Deficiency of Mettl3 in Bladder Cancer Stem Cells Inhibits Bladder Cancer Progression and Angiogenesis

RNA N6-methyladenosine is a key step of posttranscriptional modulation that is involved in governing gene expression. The m6A modification catalyzed by Mettl3 has been widely recognized as a critical epigenetic regulation process for tumorigenic properties in various cancer cell lines, including bladder cancer. However, the in vivo function of Mettl3 in bladder cancer remains largely unknown. In our study, we found that ablation of Mettl3 in bladder urothelial attenuates the oncogenesis and tumor angiogenesis of bladder cancer using transgenic mouse model. In addition, conditional knockout of Mettl3 in K14+ bladder cancer stem cell population leads to inhibition of bladder cancer progression. Coupled with the global transcriptome sequencing and methylated RNA immunoprecipitation sequencing results, we showed that deletion of Mettl3 leads to the suppression of tyrosine kinase endothelial (TEK) and vascular endothelial growth factor A (VEGF-A) through reduced abundance of m6A peaks on a specific region. In addition, the depletion of Mettl3 results in the decrease in both messenger RNA (mRNA) and protein levels of TEK and VEGF-A in vitro. Taken together, Mettl3-mediated m6A modification is required for the activation of TEK–VEGF-A-mediated tumor progression and angiogenesis. Our findings may provide theoretical basis for bladder cancer treatment targeting Mettl3.


INTRODUCTION
Bladder cancer (BCa) is one of the most common malignancies worldwide, with approximately 200,000 death and 550,000 new cases yearly (Bray et al., 2018). Patients with primary tumor could be treated by surgical resection, but it is limited by the several major progressions like metastasis and recurrence (Griffiths and Action on Bladder Cancer, 2013;Crabb and Douglas, 2018). Furthermore, advanced BCa is characterized by high recurrence rate and poor prognosis, making it a growing social and medical challenge. During the past decades, despite great efforts on larger range of novel therapies for advanced BCa, limited clinical efficacy is obtained (Powles et al., 2017;Powles and Morrison, 2018). Therefore, a deeper understanding of specific mechanism in BCa metastasis and recurrence as well as the exploration of effective targets for better disease control has become major goals to be attained.
N6-methyladenosine (m 6 A), a predominantly internal modification of RNA in mammalian cells, is demonstrated to regulate RNA translation, degradation, splicing, export, and folding (Wang et al., , 2015Zhao et al., 2014;He et al., 2019). m 6 A regulation is dynamic and reversible epigenetic modification mediated by three components: "writer" complex, "eraser, " and "reader" (Jia et al., 2011;Zheng et al., 2013;Liu et al., 2014;Li et al., 2017). Methyltransferase-like 3 and 14 (Mettl3 and Mettl14) and their cofactors, Wilms tumor 1 associated protein (WTAP), VIRMA (KIAA1429), and RBM15 function as writers and form m 6 A methyltransferase complex (MTC), which catalyzes A (adenosine) to become m 6 A. Emerging insights into the role of m 6 A regulation in biological processes and pathogenesis of diseases has been uncovered in recent years, mainly including cell fate determination, embryonic development, and tumor progression (Roundtree et al., 2017;Frye et al., 2018). For example, data show that m 6 A modification pathway could exert carcinogenic or anticancer influence in various tumors for the expression pattern of the same m 6 Aregulated proteins, and their target messenger RNAs (mRNAs) may differ with the types of tumor Sun et al., 2019). Previous studies have demonstrated that Mettl3-mediated m 6 A modification directly regulates AFF4/NF-κB/MYC signaling network and ITGA6 mRNA, hence promoting BCa progression Jin et al., 2019). However, the understanding of the molecular mechanism of m 6 A in BCa in vivo remains largely undefined. N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) is a wellknown carcinogen that can induce mouse bladder cancer, which resembles human bladder cancer (Kelsey, 2018). In addition, Upk3a creER transgenic mouse expresses a tamoxifen-inducible Cre recombinase in bladder urothelium and has been widely used for in vivo study of BCa (Korac-Prlic et al., 2020). Emerging evidence have shown that cancer stem cells can be used as a predictor for overall patient survival and process properties that are correlated with tumor progression, therapy resistance, and recurrence (Kreso and Dick, 2014). Cancer stem cells (CSCs) in BCa are responsible for unresponsive chemotherapy, and this unexpected consequence can be abrogated by celecoxib (Kurtova et al., 2015). Recently, an increased level of K5/6 and K14 has been found in basal-like BCa . K14 + bladder cancer cells play an important role in urothelial homeostasis and are described as a marker for the BCa stem cell population, which facilitates tumorigenesis and chemotherapy resistance in vivo (Volkmer et al., 2012;Kurtova et al., 2015;Papafotiou et al., 2016).
Once solid tumor grows up to a diameter of 2 mm, vascular system is a necessity for continued growth. Then, angiogenesis is acquired for tumor cells and function as a selective way to push the limits. Moreover, for tumor growth and invasion, angiogenesis has been considered to be the basic pathological feature during the process. Tyrosine kinase endothelial (TEK), also called TIE2, is an effective target for malignancy treatment by inhibition of tumor angiogenesis through ANG2/TIE2 axis (Mazzieri et al., 2011). Vascular endothelial growth factor A (VEGF-A) is a well-known growth factor that activates angiogenesis to promote cell migration, therefore collaborating with tumorigenesis (Goel and Mercurio, 2013). As part of tumor comprehensive therapy, antiangiogenesis therapy has been widely used in clinical practice. Furthermore, markers of angiogenesis such as CD31, CD34, and CD105 could serve as an independent prognostic factor in cancers (Tanaka et al., 2001;Randall et al., 2009). Thus, understanding the specific pathway of regulating angiogenesis in BCa is essential for developing future diagnostic and therapeutic strategies.
In this study, we aim to identify the role that m 6 A promotes BCa progression via focusing on the key methyltransferase Mettl3. Conditional knockout Mettl3 among K14 + or Upk3a + cells in vivo is found to restrain BCa development through disabling angiogenesis, which is further confirmed by RNA sequencing and analyzing the expression pattern of angiogenesis key factors. Collectively, we first verify the function of m 6 A mediated by Mettl3 for BCa and expatiate on the network of Mettl3 in governing tumor angiogenesis in vivo.

Animals
The transgenic mice were maintained under specific pathogenfree conditions at the Center of Experimental Animal of Sun Yat-sen University. All procedures and use of animal experiments were approved by Sun Yat-sen University Animal Care and Use Committee.
Upk3a CreER (JAX stock: 015855), K14 CreER (JAX stock: 005107), and C57BL/6J background mice were obtained from the Jackson Laboratory. Mettl3 flox/flox and Mettl3 KI mice were obtained as previously described (Liu et al., 2020). For induction of BCa, 6-8-week-old mice were treated with drinking water containing 500 µg/ml BBN for 16 weeks and then given normal water for another 10 weeks. Tamoxifen was intraperitonelly injected to the mice with 0.08 mg/g of body weight each day for 3 days in order to inductively knock out the target gene. Then, the experimental mice were sacrificed for collection of BCa samples.

Immumohistochemical Staining and IHC Scoring
All the BCa specimens collected were embedded in paraffin and then cut into 5-µm thick sections. The samples were deparaffinized for 10 min in two different tanks with Histoclear fluid after heating at 65 • C for 30 min; then, these sections were hydrated in different gradients of ethanol. The samples were incubated for 10 min in 3% H 2 O 2 to block endogenous peroxidases. After blocking, antigen retrieval was performed with ethylenediaminetetraacetic acid (EDTA) buffer (Beyotime Biotechnology, Shanghai, China) in a microwave oven with medium power for 5 min and high power for another 5 min (avoid complete drying of sections). Specific primary antibody (1:200) was used at 4 • C overnight in a wet box. Then, antiprimary secondary antibody was applied and incubated for 30 min at room temperature. The secondary antibody was removed and 3,3 -diaminobenzidine (DAB) dripped upon sections, and the color development index was observed. Hematoxylin was conducted for counterstaining cell nucleus. The immunohistochemistry (IHC) score was calculated by the product of staining intensity and frequency. The intensity was defined as 0 (negative), 1 + (weakly positive), 2 + (positive), or 3 + (strongly positive), and the frequency was considered to be percentage of positive cells.

H&E Staining
The sections were prepared by the same method as described above and stained with hematoxylin for 5 min. Next, hydrochloric acid alcohol was utilized to separate color for 5 s, and the sections were further stained with eosin for 2 min. After that, all samples were dehydrated by gradient alcohol and sealed with neutral balsam medium.

Immunofluorescence Staining
The BCa specimens were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Sigma, United States), followed by blocking with 1% bovine serum in phosphatebuffered saline (PBS) at room temperature. The sections were stained with primary antibody at 1:100 and were incubated at 4 • C overnight, followed by application of the corresponding secondary antibody conjugated with DyLight 488 and Fluor 594 (Invitrogen, United States). At the same time, normal serum was used as a control to guarantee the specificity of the primary antibody. The nuclei were counterstained with 4 ,6-diamidino-2phenylindole (DAPI) at 1:1000 for 1 min. Images were captured with an upright fluorescence microscope (Nikon, Japan).

RNA-Sequencing
Oligo (dT)-attached magnetic beads were used to purified mRNA from BBN-induced K14 CreER ; Mettl3 flox/flox mice or their control littermates carrying bladder tumors. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then, first-strand complementary DNA (cDNA) was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. Afterward, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and products were purified by Ampure XP Beads and then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The doublestranded PCR products from the previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single-strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB), which had more than 300 copies of one molecular; DNBs were loaded into the patterned nanoarray, and single-end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

Western Blot Analysis
The cell culture medium was discarded, and the cells were rinsed with 4 • C precooled phosphate-buffered saline (PBS) buffer. Total cell protein lysates were obtained from T24 and UMUC-3 cells after lysing in radioimmunoprecipitation assay (RIPA) (Beyotime Biotechnology, China) for 30 min. Quantified with a BCA assay kit (Cwbiotech, Beijing, China), the samples were mixed with 5 × loading buffer and conducted thermal denaturation at 95 • C for 5 min. Then, the protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to the polyvinylidene fluoride (PVDF) membrane with a constant current of 300 mA for 1.5 h (Millipore, Germany). Then, 5% skimmed milk was used for blocking, followed by incubation with primary antibody (1:1000) overnight. Trisbuffered saline with Tween 20 (TBST)-diluted secondary antibody was applied and incubated at room temperature for 1 h. The blots were visualized using the enhanced chemiluminescence (ECL) method with Tanon 5200 Multi intelligent imaging system.

m 6 A-Sequencing
For m 6 A-seq, total RNA was isolated from the bladder tissue with or without Mettl3 knockout using TRIzol reagent. mRNA was further purified from prepared total RNA with the use of the Dynabeads mRNA DIRECT kit (Thermo Fisher) according to the manufacturer's instructions. The purified RNA was used to interact with anti-m 6 A or control rabbit immunoglobulin G (IgG) antibodies, and the antibody-conjugated samples were incubated with IP buffer-washed Protein G Dynabeads at 4 • C overnight. Next day, the isolated products were applied for constructing libraries of inputs (control IgG) and methylated RNA immunoprecipitation sequencing (meRIPs) (anti-m 6 A antibody) with TruSeq Stranded mRNA Sample Prep Kits (Illumina RS-122-2101). Libraries were multiplexed on an Illumina HiSeq 2000 platform. Mapping of reads, calling of m6A peaks, recognition of specific motifs, and further analysis were carried out based on previous study (Dominissini et al., 2013).

KEGG and Gene Ontology Analysis
To explore the link between Mettl3 and the underlying mechanisms in BCa, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology Analysis were conducted using ToppGene Suite 1 and KEGG PATHWAY Database 2 to analyze the differentially expressed genes (DEGs). The statistical significance of difference was evaluated by P value, and P < 0.05 was considered statistically significant.

Gene Set Enrichment Analysis
Gene Set Enrichment Analysis (GSEA) software downloaded from the GSEA website 3 was performed to determine signaling pathways that were correlated between the Mettl3 knockout and control groups. The annotated gene sets of c5.go.v7.2.symbols.gmt in the Molecular Signatures Database (MSigDB) were selected in GSEA version 4.0. One thousand times of permutations were performed. The normalized enrichment score (NES) was served as the GSEA main statistic. Gene sets with nominal P < 0.05, false discovery rate (FDR) < 0.25, and normalized enrichment scores (NES) > 1.0 were considered significantly enriched.

Statistical Analysis
All the data were analyzed and presented as mean ± SD. Significant tests were conducted with unpaired parametric twotailed Student's t test for difference analysis between two groups or chi-square for tumor grade analysis. P < 0.05 was defined as statistical significance ( * P < 0.05, * * P < 0.01, * * * P < 0.001, and * * * * P < 0.0001). Data management and statistical analyses were performed using GraphPad Prism software, version 7.04.

Deletion of Mettl3 in Urothelium Diminishes the Development of Mouse BCa
To explore the role of Mettl3 in BCa initiation and development, we first crossed Upk3a CreER mice with Mettl3 flox/flox mice to generate Upk3a CreER mice; Mettl3 flox/flox mice that allow us to conditional ablate the expression of Mettl3 in umbrella, intermediate, and basal cells of mouse bladder (Johnson et al., 2016). Upk3a CreER mice; Mettl3 flox/flox and their control littermates were treated with BBN in their drinking water for 16 weeks before feeding normal water for another 10 weeks, as described previously (Irving et al., 1984). Then, mice were sacrificed for the collection of bladders. Measurement of bladder tumor indicated significantly decreased volume in Upk3a CreER ; Mettl3 flox/flox mice compared with the wild-type group ( Figure 1A). We further assessed the effect of Mettl3 conditional knockout on tumor weight in vivo and found a marked tendency to reduce in tumor weight ( Figure 1B). Observation from the H&E staining specimens demonstrated that mice developed lesions on the urothelium of the bladder upon the treatment with BBN, while Upk3a CreER ; Mettl3 flox/flox mice carried tumors that were less malignant than Upk3a CreER ; Mettl3 wt/wt mice ( Figure 1C). The result of BCa grading indicated a higher likelihood to develop into a high-grade tumor in wild-type mice ( Figure 1D). IHC staining was applied to examine the protein levels of Mettl3, Ki67, and Caspase-3 protein in BCa (Figure 1E). The reduced expression of Mettl3 and Ki67 with the increased level of Caspase-3 was detected by randomized double-blind IHC scoring in Upk3a CreER ; Mettl3 flox/flox mice, manifesting the role of Mettl3 in promoting proliferation and inhibiting apoptosis in vivo ( Figure 1F). A significant reduction in AKT1 (a proliferation-related factor)  and BCL9L (an inhibitor of apoptosis) (Huge et al., 2020) was observed by the loss of Mettl3 in Upk3a + BCa cells ( Figure 1G). These results indicate that Mettl3 is essential for cellular proliferation and survival in BBN-induced BCa.

Ablation of Mettl3 in K14 + Cancer Stem Cells Inhibits Tumorigenicity and Progression of BCa
We next investigated the function of Mettl3 in K14 + CSCs by generation of BBN-induced K14 CreER , Mettl3 flox/flox and K14 CreER , Mettl3 wt/wt mice. Bladder tumor size was obviously decreased after inducible knockout of Mettl3 in K14 + CSCs (Figure 2A). Similar to the results above, a notably reduced tumor weight was observed with the knockout of Mettl3 ( Figure 2B). Then, BCa sections taken from transgenic mice were stained with H&E kit to show the tumor histological morphology. The atypia of cells and tissues was more remarkable in wild-type mice compared with K14 CreER , Mettl3 flox/flox mice, revealing that ablation of Mettl3 in K14 + CSCs suppressed the malignant transformation of BCa ( Figure 2C). Additionally, bladder tumor grading described Mettl3 as a driving factor of cancer progression ( Figure 2D). We then performed immunofluorescence staining to detect the expression of K14 and expression level of Mettl3, Ki67, and Caspase-3 in our transgenic mice model. The data showed that Mettl3 was mostly knocked out after treatment with tamoxifen in K14-derived BCa cells followed by the suppression of Ki67 and enhancement of apoptosis, proving the effect of Mettl3 on elevating cell proliferative potential and limiting apoptosis (Figures 2E,F). The expression of genes associated with proliferation and survival as AKT1 and BCL9L were evaluated by IHC staining in specimens. Yet, the deficiency of Mettl3 led to the decrease in AKT1, while it increased the level of BCL9L ( Figure 2G). These data demonstrate that conditional knockout of Mettl3 in K14 + CSCs reduced the tumor aggressiveness in mice with BBN induction.

Overexpression of Mettl3 in Urothelium or K14 + CSCs BCa Cells Promotes the Growth of BCa
Complementarily, we constructed mice models with inducible conditional overexpression of Mettl3 (Mettl3 KI ) in the urothelium or K14 + CSCs using Upk3a CreER or K14 CreER . Comparing tumor bulk in Upk3a CreER wild-type and KI mice, the mice bearing BCa with overexpressed Mettl3 in Upk3a-derived cells developed tumors more aggressively (Figure 3A). It was also found that excessive Mettl3 in cells of Upk3a origin contributed to promoting tumor growth by measuring tumor mass in mice ( Figure 3B). Likewise, overexpression of Mettl3 in K14 + CSCs cells accelerated tumor growth and progression (Figures 3C,D). We then assessed the level of SOX2, the BCa stem cell marker, in K14 CreER wild type and Mettl3 KI mice, respectively, and a significantly elevated expression of SOX2 was observed after overexpressing Mettl3 (Figures 3E,F) (Zhu et al., 2017). IHC was performed to confirm the knock-in efficiency of Mettl3 (left), and there was an increased Ki67-positive cell along with enhanced staining intensity (right) after overexpression of Mettl3 ( Figure 3G). The level of Mettl3 and Ki67 was found to be elevated in Upk3a CreER KI mice by estimating IHC score ( Figure 3F). Subsequently, Ki67 was also discovered to be upregulated in K14 CreER , Mettl3 knock-in mice (Figures 3I,J). Taken together, our data demonstrate that overexpressed Mettl3 in bladder urothelium cells or K14 + CSCs escalate the tumor aggressiveness in mouse BCa model.

Mettl3 Regulates the PI3K/AKT Pathway and Tumor Angiogenesis in BCa
To determine the downstream mechanism of how Mettl3 regulates BCa progression, RNA-sequencing was conducted to investigate the mechanisms of Mettl3 in modulating downstream targets upon control and Upk3a/K14 CreER ; Mettl3 cKO flox/flox mice, and differentially expressed genes were shown by separating into down-, not significant (not sig.), and upregulation with the use of the volcano scatterplot ( Figure 4A). Gene Ontology of analysis in DEGs associated with biological process enrichment upon control and Mettl3 KO mice revealed that those genes were selectively enriched by the regulation of angiogenesis, ameboidal-type cell migration, regulation of vasculature development, and endothelial cell proliferation, which was likely involved in construction of tumor microenvironment and sustaining tumor metastasis ( Figure 4B). Next, we performed the KEGG enrichment analysis of down DEGs to systematically examine the cellular pathways affected by exhaustion of Mettl3, and the PI3K-Akt signaling pathway was discovered to be the top enrichment of 13 pathways ( Figure 4C). In addition, some representative oncogenic features, such as PI3K signaling, epithelial-mesenchymal transition, and angiogenesis were statistically enriched in Mettl3 conditional knockout (KO) mice, elucidating that Mettl3 might regulate BCa in the aspect of oncogenesis, metastasis, and tumor angiogenesismediated chemoresistance (Figures 4D-F). To further test this hypothesis, shRNAs targeting Mettl3 were applied to inhibit Mettl3 expression in vitro. Consistent with the results in vivo, downregulation of Mettl3 in T24 or UMUC-3 cells could suppress the transcripts and proteins of TEK and VEGF-A, which were the participants of PI3K/AKT pathway (Figures 4G,H).

Mettl3-Dependent m 6 A Modification Upon Target mRNAs Correlated With Tumor Angiogenesis and Pathways in Cancer
To investigate whether Mettl3-mediated m 6 A modification on direct targets could effectively influence biological process of BCa, we conducted methylated meRIP-seq to identify the role of Mettl3 in BCa. First, the Pathway Analysis displaying an obviously strong enrichment for pathways in cancer proved the great potential of Mettl3 in regulating BCa progression ( Figure 5A). The GO analysis on the gene set enriched by m 6 A indicated that Mettl3-medited methylation might participate in transcription regulation, protein,phosphorylation   and angiogenesis (Figure 5B). We then applied Integrative Genomics View (IGV) software to search for the possible targets based on our meRIP-seq datasets and found that m 6 A peaks were abundant upon TEK and VEGF-A genes that were responsible for tumor angiogenesis ( Figure 5C). Moreover, to investigate the possible m 6 A modification sites of the target genes, we analyzed online database with Mettl3 PAR CLIP and m 6 A CLIP using HeLa cell lines and identify specific modified base sites ( Figure 5D) Liu et al., 2014;Wang et al., 2014). Consistent with the previous reports, our results showed the most enriched motif "GGAC" in the specific regions of corresponding mRNAs ( Figure 5E).

Mettl3 Promotes Bladder Tumor Angiogenesis via Modulating TEK and VEGF-A
For comprehensive understanding of the internal regulatory mechanisms that Mettl3 mediated bladder tumor angiogenesis, we utilized immunostaining to detect and localize the angiogenesis-related factors in vivo. Given that CD31 and CD34 were the primary indicators of endothelial cells and vascular-associated tissue, we explored their expression pattern in BCa sections. BCa with knockout of Mettl3 in the urothelium exhibited apparent inhibition of angiogenesis (Figures 6A,B). Similarly, the angiogenesis capacity was markedly declined among K14 CreER , Mettl3 flox/flox BCa tissues (Figures 6C,D). We next quested whether the loss of Mettl3 was able to inhibit the expression of specific targets involved in tumor angiogenesis such as TEK and VEGF-A. IHC staining for assessment of downstream targets suggested that inducible knockout of Mettl3 in Upk3a-originated BCa cells significantly downregulated TEK and VEGF-A (Figures 6E,F). Coincidently, these phenomena also could be observed in tumor sections collected from K14 CreER WT and KO mice (Figures 6G,H). Concordant results were found performing RT-qPCR for detecting the mRNA level of TEK and CD34. Relative mRNA expression of TEK and VEGF-A among the Upk3a CreER , Mettl3 KO mice exerted dramatically reduced versus Upk3a CreER , Mettl3 wt/wt ( Figure 6I). Furthermore, ablation of Mettl3 in K14-derived BCa cells dramatically decreased VEGF-A level ( Figure 6J). Collectively, these data have testified that Mettl3 exerted its effect on tumor angiogenesis, supporting the role of Mettl3 as a collaborator of BCa progression by modulation of neovascularization.

DISCUSSION
Recently, RNA m 6 A methyltransferase Mettl3 has been reported to modulate AFF4/NF-κB/MYC signaling for BCa progression in vitro . However, the role of Mettl3 in BCa progression and tumor microenvironment regulation in vivo remains elusive. In this study, we show that Mettl3 facilitates BCa progression by mediating tumor angiogenesis in vivo using transgenic mouse model. Conditional depletion of Mettl3 among the urothelium or K14 + CSCs significantly restricts oncogenesis and prevents BBN-induced BCa from progressing to more aggressive stage. In addition, Mettl3 ablation leads to downregulation of proliferative index protein Ki67 and upregulation of apoptosis-related Caspase-3, indicating a role of Mettl3 in cell proliferation and survival of BCa. Consistent with the data from BCa mouse model, these phenotypes were also observed in vitro by impairment of Mettl3, which induced the inhibition of corresponding downstream targets like AKT1 or BCL9L. The global transcriptome sequencing with KEGG analysis reveals that Mettl3 is closely related to tumor angiogenesis. Furthermore, data generated from m 6 A-squencing portray a regulatory network of Mettl3-modulated TEK/PI3K/VEGF axis, which propels the angiogenesis surrounding tumor cells. Indeed, the IHC staining of blood vessel markers such as CD31 and CD34 confirmed that the absence of Mettl3 in the urothelium or K14 + CSCs reduces the level of angiogenesis in BCa tissues. Our findings offer convincing evidence of Mettl3 in epigenetically modifying TEK/PI3K/VEGF cascades involving in angiogenesis, suggesting that therapeutically targeting Mettl3 may provide one of the alternatives for antiangiogenesis therapy in bladder carcinoma.
The m 6 A modification catalyzed by Mettl3 has been proven to control specific target mRNAs translation efficiency and stability, which plays a key role in promoting progression of various cancers including lung cancer, endometrial cancer, leukemia, and colorectal cancer (Barbieri et al., 2017;Choe et al., 2018;Liu et al., 2018;Li et al., 2019). Mechanically, Mettl3 could activate zinc finger MYM-type containing 1 (ZMYM1) toward genes complex of chromatin via m 6 A modification, hence promoting epithelialmesenchymal transition (EMT) and metastasis of gastric cancer (Yue et al., 2019). Herein, we report that the mRNAs enriched by m 6 A peaks are downregulated by loss of Mettl3 likely as a result of mRNA instability. Furthermore, combining analyses of Mettl3 PAR CLIP and m 6 A CLIP sequencing, we have found possible sites bound by Mettl3 for m 6 A modification upon genes of TEK and VEGF-A. However, whether the extinction of Mettl3 interferes the translation efficiency of these targets is still unclear, which calls for further research.
Insights of regulatory mechanisms in tumor angiogenesis expand our horizons of the crosstalk between tumor cells and surrounding microenvironment. In our study, we have revealed a role of Mettl3 in angiogenesis of bladder cancer. A previous study has shown that human adipose stem cells (hASCs) can be activated by breast cancer cells, hence facilitating tumor growth and aggressiveness by promoting angiogenesis (Paino et al., 2017). However, whether Mettl3 promotes angiogenesis via interaction with preexisting vasculature endothelial cells or other mechanism, such as hASCs activation, will require further investigation. Previous study has shown Ang2-TEK signaling as a key regulator in lymphogenous metastasis (Gengenbacher et al., 2020). VEGF-A as a downstream factor of Zeb1 enhances breast cancer cells' aggressiveness through sustaining stemness (Jiang et al., 2020). Indeed, the regulatory networks of epigenomics, angiogenesis, and stemness maintenance are intricate. A recent study describes a more faithful murine BCa model that carries Upk3a-Cre ERT 2 ; Trp53 L/L ; Pten L/L ; Rosa26 LSL−Luc tumor applied to excavate the biology of BCa (Saito et al., 2018). Another research indicates that the subpopulation of bladder basal cells characterized by K14 possesses self-renewal ability, and these cells are defined as the origin of urothelial cancer, which contributes to BCa progression (Papafotiou et al., 2016). Encouragingly, a combination of these rationales of signal networks in BCa may depict the extensive interactions of stemness maintenance, epigenetic modulation, and tumor angiogenesis. Our results have revealed a novel mechanism of Mettl3 by which it mediates m 6 A modulation of TEK and VEGF-A to promote tumor angiogenesis.
In summary, our data demonstrate the critical role of Mettl3 in driving BCa progression, as featured by expediting angiogenesis surrounding tumor cells, and unveil a previously unrecognized signaling axis involving Mettl3-TEK-VEGF-A-CD31/CD34 in bladder malignancy. More importantly, this study proposes that targeting Mettl3 might bring profound influence on conquering chemotherapy resistance and immunotherapy tolerance caused by tumor angiogenesis, offering therapeutic alternatives to patients suffering this treatment dilemma.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
All institutional and national guidelines for the care and use of laboratory animals were followed.