WTAP promotes proliferation of esophageal squamous cell carcinoma via m6A-dependent epigenetic promoting of PTP4A1.

Esophageal squamous cell carcinoma (ESCC) represents a frequently seen malignancy with high prevalence worldwide. Although current studies have shown that Wilms' tumor 1-associated protein (WTAP), a major part in the methyltransferase complex, is involved in various tumor pathological processes, its specific role in ESCC remains unclear. Therefore, the present work focused on exploring WTAP's function and mechanism in ESCC progression using clinical ESCC specimens, ESCC cells, and mammalian models. Firstly, we proved WTAP was significantly upregulated within ESCC, and WTAP mRNA expression showed a good diagnostic performance for ESCC. Functionally, WTAP positively regulated in-vivo and in-vitro ESCC cells' malignant phenotype through the AKT-mTOR signaling pathway. Meanwhile, WTAP positively regulated the N6-methyladenosine (m6A) modification levels in ESCC cells. Protein tyrosine phase type IVA member 1 (PTP4A1) was confirmed to be the m6A target of WTAP, and WTAP positively regulated the expression of PTP4A1. Further study revealed that PTP4A1 showed high expression within ESCC. Silencing PTP4A1 inhibited the AKT-mTOR signaling pathway to suppress ESCC cells' proliferation. Rescue experiments showed that silencing PTP4A1 partially reversed the WTAP-promoting effect on ESCC cells' proliferation ability. Mechanistically, WTAP regulated PTP4A1 expression to activate the AKT-mTOR pathway, promoting the proliferation of ESCC cells. Our study demonstrated that WTAP regulates the progression of ESCC through the m6A-PTP4A1-AKT-mTOR signaling axis and that WTAP is a potential target for diagnosing and treating ESCC.

Preparation of Oxidized and Reduced PTP4A1 for Structural and Functional Studies.

The formation of a reversible disulfide bond between the catalytic cysteine and a spatially neighboring cysteine (backdoor) in protein tyrosine phosphatases (PTPs) serves as a critical regulatory mechanism for maintaining the activity of protein tyrosine phosphatases. The failure of such protection results in the formation of irreversibly oxidized cysteines into sulfonic acid in a highly oxidative cellular environment in the presence of free radicals. Hence, it is important to develop methods to interconvert PTPs into reduced and oxidized forms to understand their catalytic function in vitro. Protein tyrosine phosphatase 4A type 1 (PTP4A1), a dual-specificity phosphatase, is catalytically active in the reduced form. Unexpectedly, also its oxidized form performs a key biological function in systemic sclerosis (SSc) by forming a kinase-phosphatase complex with Src kinases. Thus, we developed simple and efficient protocols for producing oxidized and reduced PTP4A1 to elucidate their biological function, which can be extended to study other protein tyrosine phosphatases and other recombinantly produced proteins.

Hepatic PTP4A1 ameliorates high-fat diet-induced hepatosteatosis and hyperglycemia by the activation of the CREBH/FGF21 axis.

Precise regulation of kinases and phosphatases is crucial for human metabolic homeostasis. This study aimed to investigate the roles and molecular mechanisms of protein tyrosine phosphatase type IVA1 (PTP4A1) in regulating hepatosteatosis and glucose homeostasis. Method: Ptp4a1-/- mice, adeno-associated virus encoding Ptp4a1 under liver-specific promoter, adenovirus encoding Fgf21, and primary hepatocytes were used to evaluate PTP4A1-mediated regulation in the hepatosteatosis and glucose homeostasis. Glucose tolerance test, insulin tolerance test, 2-deoxyglucose uptake assay, and hyperinsulinemic-euglycemic clamp were performed to estimate glucose homeostasis in mice. The staining, including oil red O, hematoxylin & eosin, and BODIPY, and biochemical analysis for hepatic triglycerides were performed to assess hepatic lipids. Luciferase reporter assays, immunoprecipitation, immunoblots, quantitative real-time polymerase chain reaction, and immunohistochemistry staining were conducted to explore the underlying mechanism. Results: Here, we found that deficiency of PTP4A1 aggravated glucose homeostasis and hepatosteatosis in mice fed a high-fat (HF) diet. Increased lipid accumulation in hepatocytes of Ptp4a1-/- mice reduced the level of glucose transporter 2 on the plasma membrane of hepatocytes leading to a diminution of glucose uptake. PTP4A1 prevented hepatosteatosis by activating the transcription factor cyclic adenosine monophosphate-responsive element-binding protein H (CREBH)/fibroblast growth factor 21 (FGF21) axis. Liver-specific PTP4A1 or systemic FGF21 overexpression in Ptp4a1-/- mice fed an HF diet restored the disorder of hepatosteatosis and glucose homeostasis. Finally, liver-specific PTP4A1 expression ameliorated an HF diet-induced hepatosteatosis and hyperglycemia in wild-type mice. Conclusions: Hepatic PTP4A1 is critical for regulating hepatosteatosis and glucose homeostasis by activating the CREBH/FGF21 axis. Our current study provides a novel function of PTP4A1 in metabolic disorders; hence, modulating PTP4A1 may be a potential therapeutic strategy against hepatosteatosis-related diseases.

Long noncoding RNA DLEU2 promotes growth and invasion of hepatocellular carcinoma by regulating miR-30a-5p/PTP4A1 axis.

Increasing data indicate that long noncoding RNA (lncRNA) DLEU2 is implicated in carcinogenesis in multiple malignancies including hepatocellular carcinoma (HCC). However, the role and molecular mechanism by which lncRNA DLEU2 contributes to HCC remain unknown. The association of lncRNA DLEU2 with clinicopathological characteristics and prognosis in patients with HCC was analyzed by qRT-PCR, and public TCGA dataset. CCK-8, colony formation and Transwell assays were performed to verify the role of lncRNA DLEU2 in HCC. RNA immunoprecipitation (RIP), luciferase gene report and qRT-PCR assays were employed to uncover lncRNA DLEU2-spevific binding with miR-30a-5p. The effect of lncRNA DLEU2 and (or) miR-30a-5p on PTP4A1 expression was examined by Western blot analysis. As a consequence, we found that lncRNA DLEU2 was upregulated in HCC tissue samples and associated with distant metastasis and poor survival in patients with HCC. Knockdown of lncRNA DLEU2 impaired HCC cell proliferation, colony formation and invasion, but ectopic expression of lncRNA DLEU2 abolished these effects. Furthermore, lncRNA DLEU2 harbored a negative correlation and specific binding with miR-30a-5p in HCC cells. Knockdown of lncRNA DLEU2 upregulated miR-30a-5p, but downregulated its target PTP4A1, and miR-30a-5p abrogated lncRNA DLEU2-induced tumor-promoting effects and PTP4A1 upregulation. Taken together, our findings demonstrate that lncRNA DLEU2 promotes growth and invasion of HCC cells by regulating miR-30a-5p/ PTP4A1 axis.

RP5-1148A21.3 (lncRP5) exerts oncogenic function in human ovarian carcinoma.

Ovarian cancer (OC) is a fatal gynecological malignancy that is difficult to diagnose at early stages. Various long non-coding RNAs (lncRNAs) are aberrantly expressed in OC and exert regulatory effects on OC; however, the underlying mechanism requires in-depth investigation. This work is designed to explore the molecular regulatory axis of a newly identified lncRNA in OC, that is, lncRNA RP5-1148A21.3 (lncRP5). RT-qPCR shows lncRP5 is significantly upregulated in OC patients and cell lines, and it is mainly located in the cytoplasm of OC cells. The results of CCK-8, colony formation, and transwell assays demonstrate that overexpression of lncRP5 greatly contributes to malignant behaviors of OC cells, while inhibition of lncRP5 shows the opposite effects. Moreover, the binding relationship between lncRP5 and miR-545-5p is predicted by bioinformatics and is further verified by luciferase assay. Functionally, the regulatory effects of lncRP5 and miR-545-3p are negatively related; miR-545-5p serves as a tumor suppressor in OC. Further studies demonstrate that PTP4A1 is the target gene of miR-545-5p. Overexpression of PTP4A1 abrogates the inhibitory function of miR-545-5p on OC cell growth and metastasis. The lncRP5/miR-545-5p/PTP4A1 axis is subsequently demonstrated in vivo, and knockdown of lncRP5 notably inhibits tumor growth. This study provides a novel regulatory mechanism of OC, which may contribute to the diagnosis and therapy of OC.

ERα inhibits mesenchymal and amoeboidal movement of liver cancer cell via Gα12.

Hepatocellular carcinoma (HCC) is the second most common cancer worldwide, demonstrating aggressiveness and mortality more frequently in men than in women. Despite reports regarding the inhibitory ability of estrogen receptor alpha (ERα, ESR1) in certain cancer progression, targets and the basis of underlying gender disparity in HCC worsening remain elusive. Here, we report the ability of ERα to transcriptionally inhibit G protein subunit alpha 12 (Gα12) responsible for HCC worsening. First, using human samples and public database, the expression of ERα and Gα12 in HCC was examined. Then, quantitative real-time PCR, chromatin immunoprecipitation-assay, luciferase assay and immunoblottings of liver cancer cell lines confirmed the inhibitory ability of ERα on Gα12 and HCC progression. Gα12 promoted mesenchymal characteristics and amoeboidal movement, which was antagonized by ERα overexpression. Additionally, we found microRNA-141 and microRNA-200a as downstream targets of the Gα12 signaling axis for cancer malignancy regulation under the control of ERα. As for in-depth mechanism, PTP4A1 was found to be directly inhibited by microRNA-141 and microRNA-200a. Moreover, we found the inhibitory effect of ERα on amoeboidal movement by analyzing the morphology and blebbing of liver cancer cells and the active form of MLC levels. The identified targets and ESR1 levels are inversely correlated with human specimens, as well as with sex-biased survival rates of HCC patients. Collectively, ERα-dependent repression of Gα12 and consequent changes in the Gα12 signaling may explain the gender disparity in HCC, providing pharmacological clues for the control of metastatic HCC.

LncRNA NEAT1 promotes cell proliferation, migration, and invasion via the miR-186-5p/PTP4A1 axis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a highly aggressive and malignant tumor. In this study, the effect and molecular mechanism of nuclear enriched abundant transcript 1 (NEAT1) in CCA were elucidated. The expressions of NEAT1, microRNA-186-5p (miR-186-5p), and PTP4A1 were measured by quantitative real-time PCR. The protein levels were measured by Western blotting. Kaplan-Meier analysis was performed to create survival curves. The interactions between NEAT1, miR-186-5p, and PTP4A1 were assessed through the dual luciferase reporter assay. Additionally, the cell proliferation, apoptosis, migration, and invasion were measured by colony formation, flow cytometry, the Transwell assay, and the wound healing assay, respectively. NEAT1 and PTP4A1 were significantly upregulated in CCA tissues and cells, but miR-186-5p was downregulated. NEAT1 expression was negatively correlated with the survival of CCA patients and has remarkable correlation with serum CA199 levels and lymph node metastasis. Besides, NEAT1 could act as a molecular sponge for miR-186-5p to upregulate PTP4A1 expression. More importantly, the knockdown of NEAT1 or overexpression of miR-186-5p inhibited the proliferation, migration and invasion of CCA cells, and the inhibition of miR-186-5p reversed the effects of the knockdown of NEAT1. In addition, NEAT1 could also activate the PI3K/AKT signaling pathway and regulate the epithelial-mesenchymal transition (EMT) through the miR-186-5p/PTP4A1 axis. In conclusion, NEAT1 was involved in cell proliferation, migration and invasion in CCA, and the NEAT1/miR-186-5p/PTP4A1/PI3K/AKT axis indicated novel regulatory mechanisms and therapeutics for the treatment of CCA.

Exploring the cause of the inhibitor 4AX attaching to binding site disrupting protein tyrosine phosphatase 4A1 trimerization by molecular dynamic simulation.

Ectopic overexpression of protein tyrosine phosphatase of liver regeneration-1 (PTP4A1, also called PRL-1) markedly enhanced hepatocellular carcinoma (HCC) cells migration and invasion. The PTP4A1 trimerization played a vital role in mediating cell proliferation and motility. Biochemical and structural studies have proved that the compound 4AX, a well-known inhibitor for PRL1, directly binds to the PTP4A1 trimer interface and obstructs trimer formation of PTP4A1. However, the molecular basis of the ligand-4AX inhibition on PTP4A1 trimer conformations remains unclear. In this study, the docking analysis and the molecular dynamics simulation (MD simulation) study were performed to investigate how the molecule binding at each interface disrupted the trimer formation. The results suggested that the ligand-4AX attaching to the binding site changed the conformation of A:Q131, A:Q135 in the AC interface, C:R18, C:P96 in the CA interface and B:Q131 in the BA interface, leading to the weak interactions between subunits and thus resulting in the disruption of the PTP4A1 trimerization.

lncRNA GCAWKR Promotes Gastric Cancer Development by Scaffolding the Chromatin Modification Factors WDR5 and KAT2A.

Long noncoding RNAs (lncRNAs) have been demonstrated to play a role in carcinogenesis, but their mechanisms of function remain elusive. We explored the mechanisms of the oncogenic role of GCAWKR in gastric cancer (GC) using human tissues and cell lines. The in situ hybridization analysis was utilized to determine GCAWKR levels in samples from 42 GC patients and real-time qPCR in tissues from 123 patients. The GCAWKR levels were modulated in GC cell lines, and relevant biological and molecular analyses were performed. Levels of the GCAWKR were upregulated in GC tissues compared with normal tissues and associated with tumor size, lymph node metastasis, TNM stage, and patient outcomes. GCAWKR affected cell proliferation and cell invasion in multiple GC models. Mechanistically, GCAWKR bound WDR5 and KAT2A and acted as a molecular scaffold of WDR5/KAT2A complexes, modulating the affinity for WDR5/KAT2A complexes in the target gene's promoter region. Thus, our data defined a mechanism of lncRNA-mediated carcinogenesis in GC, suggesting new therapeutic targets in GC.

Overexpression of PTP4A1 is associated with poor overall survival in non-small cell lung cancer.

OBJECTIVE: We aimed to analyze the phosphatases of regenerating liver 1 (PTP4A1) expression and its relationship with tumor invasion, metastasis, and prognosis of non-small cell lung cancer (NSCLC). METHODS: The retrospective study enrolled 150 cases who underwent radical resection for primary NSCLC during the period from January 2006 to January 2014. Baseline characteristics of patients included age, gender, smoking history, pathological type, histological grade, clinical stage, and lymphatic metastasis. Lung cancer tissues collected from 150 cases and precancerous tissues, and normal lung tissues collected from 20 cases were used for PTP4A1 detection by immunohistochemistry. RESULTS: The expression of PTP4A1 was significantly different in different tissue samples (P = 0.025). The expression level of PTP4A1 was associated with clinical stage (P = 0.022), and lymphatic metastasis (P = 0.011). Survival analysis showed the total survival rate of overexpressed PTP4A1 group was significantly lower than that of the low expressed PTP4A1 group (P = 0.006), while the recurrence rate of overexpressed PTP4A1 was significantly higher than that of the low expressed PTP4A1 group (P = 0.014). In addition, the OS was affected by lymphatic metastasis, and disease-free survival was influenced by pathological type and lymphatic metastasis. CONCLUSION: The PTP4A1 expression level is a potential prognostic biomarker for NSCLC, and overexpression of PTP4A1 suggested a high risk of poor DFS during follow-up. Also, lymphatic metastasis had an adverse effect on survival time.

Protein tyrosine phosphatase PTP4A1 promotes proliferation and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma via the PI3K/AKT pathway.

The protein tyrosine phosphatase PTP4A1 is a key molecule that activates tyrosine phosphorylation, which is important for cancer progression and metastasis. However, the clinical implications and biological function of PTP4A1 in intrahepatic cholangiocarcinoma (ICC) remains unknown. Here, we showed that PTP4A1 was frequently overexpressed in ICC versus adjacent non-tumor tissues. This overexpression significantly correlated with aggressive tumor characteristics like the presence of lymph node metastasis and advanced tumor stages. Survival analysis further indicated that high PTP4A1 expression was significantly and independently associated with worse survival and increased recurrence in ICC patients. Moreover, through forced overexpression and knock-down of PTPT4A1, we demonstrated that PTP4A1 could significantly promote ICC cells proliferation, colony formation, migration, and invasion in vitro, and markedly enhance tumor progression in vivo. Mechanistically, PTP4A1 was involved in PI3K/AKT signaling and its downstream molecules, such as phosphorylation level of GSK3ß and up-regulation of CyclinD1, in ICC cells to promote proliferation. Importantly, PTP4A1 induced ICC cells invasion was through activating PI3K/AKT signaling controlled epithelial-mesenchymal transition (EMT) process by up-regulating Zeb1 and Snail. Thus, PTP4A1 may serve as a potential oncogene that was a valuable prognostic biomarker and therapeutic target for ICC.

Suppression of cell migration is promoted by miR-944 through targeting of SIAH1 and PTP4A1 in breast cancer cells.

BACKGROUND: Aberrant expression of microRNAs has been associated with migration of tumor cells. In this study, we aimed to investigate the biological significance of miR-944 whose function is unknown in breast cancer. METHODS: MiR-944 expression in breast cancer cells and tumors was evaluated by Taqman qRT-PCR assays. Transcriptional profiling of MDA-MB-231 cells expressing miR-944 was performed using DNA microarrays. Cell viability, migration and invasion were assessed by MTT, scratch/wound-healing and transwell chamber assays, respectively. The luciferase reporter assay was used to evaluate targeting of SIAH1, PTP4A1 and PRKCA genes by miR-944. SIAH1 protein levels were measured by Western blot. Silencing of SIAH1 gene was performed by RNA interference using shRNAs. RESULTS: Our data showed that miR-944 expression was severely repressed in clinical specimens and breast cancer cell lines. Suppression of miR-944 levels was independent of hormonal status and metastatic potential of breast cancer cells. Gain-of-function analysis indicated that miR-944 altered the actin cytoskeleton dynamics and impaired cell migration and invasion. Genome-wide transcriptional profiling of MDA-MB-231 cells that ectopically express miR-944 showed that 15 genes involved in migration were significantly repressed. Notably, luciferase reporter assays confirmed the ability of miR-944 to bind the 3´UTR of SIAH1 and PTP4A1 genes, but not PRKCA gene. Congruently, an inverse correlation between miR-944 and SIAH1 protein expression was found in breast cancer cells. Moreover, SIAH1 was upregulated in 75 % of miR-944-deficient breast tumors. Finally, SIAH1 gene silencing by RNA interference significantly impaired cell migration of breast cancer cells. CONCLUSIONS: Our results pointed out that miR-944 is a novel upstream negative regulator of SIAH1 and PTP4A1 genes and provided for the first time evidence for its functional role in migration and invasion of breast cancer cells. They also suggest that miR-944 restoration may represent a potential strategy for breast cancer therapy.

miR-601 is a prognostic marker and suppresses cell growth and invasion by targeting PTP4A1 in breast cancer.

MicroRNAs (miRNA) play important roles in the initiation and progression of breast cancer. Here, we investigated the role of miR-601 in breast cancer and found that its expression was significantly down-regulated in breast cancer tissues compared with matched adjacent non-cancerous breast tissues. Moreover, we found that down-regulation of miR-601 was closely associated with distant metastasis and poor distant metastasis-free survival in breast cancer. In addition, miR-601 levels were inversely correlated with metastatic potential of human breast cancer cell lines. Further experiments showed that ectopic overexpression of miR-601 suppressed breast cancer cell proliferation, migration and invasion, whereas miR-601 knockdown promoted breast cancer cell proliferation, migration and invasion. Furthermore, protein tyrosine phosphatase type IVA 1 (PTP4A1) was identified as a direct target of miR-601. Overexpression of miR-601 repressed PTP4A1 mRNA and protein expression. Conversely, inhibition of miR-601 increased PTP4A1 mRNA and protein expression. Taken together, our data suggest that miR-601 inhibits growth and invasion of breast cancer cells by targeting PTP4A1 and that miR-601 is a potential biomarker for prognosis and therapeutic target in breast cancer.

Identification of PRL1 as a novel diagnostic and therapeutic target for castration-resistant prostate cancer by the Escherichia coli ampicillin secretion trap (CAST) method.

OBJECTIVES: Although chemotherapy for castration-resistant prostate cancer (CRPC) has been applied clinically in recent years, the effects are not sufficient. It is urgently necessary to develop novel therapeutics for CRPC. We previously generated Escherichia coli ampicillin secretion trap libraries of 2 prostate cancer (PCa) cell lines and normal prostate. By comparing the E. coli ampicillin secretion trap libraries of CRPC cell lines with those of androgen-sensitive PCa cell lines and normal prostate, we focused on the protein-tyrosine-phosphatase of regenerating liver 1 (PRL1) gene and analyzed its expression and biological function. MATERIALS AND METHODS: The expression of PRL1 was examined by quantitative reverse transcription polymerase chain reaction and immunohistochemistry in clinical PCa samples. The effects of PRL1 on PCa cells were evaluated by cell growth, migration, and invasion assays. To investigate the effect of PRL1 on epidermal growth factor receptor (EGFR) signaling, PRL1 knockdown PC3 cells were examined by Western blot and immunohistochemical analyses. RESULTS: Quantitative reverse transcription polymerase chain reaction revealed that PRL1 was expressed much more highly in PCa than in nonneoplastic prostate samples. High expression of PRL1 detected by immunohistochemistry correlated with poor prognosis after prostatectomy and combined androgen blockade therapy. Functional analysis indicated that PRL1 stimulated cell growth, migration, and invasion in PCa cell lines. Expression EGFR and matrix metalloproteinase 9 was reduced by knockdown of PRL1 in the PC3 cell line. CONCLUSIONS: PRL1 regulates expression of EGFR and modulates downstream targets. PRL1 has potential as a therapeutic target in PCa including CRPC.