Wilms' Tumor 1-Associating Protein Promotes Nonsmall-Cell Lung Cancer Through the Expression of Carcinoembryonic Antigen-Related Cell Adhesion Molecule 5.

Wilms' tumor 1-associating protein (WTAP) is ubiquitously expressed in many tissues and plays an important role in physiological processes and tumor development. Here, we investigated the specific biological role and underlying mechanism of WTAP in melanoma. We determined the expression of WTAP and its correlation with clinicopathological features in paraffin-embedded tissues. We investigated the effects of WTAP on melanoma cells via a CCK-8 assay, a colony formation assay, an EdU assay, a Transwell assay, and subcutaneous xenograft experiments. We then applied RNA sequencing to further screen candidate targets, and NT5E was selected as the downstream gene of WTAP. Finally, a series of rescue assays, together with nucleotidase assays and ELISA, were adopted to confirm the function of NT5E in melanoma progression. We demonstrated that WTAP expression was downregulated in melanoma, which was associated with a poor prognosis, and that WTAP expression served as an independent predictor of melanoma survival. Functionally, WTAP hindered the proliferation, growth, migration, and invasion of melanoma cells. Furthermore, NT5E was identified as the downstream effector of WTAP and was subsequently found to rescue the increased proliferation, migration, and invasion of melanoma cells induced by WTAP deficiency. Moreover, knockdown of WTAP increased the expression of NT5E, MMP2, and N-cadherin, and simultaneous transfection with siNT5E reversed the increased expression of MMP2 and N-cadherin. Moreover, increased NT5E expression caused by forced WTAP inhibition in melanoma promoted the hydrolysis of AMP to produce more adenosine and further abrogated the secretion of IFN-γ by PBMCs. We found that WTAP expression is significantly downregulated and restrains the progression of melanoma via the downstream effects of NT5E on immunosuppression and molecular adhesion. This study revealed that WTAP plays a crucial inhibitory role in melanoma oncogenesis and highlighted WTAP as a potential novel diagnosis and therapeutic target for melanoma.

Background: Hepatocellular carcinoma (HCC) is a primary aggressive gastrointestinal neoplasm that affects patients worldwide. It has been shown that Wilms' tumor 1-associating protein (WTAP) is frequently upregulated in various cancers. However, the potential role of WTAP in HCC remains largely unknown.Methods: The expression levels of WTAP in human HCC tissues were determined by the western blotting and immunohistochemical (IHC) staining. A correlation between the WTAP expression, clinicopathological features, and the HCC prognosis was analyzed. The WTAP expression was silenced by short hairpin RNA (shRNA), and effects of the knockdown of WTAP on the proliferation and invasion of HCC cells were assessed. The microRNAs (miRNAs) involved in the regulation of the WTAP expression were identified by a bioinformatics analysis and further confirmed by in vitro assays.Results: The expression levels of WTAP in liver cancer tissues were significantly elevated and compared with those in the adjacent normal tissues and significantly correlated with the clinical stage and prognosis in patients with HCC. Further investigation revealed that the knockdown of WTAP drastically suppressed HCC cell proliferation and invasion abilities. Luciferase reporter assay and validation experiments confirmed that WTAP was a direct target of miR-139-5p. Moreover, the overexpression of WTAP could partly abolish the inhibitory effects of miR-139-5p on the HCC cell growth and invasion. Mechanistically, we revealed that the miR-139-5p/WTAP axis regulated the HCC progression by controlling the epithelial to mesenchymal transition (EMT).Conclusions: In summary, the results indicate that WTAP is a potential oncogene in HCC and miR-139-5p negatively regulates the WTAP expression. MiR-139-5p/WTAP can be utilized as a potential therapeutic target for HCC.

Castration-resistant prostate cancer (CRPC) is one of the most prevalent cancers in men. The new generation androgen receptor (AR) inhibitor enzalutamide can improve the therapeutic effectiveness of patients with CRPC. However, these patients eventually develop acquired enzalutamide resistance (ENZR), and the mechanisms underlying resistance are not well understood. Wilms' tumor 1-associating protein (WTAP) plays an important role in m6A modification and has been reported as an oncogene in various cancers. Here, we utilized a tissue microarray and collected tissues from prostate cancer (PCa) patients to detect WTAP expression, and found that WTAP is upregulated in PCa. Meanwhile, WTAP overexpression promotes cell proliferation and accelerates tumor growth through colony formation assays and the establishment of a subcutaneous xenograft model in vivo. These findings establish the tumor promoter role of WTAP in prostatic tumorigenesis. Furthermore, we verified that WTAP is a novel responsive gene of AR via promoter activity and chromatin immunoprecipitation (ChIP) assays. Importantly, we uncovered that WTAP is upregulated in ENZR cells, and WTAP knockdown inhibited the proliferation of ENZR cells. Mechanistically, ubiquitin-specific protease (USP7) enhanced the stability of WTAP by the ubiquitin-proteasome pathway in ENZR cells, thereby WTAP increases promote AKT signaling through an m6A-mediated way, and an AKT inhibitor can abolish the pro-resistance phenotype mediated by WTAP. Together, these findings suggest that WTAP plays a key role in ENZR development of PCa cells, and WTAP may be a potential treatment target for ENZR tumors. In this manuscript, we utilized a tissue microarray and collected tissues from prostate cancer (PCa) patients to detect WTAP expression, and found that WTAP is upregulated in PCa. Meanwhile, WTAP overexpression promotes cell proliferation and accelerates tumor growth through colony formation assays and the establishment of a subcutaneous xenograft model in vivo. These findings establish the tumor promoter role of WTAP in prostatic tumorigenesis. Furthermore, we verified that WTAP is a novel responsive gene of AR via promoter activity and chromatin immunoprecipitation (ChIP) assays. Importantly, we uncovered that WTAP is upregulated in ENZR cells, and WTAP knockdown inhibited the proliferation of ENZR cells. Mechanistically, ubiquitin-specific protease (USP7) enhanced the stability of WTAP by the ubiquitin-proteasome pathway in ENZR cells, thereby WTAP increases promote AKT signaling through an m6A-mediated way, and an AKT inhibitor can abolish the pro-resistance phenotype mediated by WTAP. Together, these findings suggest that WTAP plays a key role in ENZR development of PCa cells, and WTAP may be a potential treatment target for ENZR tumors.

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. 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. 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. High WTAP expression predicts poor prognosis in AML and WTAP plays an epigenetic role in AML.

Apoptosis of vascular smooth muscle cells (SMC) is a culprit event in atherosclerotic plaque destabilization. We recently discovered that Wilms’ tumor 1-associating protein (WTAP) is a dynamically expressed transcriptional regulator that can be pro-apoptotic for human SMCs (Circ Res, 2006). To identify upstream regulators of this nuclear protein, we screened growth factors for their capacity to impact WTAP expression and found that insulin-like growth factor-1 (IGF-1), a potent survival factor for SMCs, stimulated a striking decline in WTAP protein abundance, to 10% at 12 h. We further determined that this decline in WTAP was due specifically to WTAP protein degradation, established by pulse-chase analysis of 35 S-labeled WTAP and the absence of an acute effect of IGF-1 on WTAP mRNA abundance. IGF-1-mediated WTAP degradation was blocked by two mechanistically distinct IGF-1 receptor inhibitors (picropodophyllin and PQ401) and by inhibition of phosphatidylinositol 3 (PI3)-kinase but not by MEK inhibition. In addition, IGF-1 induced the association of WTAP with ubiquitin, established by coimmunoprecipitation, and the downregulation of WTAP by IGF-1 was abrogated by inhibiting 26S proteasome activity with lactacystin or MG132. Interestingly, IGF-1 also stimulated phosphorylation of WTAP, that preceded the association of WTAP with ubiquitin, and hyperphosphorylation of WTAP through phosphatase-inhibition further accelerated WTAP degradation. Finally, to determine if WTAP downregulation was necessary for IGF-1-mediated SMC survival, surface expression of phosphatidylserine was quantified by flow cytometry of SMCs infected with retrovirus containing WTAP cDNA. Whereas IGF-1 enhanced the survival of vector-infected SMCs this was completely abrogated in WTAP-overexpressing SMCs. Conclusions: IGF-1-mediated SMC survival is dependent on the rapid depletion of WTAP from the nucleus, a degradation cascade that is heralded by WTAP phosphorylation. This WTAP phosphorylation and clearance response represents a novel consequence of PI3-kinase activation and highlights WTAP as a key negative regulator of SMC survival during vascular remodeling.

Aim: Wilms' tumor 1-associating protein (WTAP), plays a part in colorectal cancer (CRC) progression. However, it is not yet known how WTAP affects cancer progression by influencing leukocyte rich repeat containing proteins (NLR) - family members 3 (NLRP3) - related inflammasomes.Materials & methods: We first validated the expression of WTAP in CRC at the tissue and cellular levels. Subsequently, by transfecting si-NC and si-WTAP into cells, we verified functions of WTAP in proliferation, invasion, migration and apoptosis of CRC cells. Finally, we analyzed the N6-methyladenosine (m6A) modification of NLRP3 by WTAP using methylated RNA immunoprecipitation (MeRIP)-qPCR technology, confirming that WTAP mediated the repression of NLRP3 inflammasome and the malignant progression of tumor cells.Results: WTAP was substantially upregulated in CRC tissues and cells. WTAP reinforced the migration, proliferation and invasion ability of CRC cells, and repressed apoptosis. Mechanistically, WTAP mediated the m6A modification of NLRP3, which suppressed the expression of NLRP3 and dampened the NLRP3/Caspase-1/GSDMD axis activation as well as pyroptosis, thereby facilitating the malignant progression of CRC.Conclusion: WTAP mediates m6A modification to modulate the repression of the NLRP3/Caspase-1/GSDMD axis in pyroptosis, reinforcing the malignant progression of CRC.

EBV downregulates the m6A “writer” WTAP in EBV-associated gastric carcinoma

The N6 -methyladenosine (m6 A) modification is one of the most prevalent methylations in eukaryotic messenger RNA (mRNA), and it is essential for the development of many important biological processes such as multiple types of tumors. One of the most important enzymes catalyzing generation of m6 A on mRNA is Wilms' tumor 1-associating protein (WTAP); however, the potential role of WTAP in endometrial cancer (EC) still remains unknown. Here, we investigated WTAP expression level in cancer tissue and paracancerous tissue from an EC patient. Subsequently, WTAP was knocked down by small interfering RNA in EC cell line of Ishikawa and HEC-1A, respectively. Cell proliferation, migration, and invasion were studied. The expression of caveolin-1 (CAV-1) was detected by quantitative polymerase chain reaction (qPCR). The enrichments of m6 A and METTL3 on CAV-1 were detected using RNA immunoprecipitation-qPCR. The activity of nuclear factor-κB (NF-κB) was studied using Western blot. We observed that WTAP was dramatically upregulated in the cancer tissue, and there was an enhancement in cell proliferation, migration, and invasion and adecreasein ECapoptosis in vivo and in vitro, which indicated higher tumor malignancy and worse survival outcome. After WTAP was knocked down in EC cells, CAV-1 was significantly upregulatedand the enrichments of m6 A and METTL3 at 3'-untranslated region (UTR) region of CAV-1 were decreased. Moreover, the activity of NF-κB signaling pathway was inhibited by its regulator CAV-1. Taken together, we concluded that WTAP could methylate 3'-UTR of CAV-1 and downregulate CAV-1 expression to activate NF-κB signaling pathway in EC, which promoted EC progression.

Wilms' tumor 1-associating protein (WTAP) interacts with the Wilms' tumor 1 gene. Although originally classified as a tumor suppressor, WTAP was later found to be over-expressed in glioblastoma which is regarded as a grade IV astrocytoma. However, the expression in other glioma grades and the relationship between WTAP expression and the prognosis of glioma patients are still unknown. In this study, we investigated WTAP expression in 169 different types of glioma cases using western blot analysis and immunohistochemistry assay. Further, we evaluated the association of WTAP expression with clinicopathological characteristics using chi-square test and Spearman's correlation test. We used univariate and multivariate Cox regression analyses to evaluate the independency of diferent WTAP expression. Then, the survival curves were calculated using the Kaplan-Meier method. Results showed that WTAP was over-expressed in glioma tissues, and the expression was closely correlated with glioma grade. Moreover, high WTAP expression was correlated with poor postoperative survival in glioma patients. WTAP may serve as a novel prognostic marker.

N6-methyladenosine modification, especially Wilms tumor 1-associated protein (WTAP), is reportedly associated with a variety of cancers, including colorectal cancer (CRC). Angiogenesis also plays an important role in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection. Therefore, tissue microarray and public database were used to explore WTAP levels in CRC. Then, WTAP was down-regulated and over-expressed, respectively. CCK8, EdU, colony formation, and transwell experiments were performed to study the role of WTAP in CRC. Combined RNA sequencing and m6A RNA immunoprecipitation (MeRIP) sequencing, we found downstream molecules VEGFA. Moreover, a tube formation assay was executed for tumor angiogenesis. Finally, a subcutaneous tumorigenesis assay in nude mice was used to examine the tumor-promoting effect of WTAP in vivo. In the present study, WTAP was significantly upregulated in CRC cells and patients with CRC. Moreover, higher WTAP expression was observed in the TCGA and CPATC databases in CRC tissues. WTAP over-expression exacerbates cell proliferation, migration, invasion, and angiogenesis. Conversely, WTAP knockdown inhibited the malignant biological behavior of CRC cells. Mechanistically, WTAP positively regulated VEGFA, as identified using RNA sequencing and MeRIP sequencing. Moreover, we identified YTHDC1 as a downstream effector of the YTHDC1-VEGFA axis in CRC. Furthermore, increased WTAP expression activated the MAPK signaling pathway, which led to enhanced angiogenesis. In conclusion, our study revealed that the WTAP/YTHDC1/VEGFA axis promotes CRC development, especially angiogenesis, suggesting that it may act as a potential biomarker of CRC.

Multiple myeloma (MM) remains incurable, largely owing to the lack of effective therapeutic targets. Wilms’ tumor 1-associated protein (WTAP) has been implicated in MM tumorigenesis, but its underlying molecular mechanisms remain unclear. This study aimed to elucidate the role of WTAP in MM progression. Bone marrow samples from newly diagnosed MM patients were analyzed. WTAP expression was assessed via Quantitative real-time PCR (qRT-PCR) and Western blot. Cell proliferation was evaluated using EdU and flow cytometry assays, while RNA immunoprecipitation (RIP) confirmed protein interactions. WTAP was overexpressed in MM patients and correlated with poor survival. Its knockdown significantly suppressed MM cell proliferation and inflammation. RIP assays identified MAP6D1 as a potential WTAP target. RNA-seq analysis suggested WTAP regulates MM proliferation via the Hippo signaling pathway. WTAP promotes MM proliferation by targeting MAP6D1 and modulating the Hippo pathway, highlighting its potential as a therapeutic target.

Wilms' tumor 1-associated protein (WTAP), a component of the m6A methyltransferase complex, recruits the m6A methyltransferases METTL3 and METTL14 to the corresponding mRNA targets to participate in the formation of N6-methyladenosine. However, the molecular mechanism of WTAP in the tumorigenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. This study aimed to explore the prognostic value and biological function of WTAP in NPC. We assessed WTAP expression and its prognostic significance using microarray datasets from the Gene Expression Omnibus (GSE12452) database and 100 NPC tissues via bioinformatics analysis and immunohistochemistry (IHC), respectively. Moreover, gene ontology (GO) and gene set enrichment analysis (GSEA) were performed. In addition, the correlation of WTAP expression with the expression of immune cell biomarkers was analyzed. The results showed that WTAP expression was significantly overexpressed in NPC tissues in GSE12452. The overexpression of WTAP was validated by the external datasets including NPC tissues (GSE150430) and NPC cell lines (GSE39826). GO analysis suggested enrichment in the nucleoplasm (cellular component) and cell cycle (biological process). The GSEA revealed that differentially expressed genes were enriched in E2F-targets, Myc_targets_v1, G2M checkpoint, Myc_targets_v2, and Interferon-alpha-response. In IHC analysis, WTAP was upregulated in NPC tissues, and high levels of WTAP expression were significantly correlated with the advanced T stage (p=0.047) and advanced N stage (p=0.018). Cox regression demonstrated that WTAP overexpression was an independent biomarker of poor prognosis for overall survival (hazard ratio [HR], 4.747; 95% confidence interval [CI], 1.671-13.482; p=0.003). In IHC analysis, the expression of WTAP was positively correlated with CD206 (biomarker for M2 macrophages) (p=0.018) but negatively correlated with CD8a (biomarker for cytotoxic T cells) (p=0.001). In conclusion, WTAP is a promising prognostic biomarker and may participate in the regulation of immune cell infiltration in NPC.

Purpose: Wilms' tumor 1-associating protein (WTAP) which is known for its role in RNA methylation machinery, is highly upregulated in the necrotic region of glioblastoma multiforme (GBM). The highly heterogenic nature of GBM is associated with the necrotic region within the brain, where many types of cells such as the stromal cell, immune cells, and cancer stem cells co-localize. The complex cellular network within the necrotic region is believed to cause the resistance of the conventional chemotherapy and radiotherapy. Here we attempted to identify the role of WTAP in GBM and what kind of RNA modification is associated with the proliferation and stemness in GBM. Improvement of our understanding about post-transcriptional regulation in GBM will allow us to find out the key oncogenic switch in GBM, and development of therapeutics targeting this mechanism will provide better survival benefits. Methods: 23 Patient-derived cell lines, two established GBM cell lines, and astrocyte was arrayed to find out the correlation between WTAP and cancer stem cells. shWTAP and WTAP overexpression vector were infected into patient-derived GBM cancer stem cell lines to test the effect of WTAP. Western blot and polymerase chain reaction analysis was performed to find out the change in genes which are related to cell proliferation and stemness. Limiting dilution assay was performed to validate the function of WTAP in sphere-forming ability. RNA sequencing was performed on shWTAP infected patient-derived cancer stem cell to identify the type of RNA modification made by knocking down WTAP. Results: Knocking down of WTAP using shWTAP in patient-derived cancer stem cell lines, we were able to see the decrease in cell proliferation and stemness-related genes while overexpressing WTAP in these cells slightly increased cell proliferation and stemness. WTAP knockdown cells had a significant decrease in sphere-forming ability. RNA sequencing of shWTAP cells has revealed the increase in intron retention and the decrease in exon skipping, which shows that WTAP has a role in decreasing intron retention while increasing exon skipping. Conclusion: We have revealed that WTAP has an important role in increasing proliferation and maintaining stemness in GBM. We have also identified that WTAP decreases intron retention and increasing exon skipping. Citation Format: Sae Whan Park, Young Taek Oh, Jong Bae Park, Jong Heon Kim. Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4442.

Background: Wilms tumor 1-associated protein (WTAP) plays a critical role in ribonucleic acid (RNA) methylation of N6 adenosine (m6A) modification, which is closely related with varieties of biological process. However, the role of WTAP in cancers remains to be determined. This study is designed to demonstrate the prognostic landscape of WTAP in pan-cancer and explore the relationship between WTAP expression and immune infiltration.Methods: Here, we investigated the expression level and prognostic role of WTAP in pan-cancer using multiple databases, including PrognoScan, GEPIA, and Kaplan-Meier Plotter. Then, applying the GEPIA and TIMER databases, we illustrated the correlations between WTAP expression and immune infiltration in tumors, especially liver hepatocellular carcinoma (LIHC), and esophageal carcinoma (ESCA).Results: WTAP had significant higher expression levels in tumor tissues of ESCA, LIHC, etc., while lower expression levels in those of bladder urothelial carcinoma (BLCA), breast invasive carcinoma (BRCA), etc. And WTAP demonstrated multifaceted prognostic value in cancers. Of our interests, WTAP exerted a harmful effect on LIHC patient for overall survival (OS) and progression free survival (PFS). WTAP expression also significantly associated with the infiltration levels of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells (DC) in LIHC but not ESCA. Furthermore, combined analysis about WTAP expression level and immune cell specific gene markers implied WTAP correlates with regulatory cells (T reg) infiltration in LIHC and ESCA.Conclusion: The m6A regulator WTAP can serve as a prognostic biomarker for certain tumor types in pan-cancer and potentially result from immune cell infiltration.

Wilms' tumor 1-associating protein (WTAP), a core component of the N6-methyladenosine (m6A) methyltransferase complex, plays a crucial role in various biological processes. However, functional studies of WTAP in atherosclerosis development are largely unknown. Here, we demonstrate that WTAP expression was elevated in lipopolysaccharide (LPS)-stimulated macrophages and atherosclerotic lesions of apolipoprotein E-deficient (ApoE-/-) mice. To explore its functional role, we employed AAV8-mediated invivo knockdown and siRNA-mediated invitro silencing, which revealed that WTAP deficiency attenuated macrophage apoptosis and reduced atherosclerotic plaque formation. Mechanistically, we identified myosin heavy chain 11 (MYH11) as a mediator of WTAP-induced macrophage apoptosis. Notably, WTAP upregulated MYH11 expression in macrophages through an m6A-independent mechanism. These results delineate a new molecular paradigm that macrophage WTAP promotes macrophage apoptosis and atherosclerosis by increasing MYH11 expression, indicating that WTAP may be a potential therapeutic target against atherosclerosis.