and/or fulvestrant in cell culture and in a xenograft model. Further analysis revealed that PTPH1 depends on its catalytic activity to stimulate ER nuclear accumulation and to enhance breast cancer antihormone sensitivity. These studies thus identified PTPH1 as a novel ER phosphatase and further demonstrate a therapeutic potential of enhancing breast cancer sensitivity to antiestrogens through dephosphorylating ER by PTPH1.

P38γ Mitogen-activated Protein Kinase Signals through Phosphorylating Its Phosphatase PTPH1 in Regulating Ras Protein Oncogenesis and Stress Response

Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.

PTPH1 Dephosphorylates and Cooperates with p38γ MAPK to Increase Ras Oncogenesis through PDZ-Mediated Interaction

Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.

Characterization of protein tyrosine phosphatase H1 knockout mice in animal models of local and systemic inflammation

BackgroundPTPH1 is a protein tyrosine phosphatase expressed in T cells but its effect on immune response is still controversial. PTPH1 dephosphorylates TCRzeta in vitro, inhibiting the downstream inflammatory signaling pathway, however no immunological phenotype has been detected in primary T cells derived from PTPH1-KO mice. The aim of the present study is to characterize PTPH1 phenotype in two in vivo inflammatory models and to give insights in possible PTPH1 functions in cytokine release.MethodsWe challenged PTPH1-KO mice with two potent immunomodulatory molecules, carrageenan and LPS, in order to determine PTPH1 possible role in inflammatory response in vivo. Cytokine release, inflammatory pain and gene expression were investigated in challenged PTPH1-WT and KO mice.ResultsThe present study shows that carrageenan induces a trend of slightly increased spontaneous pain sensitivity in PTPH1-KO mice compared to WT (wild-type) littermates, but no differences in cytokine release, induced pain perception and cellular infiltration have been detected between the two genotypes in this mouse model. On the other hand, LPS-induced TNFα, MCP-1 and IL10 release was significantly reduced in PTPH1-KO plasma compared to WTs 30 and 60 minutes post challenge. No cytokine release modulation was detectable 180 minutes post LPS challenge.ConclusionIn conclusion, the present study points out a slight potential role for PTPH1 in spontaneous pain sensitivity and it indicates that this phosphatase might play a role in the positive regulation of the LPS-induced cytokines release in vivo, in contrast to previous reports indicating PTPH1 as potential negative regulator of immune response.

Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition

BackgroundThe present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions.MethodsWe have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions.ResultsIn CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females.ConclusionThese results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.

Protein tyrosine phosphatase H1 is a target of the E6 oncoprotein of high-risk genital human papillomaviruses

The E6 proteins of high-risk genital human papillomaviruses (HPV), such as HPV types 16 and 18, possess a conserved C-terminal PDZ-binding motif, which mediates interaction with some cellular PDZ domain proteins. The binding of E6 usually results in their ubiquitin-mediated degradation. The ability of E6 to bind to PDZ domain proteins correlates with the oncogenic potential. Using a yeast two-hybrid system, GST pull-down experiments and coimmunoprecipitations, we identified the protein tyrosine phosphatase H1 (PTPH1/PTPN3) as a novel target of the PDZ-binding motif of E6 of HPV16 and 18. PTPH1 has been suggested to function as tumour suppressor protein, since mutational analysis revealed somatic mutations in PTPH1 in a minor fraction of various human tumours. We show here that HPV16 E6 accelerated the proteasome-mediated degradation of PTPH1, which required the binding of E6 to the cellular ubiquitin ligase E6-AP and to PTPH1. The endogenous levels of PTPH1 were particularly low in HPV-positive cervical carcinoma cell lines. The reintroduction of the E2 protein into the HPV16-positive cervical carcinoma cell line SiHa, known to lead to a sharp repression of E6 expression and to induce growth suppression, resulted in an increase of the amount of PTPH1. Our data suggest that reducing the level of PTPH1 may contribute to the oncogenic activity of high-risk genital E6 proteins.

Engineering non-natural inhibitor sensitivity in protein tyrosine phosphatase H1

Protein tyrosine phosphatase H1, a member of the ubiquitous protein tyrosine phosphatase (PTP) superfamily of enzymes, is an important signaling molecule, mutant forms of which have been found in human colorectal cancers. Selective PTPH1 inhibitors would be valuable tools for investigating PTPH1’s roles in cellular regulation. However, no PTPH1-specific inhibitors are known. To identify target-selective inhibitors of human PTPH1, we have redesigned a PTPH1/inhibitor interface. Structure-based protein design was used to identify two amino-acid residues, isoleucine 846 and methionine 883, that control PTPH1’s sensitivity to oxalylaminoindole PTP inhibitors. Mutation of residues 846 and 883 to alanine and glycine, respectively, conferred novel inhibitor sensitivity onto PTPH1. From a small panel of putative inhibitors, compounds that potently and selectively target the inhibitor-sensitized PTPH1 mutants were identified.