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Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

ABSTRACT: Oncogenic KRAS mutations combined with the loss of the LKB1 tumor-suppressor gene (KL) are strongly associated with aggressive forms of lung cancer. N6-methyladenosine (m6A) in mRNA is a crucial epigenetic modification that controls cancer self-renewal and progression. However, the regulation and role of m6A modification in this cancer are unclear. We found that decreased m6A levels correlated with the disease progression and poor survival for KL patients. The correlation was mediated by a special increase in ALKBH5 (AlkB family member 5) levels, an m6A demethylase. ALKBH5 gain- or loss-of function could effectively reverse LKB1 regulated cell proliferation, colony formation, and migration of KRAS-mutated lung cancer cells. Mechanistically, LKB1 loss upregulated ALKBH5 expression by DNA hypermethylation of the CTCF-binding motif on the ALKBH5 promoter, which inhibited CTCF binding but enhanced histone modifications, including H3K4me3, H3K9ac, and H3K27ac. This effect could successfully be rescued by LKB1 expression. ALKBH5 demethylation of m6A stabilized oncogenic drivers, such as SOX2, SMAD7, and MYC, through a pathway dependent on YTHDF2, an m6A reader protein. The above findings were confirmed in clinical KRAS-mutated lung cancer patients. We conclude that loss of LKB1 promotes ALKBH5 transcription by a DNA methylation mechanism, reduces m6A modification, and increases the stability of m6A target oncogenes, thus contributing to aggressive phenotypes of KRAS-mutated lung cancer.

SUBMITTER: Zhang D

PROVIDER: S-EPMC8137886 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundN6-methyladenosine (m6A) modification is the most abundant reversible methylation modification in eukaryotes, and it is reportedly closely associated with a variety of cancers progression, including colorectal cancer (CRC). This study showed that activated lipid metabolism and glycolysis play vital roles in the occurrence and development of CRC. However, only a few studies have reported the biological mechanisms underlying this connection.MethodsProtein and mRNA levels of FTO and ALKBH5 were measured using western blot and qRT-PCR. The effects of FTO and ALKBH5 on cell proliferation were examined using CCK-8, colony formation, and EdU assays, and the effects on cell migration and invasion were tested using a transwell assay. m6A RNA immunoprecipitation (MeRIP) and RNA-seq was used to explore downstream target gene. RIP was performed to verify the interaction between m6A and HK2. The function of FTO and ALKBH5 in vivo was determined by xenograft in nude mice.ResultsIn this study, FTO and ALKBH5 were significantly down-regulated in CRC patients and cells both in vivo and in vitro in a high-fat environment. Moreover, FTO and ALKBH5 over-expression hampered cell proliferation both in vitro and in vivo. Conversely, FTO and ALKBH5 knockdown accelerated the malignant biological behaviors of CRC cells. The mechanism of action of FTO and ALKBH5 involves joint regulation of HK2, a key enzyme in glycolysis, which was identified by RNA sequencing and MeRIP-seq. Furthermore, reduced expression of FTO and ALKBH5 jointly activated the FOXO signaling pathway, which led to enhanced proliferation ability in CRC cells. IGF2BP2, as a m6A reader, positively regulated HK2 mRNA in m6A dependent manner. Additionally, down-regulation of FTO/ALKBH5 increased METTL3 and decreased METTL14 levels, further promoting CRC progression.ConclusionIn conclusion, our study revealed the FTO-ALKBH5/IGF2BP2/HK2/FOXO1 axis as a mechanism of aberrant m6A modification and glycolysis regulation in CRC.

Project description:Introduction: Middle ear cholesteatoma is characterized by the hyperproliferation of keratinocytes. In recent decades, N6-methyladenosine (m6A) modification has been shown to play an essential role in the pathogenesis of many proliferative diseases. However, neither the m6A modification profile nor its potential role in the pathogenesis of middle ear cholesteatoma has currently been investigated. Therefore, this study aimed to explore m6A modification patterns in middle ear cholesteatoma. Materials and methods: An m6A mRNA epitranscriptomic microarray analysis was performed to analyze m6A modification patterns in middle ear cholesteatoma tissue (n = 5) and normal post-auricular skin samples (n = 5). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to predict the potential biological functions and signaling pathways underlying the pathogenesis of middle ear cholesteatoma. Subsequently, m6A modification levels were verified by methylated RNA immunoprecipitation–qPCR (MeRIP–qPCR) in middle ear cholesteatoma tissue and normal skin samples, respectively. Results: A total of 6,865 distinctive m6A-modified mRNAs were identified, including 4,620 hypermethylated and 2,245 hypomethylated mRNAs, as well as 9,162 differentially expressed mRNAs, including 4,891 upregulated and 4,271 downregulated mRNAs, in the middle ear cholesteatoma group relative to the normal skin group. An association analysis between methylation and gene expression demonstrated that expression of 1,926 hypermethylated mRNAs was upregulated, while expression of 2,187 hypomethylated mRNAs and 38 hypermethylated mRNAs was downregulated. Moreover, GO analysis suggested that differentially methylated mRNAs might influence cellular processes and biological behaviors, such as cell differentiation, biosynthetic processes, regulation of molecular functions, and keratinization. KEGG pathway analysis demonstrated that the hypermethylated transcripts were involved in 26 pathways, including the Hippo signaling pathway, the p53 signaling pathway, and the inflammatory mediator regulation of transient receptor potential (TRP) channels, while the hypomethylated transcripts were involved in 13 pathways, including bacterial invasion of epithelial cells, steroid biosynthesis, and the Hippo signaling pathway. Conclusion: Our study presents m6A modification patterns in middle ear cholesteatoma, which may exert regulatory roles in middle ear cholesteatoma. The present study provides directions for mRNA m6A modification-based research on the epigenetic etiology and pathogenesis of middle ear cholesteatoma.

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Suppression of m6A mRNA modification by DNA hypermethylated ALKBH5 aggravates the oncological behavior of KRAS mutation/LKB1 loss lung cancer.

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