WTAP-induced N6-methyladenosine of PD-L1 blocked T-cell-mediated antitumor activity under hypoxia in colorectal cancer.

N6-Methyladenosine (m6A) is a important process regulating gene expression post-transcriptionally. Programmed death ligand 1 (PD-L1) is a major immune inhibitive checkpoint that facilitates immune evasion and is expressed in tumor cells. In this research we discovered that Wilms' tumor 1-associated protein (WTAP) degradation caused by ubiquitin-mediated cleavage in cancer cells (colorectal cancer, CRC) under hypoxia was inhibited by Pumilio homolog 1 (PUM1) directly bound to WTAP. WTAP enhanced PD-L1 expression in a way that was m6A-dependent. m6A "reader," Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) identified methylated PD-L1 transcripts and subsequently fixed its mRNA. Additionally, we found that T-cell proliferation and its cancer cell-killing effects were prevented by overexpression of WTAP in vitro and in vivo. Overexpression prevented T cells from proliferating and killing CRC by maintaining the expression of PD-L1. Further evidence supporting the WTAP-PD-L1 regulatory axis was found in human CRC and organoid tissues. Tumors with high WTAP levels appeared more responsive to anti-PD1 immunotherapy, when analyzing samples from patients undergoing treatment. Overall, our findings demonstrated a novel PD-L1 regulatory mechanism by WTAP-induced mRNA epigenetic regulation and the possible application of targeting WTAP as immunotherapy for tumor hypoxia.

WTAP-mediated abnormal m6A modification promotes cancer progression by remodeling the tumor microenvironment: bibliometric and database analyses.

Background: Wilms' tumor 1 associated protein (WTAP) is an essential component of the m6A methyltransferase complex. The research on WTAP has been continuously developed and promoted in recent years. It is needed to make systematic specific bibliometric and database analyses on WTAP, to identify the cooperation and impact of authors, countries, institutions and journals, to evaluate the knowledge base and find the hotspot trends, and to detect the emerging topics regarding WTAP research. Methods: The related articles and reviews of WTAP in the Web of Science Core Collection from January 1999 to June 2022 were retrieved, and CiteSpace and VOSviewer were used to perform bibliometric and knowledge-map analyses. Multiple databases were used to explore the expression level of WTAP in pan-cancers and its correlation with prognosis and immune infiltration. Results: In recent years, the number of publications on WTAP research has increased rapidly. Among the journals publishing WTAP research, Frontiers in Cell and Developmental Biology had the most papers, while Nature papers were most cited. The country with the highest number of publications on WTAP was China, and China Medical University was the institution with the most publications. The most prominent author was Haruo Sugiyama from Japan. Four main aspects of WTAP research included m6A modification, tumor association, cancer therapy, and regulatory mechanisms. The research frontiers and hotspots were m6A modification, methyltransferase, demethylation, tumor microenvironment (TME), and immunotherapy. Bioinformatics analysis showed that the expression of WTAP was up-regulated in a variety of tumors and closely related to TME and survival prognosis. Conclusions: From the bibliometric and database analyses on the researches on WTAP, it is suggested that up-regulated WTAP in cancers may promote cancer progression by mediating abnormal m6A modifications to reshape the TME, thereby affecting the survival prognosis of the patients. The information would provide helpful references for scholars focusing on WTAP and provide new insights for WTAP as a prognostic evaluation and immunotherapy for tumors in the future.

RNA N6­methyladenosine methyltransferase WTAP promotes the differentiation of endothelial progenitor cells.

N6-methyladenosine (m6A) serves a critical role in regulating gene expression and has been associated with various diseases; however, its role in the differentiation of endothelial progenitor cells (EPCs) remains unclear. The present study used liquid chromatography with tandem mass spectrometry and immunofluorescence assays to quantify the levels of m6A in human peripheral blood-derived EPCs (HPB-EPCs) before and after differentiation into mature cells. The present study performed Cell Counting Kit 8, Transwell, and tube formation assays to determine the effects of overexpression and knockdown of Wilms' tumor 1-associated protein (WTAP) on HPB-EPCs. The results revealed that the level of m6A modification was significantly increased during HPB-EPCs differentiation, and WTAP exhibited the most significant alteration among the enzymes involved in m6A regulation. When WTAP was overexpressed in HPB-EPCs, cell proliferation, invasion, and the formation of tubes were improved, whereas WTAP knockdown yielded the opposite effects. In conclusion, the present study highlighted the involvement of m6A in regulating EPC differentiation, with WTAP acting as a promoter of EPC differentiation.

WTAP-Involved the m6A Modification of lncRNA FAM83H-AS1 Accelerates the Development of Gastric Cancer.

The long noncoding RNA FAM83H-antisense RNA 1 (FAM83H-AS1) is involved in gastric cancer (GC) development. This study determined whether FAM83H-AS1 was regulated by N6-methyladenosine (m6A) modifications in GC. Real-time quantitative polymerase chain reaction was performed to determine the expression levels of FAM83H-AS1 and Wilms' tumor 1 associated protein (WTAP). The protein content of WTAP was evaluated using western blotting. To assess the m6A alterations in FAM83H-AS1, methylated RNA immunoprecipitation was performed to identify interactions between WTAP and FAM83H-AS1. Functionally, the proliferation, migration, and invasion of GC cells were measured using a Cell Counting Kit-8 and transwell assays, respectively. High expression levels of FAM83H-AS1 and WTAP were detected in GC samples and there was a positive correlation between them. In addition, WTAP mediates FAM83H-AS1 expression in an m6A-dependent manner. Further investigations indicated that WTAP silencing reversed the cancer-promoting role of FAM83H-AS1 overexpression in GC cell migration, proliferation, and invasion. Our results suggest that WTAP-mediated FAM83H-AS1 promotes GC development via m6A modification. Our findings provide new biomarkers for GC diagnosis and targeted therapy. Wilms' tumor 1 associated protein (WTAP) promotes gastric cancer (GC) by accelerating cell proliferation, migration, and invasion of GC cells via the N6-methyladenosine(m6A) modification of long non coding RNA FAM83H-AS1.

Changes of plasma METTL3, WTAP and FTO levels in patients with systemic lupus erythematosus and their correlation with clinical indicators / 中国医师杂志

Objective:To explore the clinical significance of N6-methyladenine (m6A) in systemic lupus erythematosus (SLE) by comparing the changes in plasma levels of m6A modification related proteins [methyltransferase 3 (METTL3), methyltransferase 14 (METTL14), Wilms tumor 1 associated protein (WTAP), AlkB homologous protein 5 (ALKBH5), and fat mass and obesity-associated protein (FTO)] and m6A between patients with systemic lupus erythematosus (SLE) and healthy controls.Methods:A total of 64 SLE patients admitted to the Seventh Affiliated Hospital of Sun Yat-Sen University from May 2020 to June 2022 and 24 healthy volunteers during the same period were selected to compare and analyze the plasma levels of METTL3, METTL14, WTAP, ALKBH5, FTO and m6A between the two groups. The correlation between METTL3, WTAP, FTO levels and clinical indicators was analyzed.Results:The plasma METTL3 level of SLE patients was significantly higher than that of control group ( P<0.05), and the plasma WTAP and FTO levels were significantly lower than those of control group (all P<0.05). In SLE patients, plasma METTL3 level was negatively correlated with hemoglobin level ( r=-0.344, P<0.05), plasma FTO level was positively correlated with plasma IgM level ( r=0.337, P<0.05), and plasma IgA level was negatively correlated with SLE patients ( r=-0.286, P<0.05). The incidence of renal involvement and positive rate of plasma anti-histone antibody were higher in SLE patients with high METTL3 level (all P<0.05). The positive rates of plasma anti-dsDNA antibody, anti-SM antibody and AuaA antibody were higher in SLE patients with low FTO level (all P<0.05). Conclusions:The plasma METTL3 level in SLE patients are significantly increased, while the plasma WTAP and FTO levels are significantly reduced, which are related to various clinical indicators and may be related to the onset of SLE.

A potential biomarker of esophageal squamous cell carcinoma WTAP promotes the proliferation and migration of ESCC.

This study focuses on the function of WTAP in esophageal squamous cell carcinoma (ESCC) samples and cell lines. The results showed that WTAP expression in ESCC tissues was significantly upregulated in 78.1% (57 of 73) of the ESCC tissues at the protein level compared with adjacent non-cancerous tissues via immunohistochemical staining. The WTAP protein expression level was positively correlated with the lymph node metastasis and TNM stage, and patients with higher WTAP protein expression level exhibited a shorter overall survival interval. Knocking down WTAP significantly reduced cell proliferation and migration but promoted cell apoptosis of TE-1and KYSE150 cells. Moreover, WTAP inhibition reduced the expression of ki67 and Snail related to cell proliferation and migration but increased the expression of Bax and Caspase-3 which were involved in cell apoptosis. In conclusion, our results suggest that the WTAP, a potential biomarker of ESCC, maybe play an important role in ESCC-genesis through regulating expression of genes related to cell proliferation, migration and apoptosis.

Wilms' Tumor 1-Associated Protein Contributes to Chemo-Resistance to Cisplatin Through the Wnt/ß-Catenin Pathway in Endometrial Cancer.

BACKGROUND: Cisplatin remains the mainstay of endometrial cancer (EC) chemotherapy. Wilms' tumor 1-associated protein (WTAP), playing a critical role in transcriptional and post-transcriptional regulation, has been reported as an oncogene, and its expression is elevated in multiple types of human tumors. Recent evidence has shown that the increased expression of WTAP is also closely related to chemo-resistance. However, its specific role in the susceptibility of human EC cells to cisplatin remains largely unexplored. METHODS: WTAP over-expression and WTAP depletion cell lines as well as their corresponding controls were constructed by transfection with lentivirus. Western blotting analysis and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to detect the expression of WTAP. Cell proliferation assay, colony formation assay, cell cycle assay, and apoptosis analysis were adopted to evaluate the effect of WTAP on the chemo-sensitivity of EC cells to cisplatin as well as its underlying mechanism. Immunofluorescence staining was used to assess the translocation of ß-catenin. Moreover, a subcutaneous xenograft tumor model was established to assess the effect of WTAP on tumor growth after cisplatin treatment. RESULTS: Depletion of WTAP in RL95-2 cells significantly enhanced the chemo-susceptibility of cells to cisplatin and increased the cell apoptosis, while WTAP over-expression in ARK-2 cells exhibited the opposite effects. Additionally, WTAP depletion significantly suppressed xenograft-tumor growth and enhanced sensitivity and apoptosis of tumor cells in vivo. Mechanistic analysis exhibited that WTAP over-expression facilitated the cytoplasm-to-nucleus translocation of ß-catenin and enhanced the GSK3ß phosphorylation at Ser9, while WTAP depletion revealed the opposite results, indicating that WTAP rendered chemo-resistance of EC cells to cisplatin by promoting the Wnt/ß-catenin pathway. CONCLUSIONS: WTAP might promote the chemo-resistance of EC cells to cisplatin through activating the Wnt/ß-catenin pathway. Collectively, our findings offered novel insights into EC treatment.

High Wilms' tumor 1 associating protein expression predicts poor prognosis in acute myeloid leukemia and regulates m6A methylation of MYC mRNA.

PURPOSE: Acute myeloid leukemia (AML) is a heterogenous disease and the survival of AML patients is largely attributed to the improvement of supportive treatment. Wilms' tumor 1-associated protein (WTAP) is a nuclear protein functions in many physiological and pathological processes. Although its expression and function in many malignant diseases have been reported, its prognostic and epigenetic roles in AML are largely unknown. METHODS: Peripheral blood or bone marrow samples were collected from AML patients. The WTAP expression was detected by western blot. WTAP expression level and patients clinical features were analyzed using statistical methods. WTAP knockdown AML cells were constructed. The experiments on proliferation, tumorigenic ability, cell cycle, and apoptosis were performed. Transcriptome sequencing was performed and analyzed. M6A methylation level was measured and m6A-RIP was performed to quantify m6A methylation level of MYC mRNA. RNA stability assay was performed to measure the half-life of mRNA. RESULTS: WTAP was overexpressed in AML patients and was an independent poor-risk factor in AML (p = 0.0140). Moreover, we found that WTAP regulated proliferation, tumorigenesis, cell cycle, and differentiation of AML cells. Furthermore, WTAP made AML cells resistant to daunorubicin. In further investigations, m6A methylation level was downregulated when knocking down WTAP, and c-Myc was upregulated due to the decreased m6A methylation of MYC mRNA. CONCLUSION: High WTAP expression predicts poor prognosis in AML and WTAP plays an epigenetic role in AML.

WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1.

BACKGROUND: N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer. METHODS: We determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay. RESULTS: We demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1. CONCLUSION: We have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment.

WT1-associated protein is a novel prognostic factor in pancreatic ductal adenocarcinoma.

Although Wilms tumor 1 (WT1)-associated protein (WTAP) was initially found to be a specific WT1-binding protein, it has increasingly attracted attention because of its oncogenic role in various types of malignancies, including cholangiocarcinoma, glioblastoma and acute myeloid leukemia. However, the clinical impact of WTAP on pancreatic ductal adenocarcinoma (PDAC) is still unknown. A total of 145 patients who underwent surgical treatment from 2004 to 2008 were enrolled in the present study. The cytoplasmic and nuclear expression of WTAP in tumor and adjacent normal tissues was examined by immunohistochemical analysis in order to investigate the relationship between WTAP and the clinicopathological factors and prognosis of patients with PDAC. The nuclear and cytoplasmic expression of WTAP in tumor tissues was significantly higher compared with non-tumor tissues (P<0.001). High expression of WTAP in the nucleus was significantly associated with gender (P=0.010) and tumor stage (P=0.020), while high expression of WTAP in the cytoplasm was significantly associated with gender (P=0.018), histological grade (P=0.047) and perineural invasion (P=0.028). In addition, a univariate analysis revealed that high nuclear expression of WTAP in tumor tissues was significantly associated with poor overall survival (P<0.001), as well as several clinicopathological variables, including gender and N stage. In a multivariate Cox regression analysis, nuclear WTAP expression was identified as an independent prognostic indicator for PDAC (relative risk, 1.855; 95% confidence interval, 1.033-3.333; P=0.039). The results of the present study indicated that high nuclear expression of WTAP is a valuable molecular biomarker of a poor prognosis among patients with PDAC.

Wilms tumor 1 gene, CD97, and the emerging biogenetic profile of glioblastoma.

Glioblastoma multiforme (GBM) is the most common type of primary brain tumor, and current treatment regimens are only marginally effective. One of the most vexing and malignant aspects of GBM is its pervasive infiltration into surrounding brain tissue. This review describes the role of the Wilms tumor 1 gene (WT1) and its relationship to GBM. WT1 has several alternative splicing products, one of which, the KTS(+) variant, has been demonstrated to be involved in the transcriptional activation of a variety of oncogenes as well as the inhibition of tumor suppressor genes. Further, this paper will examine the relationship of WT1 with CD97, a gene that codes for an epidermal growth factor receptor family member, an adhesion G-protein-coupled receptor, thought to promote tumor invasiveness and migration. The authors suggest that further research into WT1 and CD97 will allow clinicians to begin to deal more effectively with the infiltrative behavior displayed by GBM and design new therapies that target this deadly disease.