METTL3 in cancer-associated fibroblasts-derived exosomes promotes the proliferation and metastasis and suppresses ferroptosis in colorectal cancer by eliciting ACSL3 m6A modification.

Cancer-associated fibroblasts (CAFs) have been reported that can affect cancer cell proliferation, metastasis, ferroptosis, and immune escape. METTL3-mediated N6-methyladenine (m6A) modification is involved in the tumorigenesis of colorectal cancer (CRC). Herein, we investigated whether METTL3-dependent m6A in CAFs-derived exosomes (exo) affected CRC progression. qRT-PCR and western blotting analyses detected levels of mRNAs and proteins. Cell proliferation and metastasis were evaluated using MTT, colony formation, transwell, and wound healing assays, respectively. Cell ferroptosis was assessed by detecting cell viability and the levels of Fe+, reactive oxygen species, and glutathione after erastin treatment. Exosomes were isolated from CAFs by ultracentrifugation. The m6A modification profile was determined by methylated RNA immunoprecipitation assay and the interaction between METTL3 and ACSL3 (acyl-CoA synthetase 3) was verified using dual-luciferase reporter assay. Animal models were established for in vivo analysis. CAFs promoted CRC cell proliferation and metastasis, and suppressed cell ferroptosis. METTL3 was enriched in CAFs and was packaged into exosomes. The m6A modification and METTL3 expression were increased in CRC samples. Knockdown of METTL3 in CAFs-exo suppressed CRC cell proliferation and metastasis, and induced cell ferroptosis. Mechanistically, METTL3 induced ACSL3 m6A modification and stabilized its expression. The anticancer effects mediated by METTL3-silenced CAFs-exo could be rescued by ACSL3 overexpression. Moreover, in vivo assay also showed that CAFs-exo with decreased METTL3 could hinder CRC growth and metastasis in mice models. CAFs promoted the proliferation and metastasis, and restrained the ferroptosis in CRC by exosomal METTL3-elicited ACSL3 m6A modification.

Ren H ，Wang M ，Ma X ，An L ，Guo Y ，Ma H ... -  《Biology Direct》

Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

Fan Y ，Yu Y 《Human Cell》

METTL3 promotes colorectal carcinoma progression by regulating the m6A-CRB3-Hippo axis.

Colorectal carcinoma (CRC) is the third most common cancer and second most common cause of cancer-related deaths worldwide. Ribonucleic acid (RNA) N6-methyladnosine (m6A) and methyltransferase-like 3 (METTL3) play key roles in cancer progression. However, the roles of m6A and METTL3 in CRC progression require further clarification. Adenoma and CRC samples were examined to detect m6A and METTL3 levels, and tissue microarrays were performed to evaluate the association of m6A and METTL3 levels with the survival of patients with CRC. The biological functions of METTL3 were investigated through cell counting kit-8, wound healing, and transwell assays. M6A epitranscriptomic microarray, methylated RNA immunoprecipitation-qPCR, RNA stability, luciferase reporter, and RNA immunoprecipitation assays were performed to explore the mechanism of METTL3 in CRC progression. M6A and METTL3 levels were substantially elevated in CRC tissues, and patients with CRC with a high m6A or METTL3 levels exhibited shorter overall survival. METTL3 knockdown substantially inhibited the proliferation, migration, and invasion of CRC cells. An m6A epitranscriptomic microarray revealed that the cell polarity regulator Crumbs3 (CRB3) was the downstream target of METTL3. METTL3 knockdown substantially reduced the m6A level of CRB3, and inhibited the degradation of CRB3 mRNA to increase CRB3 expression. Luciferase reporter assays also showed that the transcriptional level of wild-type CRB3 significantly increased after METTL3 knockdown but not its level of variation. Knockdown of YT521-B homology domain-containing family protein 2 (YTHDF2) substantially increased CRB3 expression. RNA immunoprecipitation assays also verified the direct interaction between the YTHDF2 and CRB3 mRNA, and this direct interaction was impaired after METTL3 inhibition. In addition, CRB3 knockdown significantly promoted the proliferation, migration, and invasion of CRC cells. Mechanistically, METTL3 knockdown activated the Hippo pathway and reduced nuclear localization of Yes1-associated transcriptional regulator, and the effects were reversed by CRB3 knockdown. M6A and METTL3 levels were substantially elevated in CRC tissues relative to normal tissues. Patients with CRC with high m6A or METTL3 levels exhibited shorter overall survival, and METTL3 promoted CRC progression. Mechanistically, METTL3 regulated the progression of CRC by regulating the m6A-CRB3-Hippo pathway.

Pan J ，Liu F ，Xiao X ，Xu R ，Dai L ，Zhu M ，Xu H ，Xu Y ，Zhao A ，Zhou W ，Dang Y ，Ji G ... -  《JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH》

Cancer-associated fibroblasts-derived exosome-mediated transfer of miR-345-5p promotes the progression of colorectal cancer by targeting CDKN1A.

Colorectal cancer (CRC) is the second leading cause of cancer-induced death in the world. Cancer-associated fibroblasts (CAFs) released exosomes that contributed to cancer progression. This research was carried out to study the influence of CRC-associated fibroblasts-derived exosomes on the phenotype of CRC cells and the underlying mechanism. CAFs-derived exosomes (CAFs-exo) and normal fibroblasts (NFs)-derived exosomes (NFs-exo) were recognized by transmission electronic microscopy, nanoparticle tracking analysis and western blot analysis. Cell counting kit-8, flow cytometry analysis, colony formation assay, Transwell, qRT-PCR, immunofluorescence, immunohistochemistry staining and xenografts model were carried out to proceed with function studies in vitro and in vivo. The results showed that CAFs-exo induced cell proliferation, migration and invasion, while NFs-exo did not influence the tumor biological properties of CRC cells. Using qRT-PCR, miR-345-5p was observed to be a notably up-regulated miRNA in CAFs-exo compared to NFs-exo. CAFs-exo could mediate the transfer of miR-345-5p to CRC cells, and downregulation of miR-345-5p in CAFs notably reversed the pro-tumoral effect of CAFs-exo on CRC cells. Based on online prediction database, CDKN1A was proved as a direct downstream target of miR-345-5p in CRC cells, which was lowly expressed and negatively associated with miR-345-5p in CRC tumors. Furthermore, miR-345-5p upregulation-mediated tumor biological behaviors were abrogated by exogenous CDKN1A. In CRC cells-beared tumor xenograft, CAFs-exo administration promoted tumor growth and decreased CDKN1A expression, whereas miR-345-5p inhibition reversed these effects. The present study revealed that by interacting with CDKN1A, CAF-derived exosomal miR-345-5p promotes CRC progression and metastasis.

Shi W ，Liu Y ，Qiu X ，Yang L ，Lin G ... -  《-》

Exosomal LncRNA LINC00659 transferred from cancer-associated fibroblasts promotes colorectal cancer cell progression via miR-342-3p/ANXA2 axis.

Zhou L ，Li J ，Tang Y ，Yang M ... -  《Journal of Translational Medicine》