Abstract 4442: Wilms' tumor 1-associating protein increases cancer cell proliferation and stem-cell like properties through RNA modification

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.

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.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

Interactions between mesenchymal glioblastoma stem cells (MES GSCs) and myeloid-derived macrophages (MDMs) shape the tumor-immunosuppressive microenvironment (TIME), promoting the progression of glioblastoma (GBM). N6-methyladenosine (m6A) plays important roles in the tumor progression. However, the mechanism of m6A in shaping the TIME of GBM remains elusive. Single-cell RNA sequencing and bulk RNA-seq datasets were employed to identify the critical role of WTAP in interactions between MES GBM and MDMs. The biological function of WTAP was confirmed both in vitro and in vivo. Mechanistically, mass spectrum, RNA immunoprecipitation (RIP), and co-immunoprecipitation assays were conducted. Here, we identified that m6A methyltransferase Wilms' tumor 1-associated protein (WTAP), whose protein stability could be synergistically enhanced via OGT-mediated O-GlcNAcylation and USP7-mediated de-ubiquitination, promoted LOXL2 m6A modification to enhance its mRNA stabilization in an IGF2BP2-dependent manner, upregulating secretion of LOXL2 protein (sLOXL2). sLOXL2 then interacted with integrin α5β1 on GSCs to activate FAK-ERK signaling, inducing mesenchymal transition of GSCs in an autocrine manner. Meanwhile, sLOXL2 also activated the integrin α5β1-FAK-ERK axis in MDMs, which promoted M2-like MDM phenotypes in a paracrine pathway, thereby contributing to T-cell exhaustion to induce GBM immune escape. In translational medicine, combinations of the OGT inhibitor by targeting WTAP expression and the LOXL2 antagonist by disrupting MES GSC and MDM interactions showed favorable outcomes to the anti-PD1 immunotherapy. WTAP plays critical roles in mesenchymal transition of GSCs and formation of TIME, highlighting the therapeutic potential of targeting WTAP and its downstream effectors to enhance the efficacy of immunotherapy.

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.

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.

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.

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.

This study aimed to analyze the functional roles and molecular mechanism of Wilms' tumor 1-associating protein (WTAP) in the tumorigenesis of nonsmall-cell lung cancer (NSCLC). Retrospective analysis was used. Tumor tissues and surrounding nontumor tissues of 150 patients with NSCLS who were surgically resected in the Fourth Hospital of Hebei Medical University from January 2016 to January 2018 were selected. The expression of WTAP in NSCLC tissues was detected by immunohistochemistry. Clinicopathologic parameters were then subjected to univariate and multivariate Cox regression analysis in purpose of uncovering the independent risk factors for overall survival time. MTS (3-[4,5-dimethylthiazol-zyl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazoliuzolium, inner salt) assay, colony formation assay, and transwell assays were performed to estimate cell proliferation, migration, and invasion. Meanwhile, the relationship between WTAP and the cell migration and invasion marker-related proteins were evaluated by Western blot analysis and RT-qPCR. WTAP expression was knocked-down in cell lines by shRNA, and RNA-Seq was performed to investigate the pathways regulated by WTAP. In NSCLC patients, WTAP was highly expressed in tumor tissues and the higher expression was significantly associated with poor overall survival (OS) ( P <0.01). Compared with the control group in vitro, the overexpression of WTAP could significantly promote cell proliferation, migration, and invasion ( P <0.01), while knock-down WTAP significantly reduces the above effects ( P <0.01). In a mouse orthotopic implantation model, higher WTAP abundance could significantly promote tumor enlargement compared with the control group ( P <0.01). Compared with the control group, the knock-down of WTAP significantly inhibit the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in cell lines ( P <0.01). Besides, in NSCLC, knocked-down CEACAM5 significantly reduced the impact of WTAP on cell proliferation, migration, and invasion compared with the control group ( P <0.05). This study suggests that high expression of WTAP was associated with poor clinical outcomes. CEACAM5 may play a synergistic role with WTAP to jointly promote NSCLC progression by enhancing cell proliferation, invasion, and migration.

The methyltransferase like 3 (METTL3)-containing methyltransferase complex catalyzes the N6-methyladenosine (m6A) formation, a novel epitranscriptomic marker; however, the nature of this complex remains largely unknown. Here we report two new components of the human m6A methyltransferase complex, Wilms' tumor 1-associating protein (WTAP) and methyltransferase like 14 (METTL14). WTAP interacts with METTL3 and METTL14, and is required for their localization into nuclear speckles enriched with pre-mRNA processing factors and for catalytic activity of the m6A methyltransferase in vivo. The majority of RNAs bound by WTAP and METTL3 in vivo represent mRNAs containing the consensus m6A motif. In the absence of WTAP, the RNA-binding capability of METTL3 is strongly reduced, suggesting that WTAP may function to regulate recruitment of the m6A methyltransferase complex to mRNA targets. Furthermore, transcriptomic analyses in combination with photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) illustrate that WTAP and METTL3 regulate expression and alternative splicing of genes involved in transcription and RNA processing. Morpholino-mediated knockdown targeting WTAP and/or METTL3 in zebrafish embryos caused tissue differentiation defects and increased apoptosis. These findings provide strong evidence that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and play a critical role in epitranscriptomic regulation of RNA metabolism.

Myocardial infarction (MI) is one of the leading causes of death. Wilms' tumor 1-associating protein (WTAP), one of the components of the m6A methyltransferase complex, has been shown to affect gene expression via regulating mRNA modification. Although WTAP has been implicated in various diseases, its role in MI is unclear. In this study, we found that hypoxia/reoxygenation (H/R) time-dependently increased WTAP expression, which in turn promoted endoplasmic reticulum (ER) stress and apoptosis, in human cardiomyocytes (AC16). H/R effects on ER stress and apoptosis were all blocked by silencing of WTAP, promoted by WTAP overexpression, and ameliorated by administration of ER stress inhibitor, 4-PBA. We then investigated the underlying molecular mechanism and found that WTAP affected m6A methylation of ATF4 mRNA to regulate its expression, and that the inhibitory effects of WTAP on ER stress and apoptosis were ATF4 dependent. Finally, WTAP’s effects on myocardial I/R injury were confirmed in vivo. WTAP promoted myocardial I/R injury through promoting ER stress and cell apoptosis by regulating m6A modification of ATF4 mRNA. These findings highlight the importance of WTAP in I/R injury and provide new insights into therapeutic strategies for MI.

Wilms’ tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.

N6-methyladenosine (m6A), the most prevalent posttranscriptional RNA modification, involved in various diseases and cellular processes. However, the underlying mechanisms of m6A regulation in skin aging are still not fully understood. In this study, proteomics analysis revealed a significant correlation between Wilms' tumor 1-associating protein (WTAP) expression and cellular senescence. Next, upregulated WTAP was detected in aging skin tissues and senescent human dermal fibroblasts (HDFs). Functionally, overexpressed WTAP induced senescence and knockdown of WTAP rescued senescence of HDFs. Mechanistically, WTAP directly targeted ELF3 and promoted its expression in an m6A-dependent manner. Exogenous-ELF3 overexpression evidently reversed shWTAP-suppressed fibroblast senescence. Furthermore, ELF3 induced IRF8-mediated senescence-associated secretory phenotype (SASP) by binding to the (-817 to -804) site of the IRF8 promoter directly. In vivo, overexpression of WTAP evidently increased senescence cells in skin and induced skin aging. In summary, these findings revealed the critical role of WTAP-mediated m6A modification in skin aging and identified ELF3 as an important target of m6A modification in HDFs senescence, providing a new idea for delaying the aging process.

Glioblastoma stem like cells (GSCs) are a group of cells with strong tumorigenicity that exist in glioblastoma (GBM). Wilms tumor 1-associated protein (WTAP) is thought to promote the malignant process of GBM. However, whether WTAP regulates GSCs function to mediate GBM process is still unclear. The expression levels of WTAP and CCAAT/enhancer-binding protein delta (CEBPD) were examined by qRT-PCR and western blot. GSCs stemness, proliferation, apoptosis, and migration were assessed using sphere formation assay, CCK8 assay, EdU assay, colony formation assay, flow cytometry and transwell assay. Cell glycolysis was evaluated by testing glucose consumption and lactification. The regulation of CEBPD on WTAP was confirmed by ChIP assay and dual-luciferase reporter assay. In vivo experiments were performed to explore the effect of CEBPD/WTAP on the tumorigenicity of GSCs. WTAP and CEBPD had increased expression in GBM tissues and GSCs. Silencing of WTAP suppressed GSCs stemnness, proliferation, migration, glycolysis and promoted apoptosis. CEBPD could bind to WTAP promoter region to enhance its transcription. Besides, WTAP overexpression reversed the suppressive effect of CEBPD knockdown on GSCs stemnness, growth, migration and glycolysis in vitro, as well as the reducing effect on tumorigenicity of GSCs in vivo. CEBPD/WTAP axis played a vital role in regulating GSCs function, providing a potential therapy target for GBM.

Vascular Biology and the Sex of Flies: Regulation of Vascular Smooth Muscle Cell Proliferation by Wilms’ Tumor 1–Associating Protein