Expression Of Protein Tyrosine Phosphatase Research Articles

Abstract 6972: Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer

Abstract High expression of programmed death-ligand 1(PD-L1) is associated with poor survival outcomes in epidermal growth factor receptor (EGFR) mutation non-small cell lung cancer (NSCLC) patients receiving EGFR tyrosine kinase inhibitors (EGFR-TKIs). In this study we aimed to determine how increased PD-L1 affects the signal transduction of EGFR-mutant NSCLC cells. We first established PD-L1 overexpression EGFR mutant NSCLC cells, and discovered that PD-L1 overexpression cells had upregulated c-MET phosphorylation compared with parental cells using receptor tyrosine kinase array study. Subsequently, we demonstrated that knocking out PD-L1 (CD 274) in EGFR mutant NSCLC cells resulted in down-regulation of c-MET phosphorylation. Since protein tyrosine phosphatases (PTP) are known key regulators for c-MET phosphorylation and dephosphorylation, the activity of PTP was assessed. In PD-L1 overexpression EGFR mutant NSCLC cells, the PTP activity was decreased, whereas in PD-L1 knock-out cells, the PTP activity was increased. Additionally, the downregulation of c-MET phosphorylation in PD-L1 knock-out cells could be restored by adding PTP inhibitor (Na3VO4). The gene expression analysis showed that PTP expression was down-regulated in PD-L1 overexpression cells, indicating that PD-L1 upregulates c-MET phosphorylation by reducing PTP expression. We further established xenograft animal models using NSCLC cells with strong baseline c-MET phosphorylation to determine the effect of c-MET phosphorylation on osimertinib (a 3rd-generation EGFR-TKI) treatment. We found that tumor cells with strong c-MET phosphorylation were prone to develop MET amplification as acquired resistance under osimertinib, which could be overcome with combination of osimertinib and tepotinib, a c-MET inhibitor. Taken together, our results expand our understanding of PD-L1’s role beyond an immune checkpoint in EGFR mutant NSCLC. PD-L1-regulated MET alterations could be a crucial mechanism leading to poor treatment outcome of EGFR-TKIs in PD-L1 overexpression EGFR mutant NSCLC. Citation Format: Derek De-Rui Huang, Chia-Chi Hsu, Wei-Hsun Hsu, Bin-Chi Liao, James Chih-Hsin Yang. Programmed death-ligand 1 (PD-L1) upregulates c-MET phosphorylation via protein tyrosine phosphatase and contributes to the development of MET amplification in epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6972.

Fragment-Based Discovery of Allosteric Inhibitors of SH2 Domain-Containing Protein Tyrosine Phosphatase-2 (SHP2).

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

THE FEATURES AND ROLE OF SHP2 PROTEIN IN POSTNATAL MUSCLE DEVELOPMENT

SHP-2 (encoded by PTPN11) is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with carry germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities.Shp2 is an important signaling agent for growth factors and cytokines. To investigate the Ptpn11 in postnatal myogenesis of mice  we analyzed using immunohistochemistry stem cell markers: Pax 7, Myo D, Myf 5 and Myo G in muscle cells isolated from wild type and Shp2 knockout mice. We used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Our findings indicate that all these markers gradually decreased from birth to day 14 in mouse muscle cells. Here we show that Shp2, an intracellular tyrosine phosphatase with two SH2 domains, plays a critical role in mouse muscle development after birth. Our data demonstrate a molecular difference in the control and Sh2 knockout  postnatal myogenic stem cells, and assign to Ptpn11 signaling a key function in satellite cell activity. These findings illustrate an essential role for Shp-2 in muscle growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how Shp-2 regulates muscle morphogenesis, which could lead to the development of novel therapies for the treatment of malformations in human patients with SHP-2 mutations.

KMT2D deficiency drives lung squamous cell carcinoma and hypersensitivity to RTK-RAS inhibition.

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition.

PTPRO represses colorectal cancer tumorigenesis and progression by reprogramming fatty acid metabolism.

BackgroundAbnormal expression of protein tyrosine phosphatases (PTPs) has been reported to be a crucial cause of cancer. As a member of PTPs, protein tyrosine phosphatase receptor type O (PTPRO) has been revealed to play tumor suppressive roles in several cancers, while its roles in colorectal cancer (CRC) remains to be elucidated. Hence, we aimed to explore the roles and mechanisms of PTPRO in CRC initiation and progression.MethodsThe influences of PTPRO on the growth and liver metastasis of CRC cells and the expression patterns of different lipid metabolism enzymes were evaluated in vitro and in vivo. Molecular and biological experiments were conducted to uncover the underpinning mechanisms of dysregulated de novo lipogenesis and fatty acid β‐oxidation.ResultsPTPRO expression was notably downregulated in CRC liver metastasis compared to the primary cancer, and such a downregulation was associated with poor prognosis of patients with CRC. PTPRO silencing significantly promoted cell growth and liver metastasis. Compared with PTPRO wild‐type mice, PTPRO‐knockout mice developed more tumors and harbored larger tumor loads under treatment with azoxymethane and dextran sulfate sodium. Gene set enrichment analysis revealed that PTPRO downregulation was significantly associated with the fatty acid metabolism pathways. Blockage of fatty acid synthesis abrogated the effects of PTPRO silencing on cell growth and liver metastasis. Further experiments indicated that PTPRO silencing induced the activation of the AKT serine/threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling axis, thus promoting de novo lipogenesis by enhancing the expression of sterol regulatory element‐binding protein 1 (SREBP1) and its target lipogenic enzyme acetyl‐CoA carboxylase alpha (ACC1) by activating the AKT/mTOR signaling pathway. Furthermore, PTPRO attenuation decreased the fatty acid oxidation rate by repressing the expression of peroxisome proliferator‐activated receptor alpha (PPARα) and its downstream enzyme peroxisomal acyl‐coenzyme A oxidase 1 (ACOX1) via activating the p38/extracellular signal‐regulated kinase (ERK) mitogen‐activated protein kinase (MAPK) signaling pathway.ConclusionsPTPRO could suppress CRC development and metastasis via modulating the AKT/mTOR/SREBP1/ACC1 and MAPK/PPARα/ACOX1 pathways and reprogramming lipid metabolism.

Platelet-dependent signaling and Low Molecular Weight Protein Tyrosine Phosphatase expression promote aggressive phenotypic changes in gastrointestinal cancer cells

Over the last decades, some members of the protein tyrosine phosphatase family have emerged as cancer promoters. Among them, the Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) has been described to be associated with colorectal cancer liver metastasis and poor prostate cancer prognosis. Of importance in the process of cancer progression and metastasis is the interaction between tumor cells and platelets, as the latter are thought to promote several tumor hallmarks. Here, we examine to what extent LMWPTP expression in tumor cells affects their interaction with platelets. We demonstrate that the gene encoding LMWPTP is overexpressed in upper gastrointestinal (GI) cancer cell as well as colorectal cancer, and subsequently employ cell line models to show that the level of this phosphatase may be further augmented in the presence of platelets. We demonstrate that tumor-platelet interaction promotes GI tumor cell proliferation. Additionally, using know-down/-out models we show that LMWPTP expression in cancer cells contributes to a more efficient interaction with platelets and drives platelet-induced proliferation. These data are the first to demonstrate that phosphatases play a positive role in the tumor-promoting activities of platelets, with LMWPTP emerging as a key player promoting oncogenic phenotypic changes in tumor cells.

The tyrosine phosphatase SHP2 promotes proliferation and oxaliplatin resistance of colon cancer cells through AKT and ERK

The SH2 domain-containing phosphatase 2 (SHP2) is a widely expressed protein tyrosine phosphatase, and it is proposed to act as an oncogenic protein. SHP2 is also engaged in drug resistance of a variety of cancers. However, the role of SHP2 in the proliferation and drug resistance of colon cancer cells remains elusive. In this work we determined the effect of SHP2 expression on colon cancer cell proliferation and resistance to oxaliplatin (L-OHP), a commonly used drug in the clinic. Our results show that knockdown of SHP2 decreased and overexpression of SHP2 increased the proliferation of SW480 cells, respectively. Knockdown of SHP2 increased, and overexpression of SHP2 decreased apoptosis of the cells. We selected oxaliplatin-resistant SW480(SW480/L-OHP) and HCT116(HCT116/L-OHP) cells and found that the SHP2 protein level was raised in these drug-resistant cells. The upregulated SHP2 contributed to oxaliplatin resistance of the cells, as knockdown of SHP2 decreased the IC50 of oxaliplatin and abated proliferation and survival of SW480/L-OHP and HCT116/L-OHP cells in the presence of oxaliplatin. Also, SW480/L-OHP and HCT116/L-OHP cells had increased phosphorylation of AKT and ERK. Inhibition of AKT, ERK, or SHP2 sensitized SW480/L-OHP and HCT116/L-OHP cells to oxaliplatin. Our results indicate that SHP2 contributes oxaliplatin resistance through AKT and ERK. These results also suggest that SHP2-targeting is a potential strategy for overcoming oxaliplatin resistance of colon cancer cells.

PTPN4 germline variants result in aberrant neurodevelopment and growth.

SummaryProtein-tyrosine phosphatases (PTPs) are pleomorphic regulators of eukaryotic cellular responses to extracellular signals that function by modulating the phosphotyrosine of specific proteins. A handful of PTPs have been implicated in germline and somatic human disease. Using exome sequencing, we identified missense and truncating variants in PTPN4 in six unrelated individuals with varying degrees of intellectual disability or developmental delay. The variants occurred de novo in all five subjects in whom segregation analysis was possible. Recurring features include postnatal growth deficiency or excess, seizures, and, less commonly, structural CNS, heart, or skeletal anomalies. PTPN4 is a widely expressed protein tyrosine phosphatase that regulates neuronal cell homeostasis by protecting neurons against apoptosis. We suggest that pathogenic variants in PTPN4 confer risk for growth and cognitive abnormalities in humans.

Grb2 binding induces phosphorylation-independent activation of Shp2

The regulation of phosphatase activity is fundamental to the control of intracellular signalling and in particular the tyrosine kinase-mediated mitogen-activated protein kinase (MAPK) pathway. Shp2 is a ubiquitously expressed protein tyrosine phosphatase and its kinase-induced hyperactivity is associated with many cancer types. In non-stimulated cells we find that binding of the adaptor protein Grb2, in its monomeric state, initiates Shp2 activity independent of phosphatase phosphorylation. Grb2 forms a bidentate interaction with both the N-terminal SH2 and the catalytic domains of Shp2, releasing the phosphatase from its auto-inhibited conformation. Grb2 typically exists as a dimer in the cytoplasm. However, its monomeric state prevails under basal conditions when it is expressed at low concentration, or when it is constitutively phosphorylated on a specific tyrosine residue (Y160). Thus, Grb2 can activate Shp2 and downstream signal transduction, in the absence of extracellular growth factor stimulation or kinase-activating mutations, in response to defined cellular conditions. Therefore, direct binding of Grb2 activates Shp2 phosphatase in the absence of receptor tyrosine kinase up-regulation.

Expression of protein tyrosine phosphatases and Bombyx embryonic development

Protein phosphorylation is an integral component of signal transduction pathways within eukaryotic cells, and it is regulated by coordinated interactions between protein kinases and protein phosphatases. Our previous study demonstrated differential expressions of serine/threonine protein phosphatases (PP2A and calcineurin) between diapause and developing eggs in Bombyx mori. In the present study, we further investigated expression of protein tyrosine phosphatases (PTPs) in relation to the Bombyx embryonic development. An immunoblot analysis showed that eggs contained the proteins of the 51-kDa PTP 1B (PTP1B), the 55-kDa phosphatase and tensin homologue (PTEN), and the 70-kDa Src homology 2 (SH2) domain-containing phosphatase 2 (SHP2), which undergo differential changes between diapause and developing eggs. Protein level of PTP1B and PTEN in eggs whose diapause initiation was prevented by HCl gradually increased toward embryonic development. The protein level of SHP2 also showed a dramatic increase on days 7 and 8 after HCl treatment. However, protein levels of PTP1B, PTEN, and SHP2 in diapause eggs remained at low levels during the first 9 days after oviposition. These differential changing patterns in protein levels were further confirmed using both non-diapause eggs and eggs in which diapause had been terminated by chilling of diapausing eggs at 5 °C for 70 days and then were transferred to 25 °C. Direct determination of PTP enzymatic activities showed higher activities in developing eggs (HCl-treated eggs, non-diapause eggs, and chilled eggs) compared to those in diapause eggs. Examination of temporal changes in mRNA expression levels of PTP1B, PTEN, and SHP2 did not show significant differences between diapause eggs and HCl-treated eggs except high expression in SHP2 variant B during the later embryonic development in HCl-treated eggs. These results demonstrate that higher protein levels of PTP1B, PTEN, and SHP2 and increased tyrosine phosphatase enzymatic activities in developing eggs are likely related to embryonic development of B. mori.

Growth Factor Deregulation and Emerging Role of Phosphatases in Diabetic Peripheral Artery Disease.

Peripheral artery disease is caused by atherosclerosis of lower extremity arteries leading to the loss of blood perfusion and subsequent critical ischemia. The presence of diabetes mellitus is an important risk factor that greatly increases the incidence, the progression and the severity of the disease. In addition to accelerated disease progression, diabetic patients are also more susceptible to develop serious impairment of their walking abilities through an increased risk of lower limb amputation. Hyperglycemia is known to alter the physiological development of collateral arteries in response to ischemia. Deregulation in the production of several critical pro-angiogenic factors has been reported in diabetes along with vascular cell unresponsiveness in initiating angiogenic processes. Among the multiple molecular mechanisms involved in the angiogenic response, protein tyrosine phosphatases are potent regulators by dephosphorylating pro-angiogenic tyrosine kinase receptors. However, evidence has indicated that diabetes-induced deregulation of phosphatases contributes to the progression of several micro and macrovascular complications. This review provides an overview of growth factor alterations in the context of diabetes and peripheral artery disease, as well as a description of the role of phosphatases in the regulation of angiogenic pathways followed by an analysis of the effects of hyperglycemia on the modulation of protein tyrosine phosphatase expression and activity. Knowledge of the role of phosphatases in diabetic peripheral artery disease will help the development of future therapeutics to locally regulate phosphatases and improve angiogenesis.

Abstract 1039: Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2

Abstract The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Recent advances have shown that genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signalling and inhibits proliferation of RTK-driven cancer cell lines. SHP2 is now understood to act upstream of RAS and plays a role in KRAS-driven cancers, an area of research which is rapidly growing. Considering that RTK deregulation often leads to a wide range of cancers and the newly appreciated role of SHP2 in KRAS-driven cancers, SHP2 inhibitors are therefore a promising therapeutic approach. SHP2 contains two N-terminal tandem SH2 domains (N-SH2, C-SH2), a catalytic phosphatase domain and a C-terminal tail. SHP2 switches between “open” active and “closed” inactive forms due to autoinhibitory interactions between the N-SH2 domain and the phosphatase domain. Historically, phosphatases were deemed undruggable as there had been no advancements with active site inhibitors. We hypothesised that fragment screening would be highly applicable and amenable to this target to enable alternative means of inhibition through identification of allosteric binding sites. Here we describe the first reported fragment screen against SHP2. Using our fragment-based PyramidTM approach, screening was carried out on two constructs of SHP2; a closed autoinhibited C-terminal truncated form (phosphatase and both SH2 domains), as well as the phosphatase-only domain. A combination of screening methods such as X-ray crystallography and NMR were employed to identify fragment hits at multiple sites on SHP2, including the tunnel-like allosteric site reported by Chen et al, 2016. Initial fragment hits had affinities for SHP2 in the range of 1mM as measured by ITC. Binding of these hits was improved using structure-guided design to generate compounds which inhibit SHP2 phosphatase activity and are promising starting points for further optimization. Citation Format: Philip J. Day, Valerio Berdini, Juan Castro, Gianni Chessari, Thomas G. Davies, James E. Day, Satoshi Fukaya, Chris Hamlett, Keisha Hearn, Steve Hiscock, Rhian Holvey, Satoru Ito, Yasuo Kodama, Kenichi Matsuo, Yoko Nakatsuru, Nick Palmer, Amanda Price, Tadashi Shimamura, Jeffrey D.St. Denis, Nicola G. Wallis, Glyn Williams, Christopher N. Johnson. Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1039.

Progress in the correlation between PTPN12 gene expression and human tumors.

Background:The global morbidity of cancer is rising rapidly. Despite advances in molecular biology, immunology, and cytotoxic and immune-anticancer therapies, cancer remains a major cause of death worldwide. Protein tyrosine phosphatase non-receptor type 12 (PTPN12) is a new member of the cytoplasmic protein tyrosine phosphatase family, isolated from a cDNA library of adult colon tissue. Thus far, no studies have reviewed the correlation between PTPN12 gene expression and human tumors.Methods:This article summarizes the latest domestic and international research developments on how the expression of PTPN12 relates to human tumors. The extensive search in Web of Science and PubMed with the keywords including PTPN12, tumor, renal cell carcinoma, proto-oncogenes, tumor suppressor genes was undertaken.Results:More and more studies have shown that a tumor is essentially a genetic disease, arising from a broken antagonistic function between proto-oncogenes and tumor suppressor genes. When their antagonistic effect is out of balance, it may cause uncontrolled growth of cells and lead to the occurrence of tumors. PTPN12 is a tumor suppressor gene, so inhibiting its activity will lead directly or indirectly to the occurrence of tumors.Conclusion:The etiology, prevention, and treatment of tumors have become the focus of research around the world. PTPN12 is a tumor suppressor gene. In the future, PTPN12 might serve as a novel molecular marker to benefit patients, and even the development of tumor suppressor gene activation agents can form a practical research direction.

IL-6 promotes PD-L1 expression in monocytes and macrophages by decreasing protein tyrosine phosphatase receptor type O expression in human hepatocellular carcinoma.

BackgroundWe have previously discovered a relationship between the low expression of protein tyrosine phosphatase, receptor type O (PTPRO) in tumor-infiltrating T cells and immunosuppression. The aim of the present study was to investigate the relationship between decreased PTPRO and increased programmed death ligand 1 (PD-L1) in both the peripheral monocytes and tumor-infiltrating macrophages of human hepatocellular carcinoma (HCC).MethodsThe expression and correlation of all the indices were explored in monocytes and tumor-infiltrating macrophages within both human and mice HCC. The mechanic regulations were studied by using both in vitro and in vivo studies.ResultsWe found a significant decrease in PTPRO in HCC peripheral monocytes that was associated with increased PD-L1 expression in peripheral monocytes and tumor-associated macrophages (TAMs) in HCC. Monocyte PD-L1 and PTPRO therefore could serve as valuable prognostic indicators for post-surgery patients with HCC and were associated with increased T-cell exhaustion (Tim3+T cells). A depletion of PTPRO promoted PD-L1 secretion in both monocytes and macrophages through the JAK2/STAT1 and JAK2/STAT3/c-MYC pathways. Increased IL-6 expression was associated with activation of JAK2/STAT3/c-MYC and with decreased PTPRO expression through the STAT3/c-MYC/miR-25–3 p axis. Monocytes and TAMs showed significantly increased miR-25–3 p expression, which could target the 3′ untranslated region of PTPRO. The miR-25–3 p expression positively correlated with serum IL-6 levels, but inversely correlated with PTPRO in HCC monocytes. IL-6/STAT3/c-MYC activation enhanced in vitro miR-25–3 p transcription and decreased PTPRO, while further promoting PD-L1 secretion. Adoptive cell transfer of c-MYC/miR-25–3 p–modified monocytes promoted tumor growth by downregulating PTPRO and causing a PD-L1–induced immunosuppression in an orthotopic tumor transplantation model.ConclusionsIncreased serum IL-6 downregulated PTPRO expression in HCC monocytes and macrophages by activating STAT3/c-MYC/miR-25–3 p and by further enhancing PD-L1 expression through JAK2/STAT1 and JAK2/STAT3/c-MYC signaling.

IL-6 influences PD-L1 expression in monocytes and macrophages by effecting protein tyrosine phosphatase receptor type O expression in-human breast cancer.

e15570 Background: We have previously discovered a relationship between the low expression of protein tyrosine phosphatase, receptor type O (PTPRO) in tumor-infiltrating T cells and immunosuppression. The aim of the present study was to investigate the relationship between decreased PTPRO and increased programmed death ligand 1 (PD-L1) in both the peripheral monocytes and tumor-infiltrating macrophages of human breast cancer (BC). Methods: The expression and correlation of all the indices were explored in monocytes and tumor infiltrating macrophages within both human and mice BC. The mechanic regulations were studied by using both in vitro and in vivo studies. Results: We found a significant decrease in PTPRO in BC peripheral monocytes that was associated with increased PD-L1 expression in peripheral monocytes and tumor-associated macrophages (TAMs) in BC. Monocyte PD-L1 and PTPRO therefore could serve as valuable prognostic indicators for post-surgery patients with BC and were associated with increased T cell exhaustion (Tim3+ T cells). A depletion of PTPRO promoted PD-L1 secretion in both monocytes and macrophages through the JAK2/STAT1 and JAK2/STAT3/c-MYC pathways. Increased IL-6 expression was associated with activation of JAK2/STAT3/c-MYC and with decreased PTPRO expression through the STAT3/c-MYC/miR-25-3p axis. Monocytes and TAMs showed significantly increased miR-25-3p expression, which could target the 3' untranslated region of PTPRO. The miR-25-3p expression positively correlated with serum IL-6 levels, but inversely correlated with PTPRO in BC monocytes. IL-6/STAT3/c-MYC activation enhanced in vitro miR-25-3p transcription and decreased PTPRO, while further promoting PD-L1 secretion. Adoptive cell transfer of c-MYC/miR-25-3p–modified monocytes promoted tumor growth by downregulating PTPRO and causing a PD-L1–induced immunosuppression in breast tumor model. Conclusions: Increased serum IL-6 downregulated PTPRO expression in BC monocytes and macrophages by activating STAT3/c-MYC/miR-25-3p and by further enhancing PD-L1 expression through JAK2/STAT1 and JAK2/STAT3/c-MYC signaling.

Identification and Characterization of Post-activated B Cells in Systemic Autoimmune Diseases.

Autoimmune diseases (AID) such as systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS), and rheumatoid arthritis (RA) are chronic inflammatory diseases in which abnormalities of B cell function play a central role. Although it is widely accepted that autoimmune B cells are hyperactive in vivo, a full understanding of their functional status in AID has not been delineated. Here, we present a detailed analysis of the functional capabilities of AID B cells and dissect the mechanisms underlying altered B cell function. Upon BCR activation, decreased spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk) phosphorylation was noted in AID memory B cells combined with constitutive co-localization of CD22 and protein tyrosine phosphatase (PTP) non-receptor type 6 (SHP-1) along with hyporesponsiveness to TLR9 signaling, a Syk-dependent response. Similar BCR hyporesponsiveness was also noted specifically in SLE CD27− B cells together with increased PTP activities and increased transcripts for PTPN2, PTPN11, PTPN22, PTPRC, and PTPRO in SLE B cells. Additional studies revealed that repetitive BCR stimulation of normal B cells can induce BCR hyporesponsiveness and that tissue-resident memory B cells from AID patients also exhibited decreased responsiveness immediately ex vivo, suggesting that the hyporesponsive status can be acquired by repeated exposure to autoantigen(s) in vivo. Functional studies to overcome B cell hyporesponsiveness revealed that CD40 co-stimulation increased BCR signaling, induced proliferation, and downregulated PTP expression (PTPN2, PTPN22, and receptor-type PTPs). The data support the conclusion that hyporesponsiveness of AID and especially SLE B cells results from chronic in vivo stimulation through the BCR without T cell help mediated by CD40–CD154 interaction and is manifested by decreased phosphorylation of BCR-related proximal signaling molecules and increased PTPs. The hyporesponsiveness of AID B cells is similar to a form of functional anergy.

HePTP promotes migration and invasion in triple-negative breast cancer cells via activation of Wnt/β-catenin signaling

AimCancer metastasis remains a major challenge for the clinical management of breast cancer, especially triple-negative breast cancer (TNBC), and the underlying molecular mechanisms remain largely unknown. The aim of this study was to explore the mechanism of TNBC metastasis. Main methodsThe expression of protein tyrosine phosphatase, non-receptor type 7 (HePTP) was detected using real time-PCR, western blot. Wound healing assay and transwell matrix assay were used to evaluate the pro-migration and pro-invasion potential of HePTP in vitro. Luciferase activity assay and nuclear extract analysis were used to evaluate Wnt/β-catenin signaling activity. Key findingsWe reported that HePTP was overexpressed in TNBC, where it acted to drive migration and invasion of tumor cells. We showed that knockdown of HePTP significantly suppressed metastatic capacity of TNBC cells. Moreover, HePTP promoted cells migration and invasion by dephosphorylating glycogen synthase kinase 3 beta (GSK3β), thereby activating Wnt/β-catenin signaling. Additionally, we demonstrated that overexpression of HePTP in HePTP lowly expressed cells could effectively promote the migration and invasion of breast cancer cells. SignificanceOur results suggest that HePTP plays a key role in the metastasis of TNBC via activating Wnt/β-catenin signaling. Hence, we propose that HePTP may serve as a novel prognostic marker and a potential therapeutic target for the treatment of TNBC.

Sanguinarine Induces Apoptosis Pathway in Multiple Myeloma Cell Lines via Inhibition of the JaK2/STAT3 Signaling.

Sanguinarine (SNG), a benzophenanthridine alkaloid, has displayed various anticancer abilities in several vivo and in vitro studies. However, the anticancer potential of SNG is yet to be established in multiple myeloma (MM), a mostly incurable malignancy of plasma cells. In this study, we aimed to investigate the potential anti-proliferative and pro-apoptotic activities of SNG in a panel of MM cell lines (U266, IM9, MM1S, and RPMI-8226). SNG treatment of MM cells resulted in a dose-dependent decrease in cell viability through mitochondrial membrane potential loss and activation of caspase 3, 9, and cleavage of PARP. Pre-treatment of MM cells with a universal caspase inhibitor, Z-VAD-FMK, prevented SNG mediated loss of cell viability, apoptosis, and caspase activation, confirming that SNG-mediated apoptosis is caspase-dependent. The SNG-mediated apoptosis appears to be resulted from suppression of the constitutively active STAT3 with a concomitant increase in expression of protein tyrosine phosphatase (SHP-1). SNG treatment of MM cells leads to down-regulation of the anti-apoptotic proteins including cyclin D, Bcl-2, Bclxl, and XIAP. In addition, it also upregulates pro-apoptotic protein, Bax. SNG mediated cellular DNA damage in MM cell lines by induction of oxidative stress through the generation of reactive oxygen species and depletion of glutathione. Finally, the subtoxic concentration of SNG enhanced the cytotoxic effects of anticancer drugs bortezomib (BTZ) by suppressing the viability of MM cells via induction of caspase-mediated apoptosis. Altogether our findings demonstrate that SNG induces mitochondrial and caspase-dependent apoptosis, generates oxidative stress, and suppresses MM cell lines proliferation. In addition, co-treatment of MM cell lines with sub-toxic doses of SNG and BTZ potentiated the cytotoxic activity. These results would suggest that SNG could be developed into therapeutic agent either alone or in combination with other anticancer drugs in MM.

SHP2 regulates intramembranous ossification by modifying the TGFβ and BMP2 signaling pathway.

SHP2 is a ubiquitously expressed protein tyrosine phosphatase, which is involved in many signaling pathways to regulate the skeletal development. In endochondral ossification, SHP2 is known to modify the osteogenic fate of osteochondroprogenitors and to impair the osteoblastic transdifferentiation of hypertrophic chondrocytes. However, how SHP2 regulates osteoblast differentiation in intramembranous ossification remains incompletely understood. To address this question, we generated a mouse model to ablate SHP2 in the Prrx1-expressing mesenchymal progenitors by using "Cre-loxP"-mediated gene excision and examined the development of calvarial bone, in which the main process of bone formation is intramembranous ossification. Phenotypic characterization showed that SHP2 mutants have severe defects in calvarial bone formation. Cell lineage tracing and in situ hybridization data showed less osteoblast differentiation of mesenchymal cells and reduced osteogenic genes expression, respectively. Further mechanistic studies revealed enhanced TGFβ and suppressed BMP2 signaling in SHP2 ablated mesenchymal progenitors and their derivatives. Our study uncovered the critical role of SHP2 in osteoblast differentiation through intramembranous ossification and might provide a potential target to treat craniofacial skeleton disorders.

Prediction of Time to Castration-Resistant Prostate Cancer Using Low-Molecular-Weight Protein Tyrosine Phosphatase Expression for Men with Metastatic Hormone-Naïve Prostate Cancer

Introduction: Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) expression affects carcinogenesis in various cancers and has been associated with determining the overall survival among men with metastatic hormone-naïve prostate cancer (mHNPC). In this study, we analyzed the value of LMWPTP for prediction of time to castration-resistant prostate cancer (CRPC) for men with mHNPC. Materials and Methods: We retrospectively enrolled 45 men with mHNPC who were diagnosed from 2003 to 2009. All patients had received androgen deprivation therapy as first-line treatment. Prostate cancer tissues (pre-treatment needle biopsies) were immunohistochemically stained for LMW-PTP. Multivariate analyses (Cox proportional hazard model) were used to correlate baseline clinical factors of age, prostate-specific antigen (PSA), Gleason scores, T stage, N stage, extent of disease on bone scan (EOD), LMW-PTP expression and time to CRPC. Continuous variables were classified as dichotomous. Results: Median age and PSA were 70.0 years and 87.8 ng/mL respectively. Median time to CRPC was 40.2 months. Median time to CRPC was significantly shorter in the high LMW-PTP group (14.8 months) than that in the low LMW-PTP group (86.3 months, p < 0.01). In multivariate analysis, age ≥70 years and high LMW-PTP expression were significant predictors of time to CRPC.