ALKBH5 Modulates Asthma Progression by Downregulating N6-methyladenosine Methylation.

Asthma is a chronic respiratory disease that is characterized by airway inflammation, excessive mucus production, and airway remodeling. Prevention and treatment for asthma is an urgent issue in clinical studies. In recent years, N6-methyladenosine methylation (m6A) has emerged as a promising regulatory approach involved in multiple diseases. ALKBH5 (alkB homolog 5) is a demethylase widely studied in disease pathologies. This work aimed to explore the regulatory mechanisms underlying the ALKBH5-regulated asthma. We established an interleukin-13 (IL-13)-stimulated cell model to mimic the in vitro inflammatory environment of asthma. ALKBH5 knockdown in bronchial epithelial cells was performed using siRNAs, and the knockdown efficacy was analyzed by quantitative PCR (qPCR). Cell viability and proliferation were measured by cell counting kit 8 (CCK-8) and colony formation assay. The ferroptosis was assessed by measuring the total iron, Fe2+, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD) levels. The enrichment of N6-methyladenosine methylation (m6A) modification was detected by the MeRIP assay. Knockdown of ALKBH5 significantly elevated the survival and colony formation ability of bronchial epithelial cells in the IL-13 induction model. The levels of total iron, Fe2+, lipid ROS, and MDA were remarkedly elevated, and the SOD level was reduced in IL-13-induced bronchial epithelial cells, and depletion of ALKBH5 reversed these effects. Knockdown of ALKBH5 elevated the enrichment of m6A modification and expression of glutathione peroxidase 4 (GPX4). Knockdown of GPX4 abolished the pro-proliferation and anti-ferroptosis effects of siALKBH5. Knockdown of ALKBH5 improved the proliferation of bronchial epithelial cells and alleviated cell ferroptosis.

ALKBH5 promotes non-small cell lung cancer progression and susceptibility to anti-PD-L1 therapy by modulating interactions between tumor and macrophages.

BACKGROUND: Understanding the mechanisms that mediate the interaction between tumor and immune cells may provide therapeutic benefit to patients with cancer. The N6-methyladenosine (m6A) demethylase, ALKBH5 (alkB homolog 5), is overexpressed in non-small cell lung cancer. However, its role in the tumor microenvironment is unknown. METHODS: Datasets and tissue samples were used to determine the relationship between ALKBH5 expression and immunotherapy efficacy. Bioinformatic analysis, colorimetric assay to determine m6A RNA methylation, dual luciferase reporter assay, RNA/m6A-modified RNA immunoprecipitation, RNA stability assay, and RNA sequencing were used to investigate the regulatory mechanism of ALKBH5 in non-small cell lung cancer. In vitro and in vivo assays were performed to determine the contribution of ALKBH5 to the development of non-small cell lung cancer. RESULTS: ALKBH5 was upregulated in primary non-small cell lung cancer tissues. ALKBH5 was positively correlated with programmed death-ligand 1 expression and macrophage infiltration and was associated with immunotherapy response. JAK2 was identified as a target of ALKBH5-mediated m6A modification, which activates the JAK2/p-STAT3 pathway to promote non-small cell lung cancer progression. ALKBH5 was found to recruit programmed death-ligand 1-positive tumor-associated macrophages and promote M2 macrophage polarization by inducing the secretion of CCL2 and CXCL10. ALKBH5 and tumor-associated macrophage-secreted IL-6 showed a synergistic effect to activate the JAK2/p-STAT3 pathway in cancer cells. CONCLUSIONS: ALKBH5 promotes non-small cell lung cancer progression by regulating cancer and tumor-associated macrophage behavior through the JAK2/p-STAT3 pathway and the expression of CCL2 and CXCL10, respectively. These findings suggest that targeting ALKBH5 is a promising strategy of enhancing the anti-tumor immune response in patients with NSCLC and that identifying ALKBH5 status could facilitate prediction of clinical response to anti-PD-L1 immunotherapy.

ALKBH5-mediated m6A modification of circFOXP1 promotes gastric cancer progression by regulating SOX4 expression and sponging miR-338-3p.

Circular RNAs (circRNAs) have recently been suggested as potential functional modulators of cellular physiology processes in gastric cancer (GC). In this study, we demonstrated that circFOXP1 was more highly expressed in GC tissues. High circFOXP1 expression was positively associated with tumor size, lymph node metastasis, TNM stage, and poor prognosis in patients with GC. Cox multivariate analysis revealed that higher circFOXP1 expression was an independent risk factor for disease-free survival (DFS) and overall survival (OS) in GC patients. Functional studies showed that increased circFOXP1 expression promoted cell proliferation, cell invasion, and cell cycle progression in GC in vitro. In vivo, the knockdown of circFOXP1 inhibited tumor growth. Mechanistically, we observed ALKBH5-mediated m6A modification of circFOXP1 and circFOXP1 promoted GC progression by regulating SOX4 expression and sponging miR-338-3p in GC cells. Thus, our findings highlight that circFOXP1 could serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for GC.

Astrocytic ALKBH5 in stress response contributes to depressive-like behaviors in mice.

Epigenetic mechanisms bridge genetic and environmental factors that contribute to the pathogenesis of major depression disorder (MDD). However, the cellular specificity and sensitivity of environmental stress on brain epitranscriptomics and its impact on depression remain unclear. Here, we found that ALKBH5, an RNA demethylase of N6-methyladenosine (m6A), was increased in MDD patients' blood and depression models. ALKBH5 in astrocytes was more sensitive to stress than that in neurons and endothelial cells. Selective deletion of ALKBH5 in astrocytes, but not in neurons and endothelial cells, produced antidepressant-like behaviors. Astrocytic ALKBH5 in the mPFC regulated depression-related behaviors bidirectionally. Meanwhile, ALKBH5 modulated glutamate transporter-1 (GLT-1) m6A modification and increased the expression of GLT-1 in astrocytes. ALKBH5 astrocyte-specific knockout preserved stress-induced disruption of glutamatergic synaptic transmission, neuronal atrophy and defective Ca2+ activity. Moreover, enhanced m6A modification with S-adenosylmethionine (SAMe) produced antidepressant-like effects. Our findings indicate that astrocytic epitranscriptomics contribute to depressive-like behaviors and that astrocytic ALKBH5 may be a therapeutic target for depression.

N6-Methyladenosine modified circ-NAB1 modulates cell cycle and epithelial-mesenchymal transition via CDKN3 in endometrial cancer.

Endometrial cancer (EC) is a common malignant tumor in the female reproductive system. Circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification are widely involved in cancer progression. Nevertheless, the cross-talk between circ-NAB1 and m6A as well as the biological functions of circ-NAB1 in EC remain unclear. Circ-NAB1 was observed to be upregulated in EC tissues and cells by RT-qPCR. MeRIP and RNA pull-down assays were utilized for detecting the m6A modification of circ-NAB1. The interaction between circ-NAB1 and RNAs was also detected. Colony formation, transwell, flow cytometry, and western blot were utilized for measuring EC cell behaviors. Mechanically, we proved the m6A demethylase alkylation repair homolog protein 5 (ALKBH5) can mediate circ-NAB1 expression through an m6A-YTHDF2-dependent manner. Circ-NAB1 overexpression can promote cell proliferation, migration, invasion, epithelial-mesenchymal transition (EMT) process, and cell cycle through functional assays. Circ-NAB1 knockdown exerts the opposite function on EC cells. Furthermore, we proved that circ-NAB1 can sponge miR-876-3p to upregulate the target gene cyclin-dependent kinase inhibitor 3 (CDKN3) in EC cells. CDKN3 overexpression can reverse the impacts of circ-NAB1 depletion on EC cell behaviors. Collectively, we proved that ALKBH5-mediated m6A modification of circ-NAB1 promoted EMT process and cell cycle in EC via targeting the miR-876-3p/CDKN3 axis.

The demethylase ALKBH5 mediates ZKSCAN3 expression through the m6A modification to activate VEGFA transcription and thus participates in MNNG-induced gastric cancer progression.

N-Nitroso compounds (NOCs) are recognized as important factors that promote gastric cancer development, but the specific effects and potential mechanisms by which NOC exposure promotes gastric cancer are still poorly understood. In this study, we explored the effects and potential molecular mechanisms of NOCs on the promotion of gastric cancer using methylnitronitrosoguanidine (MNNG), a classical direct carcinogen of NOC. The results of in vivo and in vitro experiments showed that chronic and low-concentration MNNG exposure significantly promoted the malignant progression of tumors, including cell migration, cell invasion, vasculogenic mimicry (VM) formation, cell spheroid formation, stem cell-like marker expression, and gastric cancer growth and metastasis. Mechanistically, we revealed that demethylase ALKBH5 regulated the level of the N6­methyladenosine (m6A) modification in the 3'UTR and CDS region of the ZKSCAN3 mRNA to promote ZKSCAN3 expression, mediated the binding of ZKSCAN3 to the VEGFA promoter region to regulate VEGFA transcription, and participated in MNNG-induced gastric cancer cell migration, invasion, VM formation, cell spheroid formation, stem cell-like marker expression and ultimately gastric cancer progression. In addition, our study revealed that ALKBH5-ZKSCAN3-VEGFA signaling was significantly activated during MNNG-induced gastric carcinogenesis, and further studies in gastric cancer patients showed that ALKBH5, ZKSCAN3, and VEGFA expression were upregulated in cancers compared with paired gastric mucosal tissues, that ALKBH5, ZKSCAN3, and VEGFA could serve as important biomarkers for determining patient prognosis, and that the molecular combination showed greater prognostic value. These findings provide a theoretical basis for developing gastric cancer interventions for NOCs and for determining gastric cancer progression.

ALKBH5 targets ACSL4 mRNA stability to modulate ferroptosis in hyperbilirubinemia-induced brain damage.

Bilirubin-induced brain damage is a serious clinical consequence of hyperbilirubinemia, yet the underlying molecular mechanisms remain largely unknown. Ferroptosis, an iron-dependent cell death, is characterized by iron overload and lipid peroxidation. Here, we report a novel regulatory mechanism of demethylase AlkB homolog 5 (ALKBH5) in acyl-coenzyme A synthetase long-chain family member 4 (ACSL4)-mediated ferroptosis in hyperbilirubinemia. Hyperdifferential PC12 cells and newborn Sprague-Dawley rats were used to establish in vitro and in vivo hyperbilirubinemia models, respectively. Proteomics, coupled with bioinformatics analysis, first suggested the important role of ferroptosis in hyperbilirubinemia-induced brain damage. In vitro experiments showed that ferroptosis is activated in hyperbilirubinemia, and ferroptosis inhibitors (desferrioxamine and ferrostatin-1) treatment effectively alleviates hyperbilirubinemia-induced oxidative damage. Notably, we observed that the ferroptosis in hyperbilirubinemia was regulated by m6A modification through the downregulation of ALKBH5 expression. MeRIP-seq and RIP-seq showed that ALKBH5 may trigger hyperbilirubinemia ferroptosis by stabilizing ACSL4 mRNA via m6A modification. Further, hyperbilirubinemia-induced oxidative damage was alleviated through ACSL4 genetic knockdown or rosiglitazone-mediated chemical repression but was exacerbated by ACSL4 overexpression. Mechanistically, ALKBH5 promotes ACSL4 mRNA stability and ferroptosis by combining the 669 and 2015 m6A modified sites within 3' UTR of ACSL4 mRNA. Our findings unveil a novel molecular mechanism of ferroptosis and suggest that m6A-dependent ferroptosis could be an underlying clinical target for the therapy of hyperbilirubinemia.

ALKBH5 modulates macrophages polarization in tumor microenvironment of ovarian cancer.

BACKGROUND: Macrophages play an essential role in regulating ovarian cancer immune microenvironment. Studies have shown that m6A methylation could influence immune microenvironment in cancer. In this study, we investigated the roles of m6A demethylase ALKBH5 and m6A recognition protein IGF2BP2 played in regulating macrophages polarization in ovarian cancer. METHODS: In this study, we first explored the differentially expressed m6A methylation enzymes in M0 and M2 macrophages according to two independent GEO datasets. TIMER2.0 and GSCA database were used to explore the immune analysis of ALKBH5 and IGF2BP2 in ovarian cancer. K-M plotter and TIMER2.0 databases were used to evaluate the prognostic role of ALKBH5 and IGF2BP2 in ovarian cancer. For CNV mutation analysis of ALKBH5 and IGF2BP2, cBioPortal and GSCA databases were used. For single-cell analysis, sc-TIME and HPA softwares were used to analyze the roles of ALKBH5 and IGF2BP2 played in immune cells in ovarian cancer. To identify the role of ALKBH5 played in macrophage polarization, RT-PCR was used to verify the macrophage polarization related markers in vitro study. The function of ALKBH5 played in ovarian cancer was further analyzed through GO and KEGG analysis. FINDINGS: In this study, we found that ALKBH5 and IGF2BP2 were up-regulated in M2 macrophages, which showed closely correlation with immune cells expressions in ovarian cancer, especially with macrophages. Ovarian cancer patients with higher expression of ALKBH5 and IGF2BP2 showed worse prognosis, possibly because of their close correlation with immune response. ALKBH5 also correlated with macrophage phenotypes in single-cell levels analysis. However, the expression level of IGF2BP2 in ovarian cancer immune microenvironment was very low. The results of RT-PCR indicated the potential role of ALKBH5 in M2 polarization of macrophages. INTERPRETATION: ALKBH5 participated in regulating macrophage M2 polarization in ovarian cancer immune microenvironment.

ALKBH5-mediated m6A demethylation of pri-miR-199a-5p exacerbates myocardial ischemia/reperfusion injury by regulating TRAF3-mediated pyroptosis.

Myocardial ischemia‒reperfusion injury (MI/RI) is closely related to pyroptosis. alkB homolog 5 (ALKBH5) is abnormally expressed in the MI/RI models. However, the detailed molecular mechanism of ALKBH5 in MI/RI has not been elucidated. In this study, rats and H9C2 cells served as experimental subjects and received MI/R induction and H/R induction, respectively. The abundance of the targeted molecules was evaluated using RT-qPCR, Western blotting, immunohistochemistry, immunofluorescence, and enzyme-linked immunosorbent assay. The heart functions of the rats were evaluated using echocardiography, and heart injury was evaluated. Cell viability and pyroptosis were determined using cell counting Kit-8 and flow cytometry, respectively. Total m6A modification was measured using a commercial kit, and pri-miR-199a-5p m6A modification was detected by Me-RNA immunoprecipitation (RIP) assay. The interactions among the molecules were validated using RIP and luciferase experiments. ALKBH5 was abnormally highly expressed in H/R-induced H9C2 cells and MI/RI rats. ALKBH5 silencing improved injury and inhibited pyroptosis. ALKBH5 reduced pri-miR-199a-5p m6A methylation to block miR-199a-5p maturation and inhibit its expression. TNF receptor-associated Factor 3 (TRAF3) is a downstream gene of miR-199a-5p. Furthermore, in H/R-induced H9C2 cells, the miR-199a-5p inhibitor-mediated promotion of pyroptosis was reversed by ALKBH5 silencing, and the TRAF3 overexpression-mediated promotion of pyroptosis was offset by miR-199a-5p upregulation. ALKBH5 silencing inhibited pri-miR-199a-5p expression and enhanced pri-miR-199a-5p m6A modification to promote miR-199a-5p maturation and enhance its expression, thereby suppressing pyroptosis to alleviate MI/RI through decreasing TRAF3 expression.

HSPA4 upregulation induces immune evasion via ALKBH5/CD58 axis in gastric cancer.

INTRODUCTION: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. Recently, targeted therapies including PD1 (programmed cell death 1) antibodies have been used in advanced GC patients. However, identifying new biomarker for immunotherapy is still urgently needed. The objective of this study is to unveil the immune evasion mechanism of GC cells and identify new biomarkers for immune checkpoint blockade therapy in patients with GC. METHODS: Coimmunoprecipitation and meRIP were performed to investigate the mechanism of immune evasion of GC cells. Cocuture system was established to evaluate the cytotoxicity of cocultured CD8+ T cells. The clinical significance of HSPA4 upregulation was analyzed by multiplex fluorescent immunohistochemistry staining in GC tumor tissues. RESULTS: Histone acetylation causes HSPA4 upregulation in GC tumor tissues. HSPA4 upregulation increases the protein stability of m6A demethylase ALKBH5. ALKBH5 decreases CD58 in GC cells through m6A methylation regulation. The cytotoxicity of CD8+ T cells are impaired and PD1/PDL1 axis is activated when CD8+ T cells are cocultured with HSPA4 overexpressed GC cells. HSPA4 upregulation is associated with worse 5-year overall survival of GC patients receiving only surgery. It is an independent prognosis factor for worse survival of GC patients. In GC patients receiving the combined chemotherapy with anti-PD1 immunotherapy, HSPA4 upregulation is observed in responders compared with non-responders. CONCLUSION: HSPA4 upregulation causes the decrease of CD58 in GC cells via HSPA4/ALKBH5/CD58 axis, followed by PD1/PDL1 activation and impairment of CD8+ T cell's cytotoxicity, finally induces immune evasion of GC cells. HSPA4 upregulation is associated with worse overall survival of GC patients with only surgery. Meanwhile, HSPA4 upregulation predicts for better response in GC patients receiving the combined immunotherapy.

Circ_0005615 enhances multiple myeloma progression through interaction with EIF4A3 to regulate MAP3K4 m6A modification mediated by ALKBH5.

BACKGROUND: Circular RNAs (circRNAs) are associated with development and progression of multiple myeloma (MM). However, the role and mechanism of circ_0005615 in MM have not been elucidated. METHODS: Circ_0005615 was determined by GEO database. quantitative RT-PCR was performed to confirm the expression of circ_0005615 in peripheral blood of MM patients and MM cells. The roles of circ_0005615 in MM were analyzed using CCK8, transwell invasion, cell apoptosis and tumor xenograft experiments. Bioinformatics tools, RIP and RNA pull down assays were conducted to explore the downstream of circ_0005615. Furthermore, the mechanism was investigated by quantitative RT-PCR, western blot, dot blot and meRIP-PCR assays. RESULTS: Circ_0005615 was upregulated in MM. Overexpression of circ_0005615 promoted cell viability and invasion, and suppressed apoptosis in vitro, which were opposite when circ_0005615 was knockdowned. Mechanistically, EIF4A3, a RNA-binding protein (RBP), could directly bind to circ_0005615 and ALKBH5, where ALKBH5 could directly combine with MAP3K4, forming a circ_0005615- EIF4A3-ALKBH5-MAP3K4 module. Furthermore, circ_0005615 overexpression increased m6A methylation of MAP3K4 by inhibiting ALKBH5, leading to decreased MAP3K4. Further functional experiments indicated that ALKBH5 overexpression weakened the promoting roles of circ_0005615 overexpression in MAP3K4 m6A methylation and tumor progression in MM. The above functions and mechanism were also verified in vivo. CONCLUSIONS: Elevated circ_0005615 decreased MAP3K4 mediated by ALKBH5 through interacting with EIF4A3, thereby accelerating MM progression. Circ_0005615 might be a promising biomarker and target of MM.

High expression of AlkB homolog 5 suppresses the progression of non-small cell lung cancer by facilitating ferroptosis through m6A demethylation of SLC7A11.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is a prevailing LC characterized by poor outcomes. AlkB homolog 5 (ALKBH5) functions as a tumor suppressor in several cancers. This study delved into the role of ALKBH5 in NSCLC development. METHODS: TCGA database predicted ALKBH5 expression in NSCLC patients. ALKBH5 levels in NSCLC and human bronchial epithelial cells were determined. pcDNA3.1-ALKBH5/NC, pcDNA3.1-SLC7A11/NC, and ferrostatin-1 were used to explore the interactions among ALKBH5, SLC7A11, and ferroptosis. SLC7A11 mRNA and its protein levels were measured by RT-qPCR and Western blot. Cell viability, apoptosis, migration, and invasion were assessed by CCK-8, flow cytometry, and Transwell. Total N6-methyladenosine (m6A) quantification and its enrichment on SLC7A11 mRNA were determined, followed by the observation of Ki67, ALKBH5 and SLC7A11-positive cell numbers. Glutathione (GSH), lipid reactive oxygen species (lipid-ROS), malondialdehyde (MDA), and iron ion contents were determined. Animal experiments further analyzed the role of ALKBH5 in tumor development and glutathione peroxidase 4 (GPX4) expression. RESULTS: Bioinformatics analysis revealed the lowly-expressed ALKBH5 in LC patients. ALKBH5 was downregulated in NSCLC cells and its upregulation repressed proliferation activity, invasion, and migration, and facilitated apoptosis. ALKBH5 upregulation decreased GSH, increased lipid-ROS, MDA, and iron ion contents, and downregulated SLC7A11 by reducing m6A modification. SLC7A11 upregulation partly annulled the effect of ALKBH5 overexpression on cell ferroptosis and malignant behaviors. In vivo assays elucidated the suppression of ALKBH5 upregulation on tumor development and GPX4 levels. CONCLUSION: ALKBH5 upregulation downregulates SLC7A11 transcription by decreasing m6A modification, thus promoting NSCLC cell ferroptosis and ultimately repressing NSCLC progression.

RNA m6A modification, signals for degradation or stabilisation?

The RNA modification N6-methyladenosine (m6A) is conserved across eukaryotes, and profoundly influences RNA metabolism, including regulating RNA stability. METTL3 and METTL14, together with several accessory components, form a 'writer' complex catalysing m6A modification. Conversely, FTO and ALKBH5 function as demethylases, rendering m6A dynamic. Key to understanding the functional significance of m6A is its 'reader' proteins, exemplified by YTH-domain-containing proteins (YTHDFs) canonical reader and insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs) non-canonical reader. These proteins play a crucial role in determining RNA stability: YTHDFs mainly promote mRNA degradation through different cytoplasmic pathways, whereas IGF2BPs function to maintain mRNA stability. Additionally, YTHDC1 functions within the nucleus to degrade or protect certain m6A-containing RNAs, and other non-canonical readers also contribute to RNA stability regulation. Notably, m6A regulates retrotransposon LINE1 RNA stability and/or transcription via multiple mechanisms. However, conflicting observations underscore the complexities underlying m6A's regulation of RNA stability depending upon the RNA sequence/structure context, developmental stage, and/or cellular environment. Understanding the interplay between m6A and other RNA regulatory elements is pivotal in deciphering the multifaceted roles m6A plays in RNA stability regulation and broader cellular biology.

A LATS2 and ALKBH5 positive feedback loop supports their oncogenic roles.

N(6)-methyladenosine (m6A) critically regulates RNA dynamics in various biological processes. The m6A demethylase ALKBH5 promotes tumorigenesis of glioblastoma, while the intricate web that orchestrates its regulation remains enigmatic. Here, we discover that cell density affects ALKBH5 subcellular localization and m6A dynamics. Mechanistically, ALKBH5 is phosphorylated by the large tumor suppressor kinase 2 (LATS2), preventing its nuclear export and enhancing protein stability. Furthermore, phosphorylated ALKBH5 reciprocally erases m6A from LATS2 mRNA, thereby stabilizing this transcript. Unexpectedly, LATS2 depletion suppresses glioblastoma stem cell self-renewal independent of yes-associated protein activation. Additionally, deficiency in either LATS2 or ALKBH5 phosphorylation impedes tumor progression in mouse xenograft models. Moreover, high levels of LATS2 expression and ALKBH5 phosphorylation are associated with tumor malignancy in patients with gliomas. Collectively, our study unveils an oncogenic positive feedback loop between LATS2 and ALKBH5, revealing a non-canonical branch of the Hippo pathway for RNA processing and suggesting potential anti-cancer interventions.

ALKBH5-mediated m6A modification of IL-11 drives macrophage-to-myofibroblast transition and pathological cardiac fibrosis in mice.

Cardiac macrophage contributes to the development of cardiac fibrosis, but factors that regulate cardiac macrophages transition and activation during this process remains elusive. Here we show, by single-cell transcriptomics, lineage tracing and parabiosis, that cardiac macrophages from circulating monocytes preferentially commit to macrophage-to-myofibroblast transition (MMT) under angiotensin II (Ang II)-induced hypertension, with accompanying increased expression of the RNA N6-methyladenosine demethylases, ALKBH5. Meanwhile, macrophage-specific knockout of ALKBH5 inhibits Ang II-induced MMT, and subsequently ameliorates cardiac fibrosis and dysfunction. Mechanistically, RNA immunoprecipitation sequencing identifies interlukin-11 (IL-11) mRNA as a target for ALKBH5-mediated m6A demethylation, leading to increased IL-11 mRNA stability and protein levels. By contrast, overexpression of IL11 in circulating macrophages reverses the phenotype in ALKBH5-deficient mice and macrophage. Lastly, targeted delivery of ALKBH5 or IL-11 receptor α (IL11RA1) siRNA to monocytes/macrophages attenuates MMT and cardiac fibrosis under hypertensive stress. Our results thus suggest that the ALKBH5/IL-11/IL11RA1/MMT axis alters cardiac macrophage and contributes to hypertensive cardiac fibrosis and dysfunction in mice, and thereby identify potential targets for cardiac fibrosis therapy in patients.

ALKBH5 facilitates the progression of skin cutaneous melanoma via mediating ABCA1 demethylation and modulating autophagy in an m6A-dependent manner.

Background: N6-methyladenosine (m6A) is the most common and abundant mRNA modification, playing an essential role in biological processes and tumor development. However, the role of m6A methylation in skin cutaneous melanoma (SKCM) is not yet clear. This study analyzed the expression of m6A-related functional genes in SKCM and aimed to explore the key demethylase ALKBH5 mediated m6A modification and its potential mechanism in human SKCM. Methods: Based on public databases, the m6A-related gene expression landscape in SKCM was portrayed. MeRIP-Seq and RNA-Seq were used to recognize the downstream target of ALKBH5. In vivo and in vitro functional phenotype and rescue functional experiments were performed to explore the mechanism of the ALKBH5-m6A-ABCA1 axis in SKCM. Results: We found ALKBH5 upregulated in SKCM, associated with poor prognosis. ALKBH5 can promote melanoma cell proliferation, colony formation, migration, and invasion and inhibit autophagy in vitro, facilitating tumor growth and metastasis in vivo. We identified ABCA1, a membrane protein that assists cholesterol efflux, as a downstream target of ALKBH5-mediated m6A demethylation. Finally, our data demonstrated that ALKBH5 promoted SKCM via mediating ABCA1 downregulation by reducing ABCA1 mRNA stability in an m6A-dependent manner. Conclusion: Our findings exhibited the functional value of the key demethylase ALKBH5 mediated m6A modification in the progression of SKCM, suggesting the ALKBH5-m6A-ABCA1 axis as a potential therapeutic target in SKCM.

FSH induces EMT in ovarian cancer via ALKBH5-regulated Snail m6A demethylation.

Background: The therapeutic benefits of targeting follicle-stimulating hormone (FSH) receptor in treatment of ovarian cancer are significant, whereas the role of FSH in ovarian cancer progresses and the underlying mechanism remains to be developed. Methods: Tissue microarray of human ovarian cancer, tumor xenograft mouse model, and in vitro cell culture were used to investigate the role of FSH in ovarian carcinogenesis. siRNA, lentivirus and inhibitors were used to trigger the inactivation of genes, and plasmids were used to increase transcription of genes. Specifically, pathological characteristic was assessed by histology and immunohistochemistry (IHC), while signaling pathway was studied using western blot, quantitative RT-PCR, and immunofluorescence. Results: Histology and IHC of human normal ovarian and tumor tissue confirmed the association between FSH and Snail in ovarian cancer metastasis. Moreover, in epithelial ovarian cancer cells and xenograft mice, FSH was showed to promote epithelial mesenchymal transition (EMT) progress and metastasis of ovarian cancer via prolonging the half-life of Snail mRNA in a N6-methyladenine methylation (m6A) dependent manner, which was mechanistically through the CREB/ALKBH5 signaling pathway. Conclusions: These findings indicated that FSH induces EMT progression and ovarian cancer metastasis via CREB/ALKBH5/Snail pathway. Thus, this study provided new insight into the therapeutic strategy of ovarian cancer patients with high level of FSH.

ALKBH5-mediated upregulation of CPT1A promotes macrophage fatty acid metabolism and M2 macrophage polarization, facilitating malignant progression of colorectal cancer.

m6A modification has been studied in tumors, but its role in host anti-tumor immune response and TAMs polarization remains unclear. The fatty acid oxidation (FAO) process of TAMs is also attracting attention. A co-culture model of colorectal cancer (CRC) cells and macrophages was used to simulate the tumor microenvironment. Expression changes of m6A demethylase genes FTO and ALKBH5 were screened. ALKBH5 was further investigated. Gain-of-function experiments were conducted to study ALKBH5's effects on macrophage M2 polarization, CRC cell viability, proliferation, migration, and more. Me-RIP and Actinomycin D assays were performed to study ALKBH5's influence on CPT1A, the FAO rate-limiting enzyme. AMP, ADP, and ATP content detection, OCR measurement, and ECAR measurement were used to explore ALKBH5's impact on macrophage FAO level. Rescue experiments validated ALKBH5's mechanistic role in macrophage M2 polarization and CRC malignant development. In co-culture, CRC cells enhance macrophage FAO and suppress m6A modification in M2 macrophages. ALKBH5 was selected as the gene for further investigation. ALKBH5 mediates CPT1A upregulation by removing m6A modification, promoting M2 macrophage polarization and facilitating CRC development. These findings indicate that ALKBH5 enhances fatty acid metabolism and M2 polarization of macrophages by upregulating CPT1A, thereby promoting CRC development.

Demethylases in tumors and the tumor microenvironment: Key modifiers of N6-methyladenosine methylation.

RNA methylation modifications are widespread in eukaryotes and prokaryotes, with N6-methyladenosine (m6A) the most common among them. Demethylases, including Fat mass and obesity associated gene (FTO) and AlkB homolog 5 (ALKBH5), are important in maintaining the balance between RNA methylation and demethylation. Recent studies have clearly shown that demethylases affect the biological functions of tumors by regulating their m6A levels. However, their effects are complicated, and even opposite results have appeared in different articles. Here, we summarize the complex regulatory networks of demethylases, including the most important and common pathways, to clarify the role of demethylases in tumors. In addition, we describe the relationships between demethylases and the tumor microenvironment, and introduce their regulatory mechanisms. Finally, we discuss evaluation of demethylases for tumor diagnosis and prognosis, as well as the clinical application of demethylase inhibitors, providing a strong basis for their large-scale clinical application in the future.

A neural m6A pathway regulates behavioral aggregation in migratory locusts.

RNA N6-methyladenosine (m6A), as the most abundant modification of messenger RNA, can modulate insect behaviors, but its specific roles in aggregation behaviors remain unexplored. Here, we conducted a comprehensive molecular and physiological characterization of the individual components of the methyltransferase and demethylase in the migratory locust Locusta migratoria. Our results demonstrated that METTL3, METTL14 and ALKBH5 were dominantly expressed in the brain and exhibited remarkable responses to crowding or isolation. The individual knockdown of methyltransferases (i.e., METTL3 and METTL14) promoted locust movement and conspecific attraction, whereas ALKBH5 knockdown induced a behavioral shift toward the solitary phase. Furthermore, global transcriptome profiles revealed that m6A modification could regulate the orchestration of gene expression to fine tune the behavioral aggregation of locusts. In summary, our in vivo characterization of the m6A functions in migratory locusts clearly demonstrated the crucial roles of the m6A pathway in effectively modulating aggregation behaviors.