Re-expression of epigenetically silenced PTPRR by histone acetylation sensitizes RAS-mutant lung adenocarcinoma to SHP2 inhibition.

Silenced protein tyrosine phosphatase receptor type R (PTPRR) participates in mitogen-activated protein kinase (MAPK) signaling cascades during the genesis and development of tumors. Rat sarcoma virus (Ras) genes are frequently mutated in lung adenocarcinoma, thereby resulting in hyperactivation of downstream MAPK signaling. However, the molecular mechanism manipulating the regulation and function of PTPRR in RAS-mutant lung adenocarcinoma is not known. Patient records collected from the Cancer Genome Atlas and Gene Expression Omnibus showed that silenced PTPRR was positively correlated with the prognosis. Exogenous expression of PTPRR suppressed the proliferation and migration of lung cancer cells. PTPRR expression and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibition acted synergistically to control ERK1/2 phosphorylation in RAS-driven lung cancer cells. Chromatin immunoprecipitation assay revealed that HDAC inhibition induced enriched histone acetylation in the promoter region of PTPRR and recovered PTPRR transcription. The combination of the HDAC inhibitor SAHA and SHP2 inhibitor SHP099 suppressed the progression of lung cancer markedly in vitro and in vivo. Therefore, we revealed the epigenetic silencing mechanism of PTPRR and demonstrated that combination therapy targeting HDAC and SHP2 might represent a novel strategy to treat RAS-mutant lung cancer.

Quercetin inhibits the proliferation of multiple myeloma cells by upregulating PTPRR expression.

Multiple myeloma (MM) is an incurable disease characterized by malignant plasma cell clonal expansion in the bone marrow; therefore, inhibiting the proliferation of plasma cells is an important approach to overcome the progression of MM. Quercetin (Que) is a promising flavonoid with broad-spectrum anti-tumor activity against various cancers, including MM; however, the underlying mechanism is not yet understood. The present study aimed to reveal the gene expression profile of Que-treated MM cells and clarify its potential mechanism. The 30% inhibitory concentration (IC30) of Que against MM cells was calculated, and the proliferation rate was significantly reduced after Que treatment. Next, 495 dysregulated genes were identified via RNA sequencing in Que-treated MM cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes analyses indicated that the dysregulated genes were enriched in various apoptosis-related GO terms and amino acid metabolism-related pathways. qPCR validation showed that protein tyrosine phosphatase receptor-type R (PTPRR) had the highest verified log2 FC (abs) among the top 15 dysregulated genes. Overexpression of PTPRR increased the sensitivity of MM cells against Que, significantly inhibiting their proliferation and colony formation ability; silencing of PTPRR showed the opposite results. Furthermore, bioinformatics analyses and PPI network construction of PTPRR indicated that dephosphorylation of ERK might be the potential pathway for the PTPRR-induced inhibition of MM cell proliferation. In summary, our study identified the gene expression profile in Que-treated MM cells and demonstrated that the upregulation of PTPRR was one of the important mechanisms for the Que-induced inhibition of MM cell proliferation.

Protein tyrosine phosphatase receptor type R (PTPRR) antagonizes the Wnt signaling pathway in ovarian cancer by dephosphorylating and inactivating ß-catenin.

Despite a lack of mutations, accumulating evidence supports an important role for the Wnt/ß-catenin pathway in ovarian tumorigenesis. However, the molecular mechanism that contributes to the aberrant activation of the Wnt signaling cascade in ovarian cancer has not been fully elucidated. Here, we found that protein tyrosine phosphatase receptor type R (PTPRR) suppressed the activation of the Wnt/ß-catenin pathway in ovarian cancer. We performed an shRNA-based biochemical screen, which identified PTPRR as being responsible for tyrosine dephosphorylation of ß-catenin on Tyr-142, a key site controlling the transcriptional activity of ß-catenin. Of note, PTPRR was down-regulated in ovarian cancers, and ectopic PTPRR re-expression delayed ovarian cancer cell growth both in vitro and in vivo Using a proximity-based tagging system and RNA-Seq analysis, we identified a signaling nexus that includes PTPRR, α-catenin, ß-catenin, E-cadherin, and AT-rich interaction domain 3C (ARID3C) in ovarian cancer. Immunohistochemistry staining of human samples further suggested that PTPRR expression is inversely correlated with disease prognosis. Collectively, our findings indicate that PTPRR functions as a tumor suppressor in ovarian cancer by dephosphorylating and inactivating ß-catenin. These results suggest that PTPRR expression might have utility as a prognostic marker for predicting overall survival.

PTPRR regulates ERK dephosphorylation in depression mice model.

BACKGROUND: The Protein tyrosine phosphatase receptor type R (PTPRR), which regulates the dephosphorylation of the downstream mitogen-activated protein kinase (MAPK) steering cell proliferation, apoptosis and synaptic plasticity, may be involved in the pathogenesis of depression. METHODS: Lentiviral vectors were utilized to express the PTPRR constitutively in the hippocampal dentate gyrus (DG) of mice before or after chronic mild stress. Behavior tests, MAPK levels, neuronal apoptosis and cell proliferation in the hippocampal DG were examined. RESULTS: Without chronic mild stress (CMS), the lenti-shPTPRR mice showed shorter immobility time in the tail suspension test than controls, while the lenti-PTPRR mice exhibited significantly less sucrose intake and increased immobility time in the forced swim tests than control mice, indicating increased prodepressant-like effects of PTPRR in lenti-PTPRR mice. Similarly, under CMS, the lenti-shPTPRR mice had more sucrose intake, shorter immobility time in the forced swim test and tail suspension test compared to controls, and lenti-PTPRR mice had less sucrose intake and longer immobility time in forced swim test and tail suspension test, exhibiting increased susceptibility to depressive-like behaviors and greater sensitivity to CMS. Besides, the Phospho-ERK1/2(p-ERK) had significant lower phosphorylation in lenti-PTPRR group and higher expression in lenti-shPTPRR group, both without CMS. The Lenti-PTPRR mice exposed to CMS had significant lower p-ERK, and the lenti-shPTPRR mice exposed to CMS had significant higher p-ERK and lower p-P38. Moreover, there were more cells underwent apoptosis in lenti-PTPRR group ,with and without CMS. In cell proliferation, less BrdU positive cells were observed in lenti-PTPRR mice than controls. CONCLUSION: The site-specific lentiviral injections resulted in the PTPRR over-expression in the hippocampal DG and subsequent increased ERK dephosphorylation, which leads to more neuron apoptosis, less cell proliferation, depression onset and increased sensitivity to CMS. PTPRR/ERK pathway could be potential target for depression therapy.

Protein tyrosine phosphatase receptor type R is required for Purkinje cell responsiveness in cerebellar long-term depression.

BACKGROUND: Regulation of synaptic connectivity, including long-term depression (LTD), allows proper tuning of cellular signalling processes within brain circuitry. In the cerebellum, a key centre for motor coordination, a positive feedback loop that includes mitogen-activated protein kinases (MAPKs) is required for proper temporal control of LTD at cerebellar Purkinje cell synapses. Here we report that the tyrosine-specific MAPK-phosphatase PTPRR plays a role in coordinating the activity of this regulatory loop. RESULTS: LTD in the cerebellum of Ptprr (-/-) mice is strongly impeded, in vitro and in vivo. Comparison of basal phospho-MAPK levels between wild-type and PTPRR deficient cerebellar slices revealed increased levels in mutants. This high basal phospho-MAPK level attenuated further increases in phospho-MAPK during chemical induction of LTD, essentially disrupting the positive feedback loop and preventing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation and endocytosis. CONCLUSIONS: Our findings indicate an important role for PTPRR in maintaining low basal MAPK activity in Purkinje cells. This creates an optimal 'window' to boost MAPK activity following signals that induce LTD, which can then propagate through feed-forward signals to cause AMPAR internalization and LTD.

Androgen-regulation of the protein tyrosine phosphatase PTPRR activates ERK1/2 signalling in prostate cancer cells.

BACKGROUND: Androgens drive the onset and progression of prostate cancer (PCa) via androgen receptor (AR) signalling. The principal treatment for PCa is androgen deprivation therapy, although the majority of patients eventually develop a lethal castrate-resistant form of the disease, where despite low serum testosterone levels AR signalling persists. Advanced PCa often has hyper-activated RAS/ERK1/2 signalling thought to be due to loss of function of key negative regulators of the pathway, the details of which are not fully understood. METHODS: We recently carried out a genome-wide study and identified a subset of 226 novel androgen-regulated genes (PLOS ONE 6:e29088, 2011). In this study we have meta-analysed this dataset with genes and pathways frequently mutated in PCa to identify androgen-responsive regulators of the RAS/ERK1/2 pathway. RESULTS: We find the PTGER4 and TSPYL2 genes are up-regulated by androgen stimulation and the ADCY1, OPKR1, TRIB1, SPRY1 and PTPRR are down-regulated by androgens. Further characterisation of PTPRR protein in LNCaP cells revealed it is an early and direct target of the androgen receptor which negatively regulates the RAS/ERK1/2 pathway and reduces cell proliferation in response to androgens. CONCLUSION: Our data suggest that loss of PTPRR in clinical PCa is one factor that might contribute to activation of the RAS/ERK1/2 pathway.

Interaction of kinase-interaction-motif protein tyrosine phosphatases with the mitogen-activated protein kinase ERK2.

The mitogen-activation protein kinase ERK2 is tightly regulated by multiple phosphatases, including those of the kinase interaction motif (KIM) PTP family (STEP, PTPSL and HePTP). Here, we use small angle X-ray scattering (SAXS) and isothermal titration calorimetry (ITC) to show that the ERK2:STEP complex is compact and that residues outside the canonical KIM motif of STEP contribute to ERK2 binding. Furthermore, we analyzed the interaction of PTPSL with ERK2 showing that residues outside of the canonical KIM motif also contribute to ERK2 binding. The integration of this work with previous studies provides a quantitative and structural map of how the members of a single family of regulators, the KIM-PTPs, differentially interact with their corresponding MAPKs, ERK2 and p38α.

Immunohistochemical and Western blot analysis of two protein tyrosine phosphatase receptors, R and Z1, in colorectal carcinoma, colon adenoma and normal colon tissues.

Two classes of proteins, namely tyrosine kinases (PTK) and phosphatases (PTP), play an important role in cell proliferation and differentiation, thus leading to an acceleration or inhibition of tumour growth. The role of the above proteins in colorectal carcinoma (CRC) growth is a well-known event. In this study we carried out immunohistochemical and Western blot analysis of colorectal carcinoma, adenoma and normal colon tissue in relation to two protein tyrosine phosphatase receptors, R and Z1. Twenty-five cases of CRC were analyzed and the results were compared with similar data obtained in non-malignant tissues. High expression of both PTP receptors was observed in all examined cases of CRC, adenoma and normal colon tissue in this study. These results are not in line with recently published data, showing that genetic coding for PTPRR and PTPRZ1 were hypermethylated in CRC's. We presume that the protein tyrosine phosphatase overexpression in colorectal carcinoma is not enough to protect from the progression of disease.

PTPBR7 binding proteins in myelinating neurons of the mouse brain.

Mouse protein tyrosine phosphatase PTPBR7 is a receptor-like, transmembrane protein that is localized on the surface of neuronal cells. Its protein phosphatase activity is reduced upon multimerization, and PTPBR7-deficient mice display motor coordination defects. Extracellular molecules that may influence PTPBR7 activity, however, remain to be determined. We here show that the PTPBR7 extracellular domain binds to highly myelinated regions in mouse brain, in particular the white matter tracks in cerebellum. PTPBR7 deficiency does not alter this binding pattern, as witnessed by RAP in situ staining of Ptprr⁻/⁻ mouse brain sections. Additional in situ and in vitro experiments also suggest that sugar moieties of heparan sulphate and chondroitin sulphate glycosaminoglycans are not critical for PTPBR7 binding. Candidate binding proteins were affinity-purified exploiting the PTPBR7 extracellular domain and identified by mass spectrometric means. Results support the suggested link between PTPRR isoforms and cerebellar calcium ion homeostasis, and suggest an additional role in the process of cell-cell adhesion.

Identification of PTPN23 as a novel regulator of cell invasion in mammary epithelial cells from a loss-of-function screen of the 'PTP-ome'.

We used an RNAi-mediated loss-of-function screen to study systematically the role of the protein tyrosine phosphatase (PTP) superfamily of enzymes in mammary epithelial cell motility in the absence or presence of the oncoprotein tyrosine kinase ERBB2. We report that although shRNAs directed against most of the PTP family were without effect, suppression of three PTPs-PRPN23, PTPRG, and PTPRR-enhanced cell motility. Furthermore, we found that suppression of PTPN23, but not PTPRG or PTPRR, induced cell invasion. Suppression of PTPN23 increased E-cadherin internalization, impaired early endosome trafficking of E-cadherin, induced the expression of mesenchymal proteins, and caused cell scattering. The activity of SRC and ß-catenin was elevated when PTPN23 was suppressed. Moreover, we identified SRC, E-cadherin, and ß-catenin as direct substrates of PTPN23. Inhibition of SRC with the small molecular inhibitor SU6656 blocked the effects of PTPN23 depletion. These findings suggest that loss of PTPN23 may increase the activity of SRC and the phosphorylation status of the E-cadherin/ß-catenin signaling complex to promote tumor growth and invasive behavior in breast cancer. In addition, our studies highlight functional specificity among PTPs and reveal new roles for PTPs in mammary epithelial cell biology.

Interface analysis of the complex between ERK2 and PTP-SL.

The activity of ERK2, an essential component of MAP-kinase pathway, is under the strict control of various effector proteins. Despite numerous efforts, no crystal structure of ERK2 complexed with such partners has been obtained so far. PTP-SL is a major regulator of ERK2 activity. To investigate the ERK2-PTP-SL complex we used a combined method based on cross-linking, MALDI-TOF analysis, isothermal titration calorimetry, molecular modeling and docking. Hence, new insights into the stoichiometry, thermodynamics and interacting regions of the complex are obtained and a structural model of ERK2-PTP-SL complex in a state consistent with PTP-SL phosphatase activity is developed incorporating all the experimental constraints available at hand to date. According to this model, part of the N-terminal region of PTP-SL has propensity for intrinsic disorder and becomes structured within the complex with ERK2. The proposed model accounts for the structural basis of several experimental findings such as the complex-dissociating effect of ATP, or PTP-SL blocking effect on the ERK2 export to the nucleus. A general observation emerging from this model is that regions involved in substrate binding in PTP-SL and ERK2, respectively are interacting within the interface of the complex.

PTPRR protein tyrosine phosphatase isoforms and locomotion of vesicles and mice.

Protein tyrosine phosphatases (PTPs) are central players in many different cellular processes and their aberrant activity is associated with multiple human pathologies. In this review, we present current knowledge on the PTPRR subfamily of classical PTPs that is expressed in neuronal cells and comprises receptor-type (PTPBR7, PTP-SL) as well as cytosolic (PTPPBSgamma-37, PTPPBSgamma-42) isoforms. The two receptor-type isoforms PTPBR7 and PTP-SL both localize in late endosomes and the Golgi area. PTPBR7, however, is additionally localized at the cell surface and on early endosomes. During cerebellar maturation, PTPBR7 expression in developing Purkinje cells ceases and is replaced by PTP-SL expression in the mature Purkinje cells. All PTPRR isoforms contain a kinase interacting motif that makes them mitogen-activated protein kinase phosphatases. The distinct subcellular localization of the different PTPRR isoforms may reflect differential roles in growth-factor-induced MAPK-mediated retrograde signaling cascades. Studies in PTPRR-deficient mice established that PTPRR isoforms are physiological regulators of MAPK phosphorylation levels. Surprisingly, PTPRR-deficient mice display defects in motor coordination and balancing skills, while cerebellar morphological abnormalities, which are often encountered in ataxic mouse models, are absent. This is reminiscent of the phenotype observed in a handful of mouse mutants that have alterations in cerebellar calcium ion homeostasis. Elucidation of the molecular mechanisms by which PTPRR deficiency imposes impairment of cerebellar neurons and motor coordination may provide candidate molecules for hereditary cerebellar ataxias that still await identification of the corresponding disease genes.

Multimerisation of receptor-type protein tyrosine phosphatases PTPBR7 and PTP-SL attenuates enzymatic activity.

Dimerisation of receptor-type protein tyrosine phosphatases (RPTPs) represents an appealing mechanism to regulate their enzymatic activity. Studies thus far mostly concern the dimerisation behaviour of RPTPs possessing two tandemly oriented catalytic PTP domains. Mouse gene Ptprr encodes four different protein isoforms (i.e. PTPBR7, PTP-SL and PTPPBSgamma-42/37) that contain a single PTP domain. Using selective membrane permeabilisation we here demonstrate that PTP-SL, like PTPBR7, is a single membrane-spanning RPTP. Furthermore, these two receptor-type PTPs constitutively formed homo- and hetero-meric complexes as witnessed in chemical cross-linking and co-immunoprecipitation experiments, in sharp contrast to the cytosolic PTPPBSgamma-42 and PTPPBSgamma-37 PTPRR isoforms. This multimerisation occurs independently of the PTP domain and requires the transmembrane domain and/or the proximal hydrophobic region. Using overexpression of a PTPBR7 mutant that essentially lacks the intracellular PTP domain-containing segment, a monomer-mimicking state was forced upon full-length PTPBR7 immunoprecipitates. This resulted in a significant increase in the enzymatic activity of the PTPRR PTP domain, which strengthens the notion that multimerisation represents a general mechanism to tone down RPTP catalytic activity.