bims-rimeca 2023-04-23 papers

bims-rimeca

Biomed News

on RNA methylation in cancer

Issue of 2023–04–23
seventeen papers selected by
Sk Ramiz Islam, Saha Institute of Nuclear Physics

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N6 -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.
METTL3 facilitates immunosurveillance by inhibiting YTHDF2-mediated NLRC5 mRNA degradation in endometrial cancer.
LRPPRC facilitates tumor progression and immune evasion through upregulation of m6A modification of PD-L1 mRNA in hepatocellular carcinoma.
KIAA1429-mediated m6A modification of CHST11 promotes progression of diffuse large B-cell lymphoma by regulating Hippo-YAP pathway.
METTL3-Mediated m6A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL.
FTO-stabilized miR-139-5p targets ZNF217 to suppress prostate cancer cell malignancies by inactivating the PI3K/Akt/mTOR signal pathway.
YTHDC2-mediated m6A mRNA modification of Id3 suppresses cisplatin resistance in non-small cell lung cancer.
N6-methyladenosine upregulates ribosome biogenesis in environmental carcinogenesis.
N6-methyladenosine (m6A)-connected lncRNAs is linked to survival and immune infiltration in glioma patients.
The structure and function of YTHDF epitranscriptomic m6A readers.
m6A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition.
A prognostic signature consisting of N6-methyladenosine modified mRNAs demonstrates clinical potential in prediction of biochemical recurrence and guidance on precision therapy in prostate cancer.
FTO overexpression expedites wound healing and alleviates depression in burn rats through facilitating keratinocyte migration and angiogenesis via mediating TFPI-2 demethylation.
YTHDC1 positively regulates PTEN expression and plays a critical role in cisplatin resistance of bladder cancer.
Identification of a m6A-related ferroptosis signature as a potential predictive biomarker for lung adenocarcinoma.
Metabolic reprogramming driven by IGF2BP3 promotes acquired resistance to EGFR inhibitors in non-small cell lung cancer.
METTL3 is essential for small intestinal epithelial proliferation via regulation of growth factor signaling including KRAS.

Adv Sci (Weinh). 2023 Apr 18. e2204784

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N6 -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.

Yang Feng, Ping Yuan, Hongjuan Guo, Liming Gu, Zhao Yang, Jian Wang, Wei Zhu, Qi Zhang, Jianping Cao, Lili Wang, Yang Jiao.

  The biological roles of epithelial-mesenchymal transition (EMT) in the pathogenesis of radiation-induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6 -methyladenosine (m6 A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6 A modification participates in ionizing radiation (IR)-induced EMT and RILI remain unclear. Here, significantly increased m6 A levels upon IR-induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase-like 3 (METTL3) expression and downregulated α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3-mediated m6 A modification suppresses IR-induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3-mediated mRNA m6 A modification in a YTH-domain family 2 (YTHDF2)-dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR-responsive METTL3 is involved in IR-induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2-dependent FOXO1 m6 A modification, which may be a novel mechanism involved in the occurrence and development of RILI.

Keywords:  N6-methyladenosine (m6A); epithelial-mesenchymal transition (EMT); forkhead box O1 (FOXO1); ionizing radiation (IR); methyltransferase-like 3 (METTL3); radiation-induced lung injury (RILI)

DOI:  https://doi.org/10.1002/advs.202204784
Biomark Res. 2023 Apr 21. 11(1): 43

METTL3 facilitates immunosurveillance by inhibiting YTHDF2-mediated NLRC5 mRNA degradation in endometrial cancer.

Lei Zhan, Jing Zhang, Jun-Hui Zhang, Xiao-Jing Liu, Bao Guo, Jia-Hua Chen, Zhen-Hai Tang, Wen-Yan Wang, Qing-Yuan Wang, Bing Wei, Yun-Xia Cao.

   BACKGROUND: N6-methyladenosine (m6A) methylation is the most abundant chemical posttranscriptional modification of mRNA, and it is associated with the regulation of the immune response to tumors. However, the function of m6A modification in the immune response to endometrial cancer (EC) remains unknown. Our study investigated the immunological role of methyltransferase-like 3 (METTL3) in EC and the underlying molecular mechanism.
METHODS: We investigated the correlation between the expression of METTL3 and CD8 by using an endometrial tissue microarray cohort. Next, we investigated the role and mechanism of METTL3 in the immune response to EC using a mouse tumor model and a CD8+ T cell-EC cell coculture system after METTL3 overexpression or depletion. Additionally, RNA immunoprecipitation (RIP), methylated RIP, and RNA stability experiments were used to investigate the mechanism underlying the function of METTL3 in immunosurveillance of EC.
RESULTS: METTL3 levels were downregulated in EC patients, low levels of METTL3 were correlated with poor prognosis in EC patients. There was a positive correlation between METTL3 expression and CD8 expression. Overexpression of METTL3 in the EC cell and CD8+ T cell coculture system inhibited EC cell proliferation, migration, and promoted CD8+ T-cell proliferation, and in vivo, METTL3 overexpression increased CD8+ T cell proportions and inhibited EC progression; however, genetic depletion of METTL3 exerted the opposite effects. NLR family CARD domain-containing 5 (NLRC5) was identified as a target of METTL3-mediated m6A modification. The degradation of NLRC5 was increased by YTH domain-containing family 2 (YTHDF2).
CONCLUSIONS: Overall, METTL3, YTHDF2, and NLRC5 have potential to be the diagnostic and prognostic biomarkers for EC. METTL3 facilitated the m6A modifications of NLRC5 and inhibited its degradation through a YTHDF2-dependent mechanism in EC. Genetic overexpression of METTL3 attenuated the immune evasion of EC by promoting NLRC5-mediated immunosurveillance, suggesting that the METTL3/YTHDF2/NLRC5 axis is a promising target of immunotherapy in EC.

Keywords:  Biomarker; Endometrial cancer; Immunosurveillance; Immunotherapy; N6-adenosine-methyltransferase-like 3; N6-methyladenosine; NLR family CARD domain-containing 5; YTH domain-containing family 2

DOI:  https://doi.org/10.1186/s40364-023-00479-4
Front Immunol. 2023 ;14 1144774

LRPPRC facilitates tumor progression and immune evasion through upregulation of m6A modification of PD-L1 mRNA in hepatocellular carcinoma.

Houhong Wang, Amao Tang, Yayun Cui, Huihui Gong, Heng Li.

   Objective: LRPPRC is a newly discovered N6-methyladenosine (m6A) modification reader, which potentially affects hepatocellular carcinoma (HCC) progression. PD-L1 in tumor cells is essential for tumor immune evasion. This work investigated the LRPPRC-mediated m6A-modification effect on PD-L1 mRNA and immune escape in HCC.
Methods: Expression and clinical implication of LRPPRC and PD-L1 were measured in human HCC cohorts. The influence of LRPPRC on malignant behaviors of HCC cells was investigated through in vitro assays and xenograft tumor murine models. The posttranscriptional mechanism of LRPPRC on PD-L1 and anti-tumor immunity was elucidated in HCC cells via RIP, MeRIP-qPCR, RNA stability, immunohistochemical staining, and so forth.
Results: LRPPRC exhibited the notable upregulated in human HCC tissues, which was in relation to advanced stage and worse overall survival and disease-free survival. Impaired proliferative capacity and G2/M phage arrest were found in LRPPRC-knockout cells, with increased apoptotic level, and attenuated migratory and invasive abilities. In HCC patients and murine models, LRPPRC presented a positive interaction with PD-L1, with negative associations with CD8+, and CD4+ T-cell infiltrations and chemokines CXCL9, and CXCL10. LRPPRC loss downregulated the expression of PD-L1 and its m6A level in HCC cells. Moreover, LRPPRC suppression mitigated tumor growth in murine models and improved anti-tumor immunity and immune infiltration in tumors.
Conclusion: This work unveiled that LRPPRC may posttranscriptionally upregulate PD-L1 partially with an m6A-dependent manner for heightening mRNA stabilization of PD-L1 and provided a new mechanism for m6A regulator-mediated immunosuppression in HCC.

Keywords:  LRPPRC; PD-L1; anti-tumor immunity; hepatocellular carcinoma; immune evasion; m6A modification; tumor progression

DOI:  https://doi.org/10.3389/fimmu.2023.1144774
Cell Mol Biol Lett. 2023 Apr 19. 28(1): 32

KIAA1429-mediated m6A modification of CHST11 promotes progression of diffuse large B-cell lymphoma by regulating Hippo-YAP pathway.

Xiaomin Chen, Tiange Lu, Yiqing Cai, Yang Han, Mengfei Ding, Yurou Chu, Xiangxiang Zhou, Xin Wang.

   BACKGROUND: N6-methyladenosine (m6A) has been shown to participate in various essential biological processes by regulating the level of target genes. However, the function of m6A modification mediated by KIAA1429 [alias virus-like m6A methyltransferase-associated protein (VIRMA)] during the progression of diffuse large B-cell lymphoma (DLBCL) remains undefined.
METHODS: The expression and clinical significance of KIAA1429 were verified by our clinical data. CRISPR/Cas9 mediated KIAA1429 deletion, and CRISPR/dCas9-VP64 for activating endogenous KIAA1429 was used to evaluate its biological function. RNA sequencing (RNA-seq), methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNA immunoprecipitation (RIP) assays, luciferase activity assay, RNA stability experiments, and co-immunoprecipitation were performed to investigate the regulatory mechanism of KIAA1429 in DLBCL. Tumor xenograft models were established for in vivo experiments.
RESULTS: Dysregulated expression of m6A regulators was observed, and a novel predictive model based on m6A score was established in DLBCL. Additionally, elevated KIAA1429 expression was associated with poor prognosis of patients with DLBCL. Knockout of KIAA1429 repressed DLBCL cell proliferation, facilitated cell cycle arrest in the G2/M phase, induced apoptosis in vitro, and inhibited tumor growth in vivo. Furthermore, carbohydrate sulfotransferase 11 (CHST11) was identified as a downstream target of KIAA1429, which mediated m6A modification of CHST11 mRNA and then recruited YTHDF2 for reducing CHST11 stability and expression. Inhibition of CHST11 diminished MOB1B expression, resulting in inactivation of Hippo-YAP signaling, reprogramming the expression of Hippo target genes.
CONCLUSIONS: Our results revealed a new mechanism by which the Hippo-YAP pathway in DLBCL is inactivated by KIAA1429/YTHDF2-coupled epitranscriptional repression of CHST11, highlighting the potential of KIAA1429 as a novel predictive biomarker and therapeutic target for DLBCL progression.

Keywords:  CHST11; Diffuse large B-cell lymphoma; Hippo–YAP; KIAA1429; N 6-methyladenosine; YTHDF2

DOI:  https://doi.org/10.1186/s11658-023-00445-w
Blood Cancer Discov. 2023 Apr 17. OF1-OF18

METTL3-Mediated m6A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL.

Yiming Wu, Meiling Jin, Mike Fernandez, Kevyn L Hart, Aijun Liao, Xinzhou Ge, Stacey M Fernandes, Tinisha McDonald, Zhenhua Chen, Daniel Röth, Lucy Y Ghoda, Guido Marcucci, Markus Kalkum, Raju K Pillai, Alexey V Danilov, Jingyi Jessica Li, Jianjun Chen, Jennifer R Brown, Steven T Rosen, Tanya Siddiqi, Lili Wang.

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL.
SIGNIFICANCE: METTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller.

DOI: https://doi.org/10.1158/2643-3230.BCD-22-0156
Arch Biochem Biophys. 2023 Apr 18. pii: S0003-9861(23)00103-0. [Epub ahead of print] 109604

FTO-stabilized miR-139-5p targets ZNF217 to suppress prostate cancer cell malignancies by inactivating the PI3K/Akt/mTOR signal pathway.

Baihetiya Azhati, Aerziguli Reheman, Diliyaer Dilixiati, Mulati Rexiati.

  As one of the most important demethylases for RNA N6-methyladenosine (m6A) modifications, fat mass and obesity-associated protein (FTO) plays anti-cancer role during prostate cancer (PC), but it is still unclear the detailed molecular mechanisms. Here, this study verified that FTO inactivated the tumor-accelerating PI3K/Akt/mTOR pathway to hamper PC development through regulating the downstream miR-139-5p/zinc finger protein 217 (ZNF217) axis. Through performing clinical analysis, it was revealed that FTO was apparently ablated in the cancerous tissues compared to the normal tissues collected from PC patients, and patients with high-expressed FTO predicted a favorable prognosis. Functional experiments confirmed that overexpression of FTO suppressed cell proliferation, mitosis, epithelial-mesenchymal transition (EMT), tumorigenesis and lung metastasis both in vitro and in vivo. The following mechanical experiments verified that FTO stabilized miR-139-5p to increase its expression levels in a m6A-dependent manner, and elevated miR-139-5p induced degradation of ZNF217 through binding to ZNF217 mRNA, resulting in the inactivation of the PI3K/Akt/mTOR signal pathway. Finally, our rescuing experiments confirmed that overexpressed FTO-induced tumor-suppressing effects on PC cells were abrogated by miR-139-5p ablation and ZNF217 overexpression. Collectively, this study firstly validated that FTO exerted its anti-tumor effects in PC through regulating the miR-139-5p/ZNF217 axis in a m6A-dependent manner, providing novel biomarkers for the advancement of anti-cancer agents for PC treatment.

Keywords:  Fat mass and obesity-associated protein; N6-methyladenosine; Prostate cancer; Zinc finger protein 217; miR-139–5p

DOI:  https://doi.org/10.1016/j.abb.2023.109604
J Thorac Dis. 2023 Mar 31. 15(3): 1247-1257

YTHDC2-mediated m6A mRNA modification of Id3 suppresses cisplatin resistance in non-small cell lung cancer.

Xifan Wang, Yu'an Hu, Xiaoqing Li, Chuandong Zhu, Fangfang Chen.

   Background: The methylation of adenosines at the N6 position (N6-methyladenosine; m6A) is one of the most conserved internal RNA modifications. m6A can modulate the expression of oncogenes or tumor suppressor genes, as well as m6A levels and the expression and activity of m6A enzymes, thus influencing tumor progression and therapeutic response. This study investigates the role of YTHDC2-mediated m6A messenger RNA (mRNA) modification of Id3 in controlling cisplatin resistance in non-small cell lung cancer (NSCLC).
Methods: The expression of the m6A reader protein YTHDC2 was detected in an NSCLC cisplatin-resistant cell line (A549/DDP) using real-time fluorescence quantitative polymerase chain reaction (qPCR). YTHDC2 overexpression plasmids were constructed and transfected into A549/DDP and A549 cells respectively. We performed qPCR and western blot (WB) to detect changes in YTHDC2 and Id3 expression, and the effects of YTHDC2 overexpression on proliferation, apoptosis, invasion, and migration of drug-resistant cells were assessed by cell counting kit-8 (CCK-8), flow cytometry, and transwell and scratch assays. The m6A modification of Id3 by YTHDC2 was clarified by m6A-immunoprecipitation-PCR (m6A-IP-PCR) assay.
Results: The CLIPdb online database predicted that YTHDC2 might bind to Id3. The results of qPCR showed that YTHDC2 was downregulated in the NSCLC cisplatin-resistant cell line A549/DDP compared to the cisplatin-sensitive cell line A549. Overexpression of YTHDC2 increased the expression of Id3, and the methylation inhibitor 3-deazaadenosine abrogated the regulatory effect of YTHDC2 on Id3. YTHDC2 overexpression significantly inhibited A549/DDP cell proliferation, migration, and invasion, and promoted apoptosis by synergistically promoting the effects of Id3. m6A-IP-PCR analysis revealed that YTHDC2 could inhibit the m6A level of Id3 mRNA.
Conclusions: To regulate the activity of Id3, YTHDC2 requires modifications to m6A, which ultimately inhibit cisplatin resistance in NSCLC.

Keywords:  Id3; N6-methyladenosine (m6A); NSCLC; RNA modifications; YTHDC2

DOI:  https://doi.org/10.21037/jtd-23-187
Sci Total Environ. 2023 Apr 13. pii: S0048-9697(23)02047-8. [Epub ahead of print]881 163428

N6-methyladenosine upregulates ribosome biogenesis in environmental carcinogenesis.

Tianhe Zhao, Donglei Sun, Keyan Long, Bernardo Lemos, Qian Zhang, Jin Man, Manyu Zhao, Zunzhen Zhang.

  Many trace metal pollutants in surface water, the atmosphere, and soil are carcinogenic, and ribosome biogenesis plays an important role in the carcinogenicity of heavy metals. However, the contradiction between upregulated ribosome biogenesis and decreased ribosomal DNA copy number in environmental carcinogenesis is not fully understood. Here, from a perspective of the most predominant and abundant RNA epigenetic modification, N6-methyladenosine (m6A), we explored the reason behind this contradiction at the post-transcriptional level using arsenite-induced skin carcinogenesis models both in vitro and in vivo. Based on the m6A microarray assay and a series of experiments, we found for the first time that the elevated m6A in arsenite-induced transformation is mainly enriched in the genes regulating ribosome biogenesis. m6A upregulates ribosome biogenesis post-transcriptionally by stabilizing ribosomal proteins and modulating non-coding RNAs targeting ribosomal RNAs and proteins, leading to arsenite-induced skin carcinogenesis. Using multi-omics analysis of human subjects and experimental validation, we identified an unconventional role of a well-known key proliferative signaling node AKT1 as a vital mediator between m6A and ribosome biogenesis in arsenic carcinogenesis. m6A activates AKT1 and transmits proliferative signals to ribosome biogenesis, exacerbating the upregulation of ribosome biogenesis in arsenite-transformed keratinocytes. Similarly, m6A promotes cell proliferation by upregulating ribosome biogenesis in cell transformation induced by carcinogenic heavy metals (chromium and nickel). Importantly, inhibiting m6A reduces ribosome biogenesis. Targeted inhibition of m6A-upregulated ribosome biogenesis effectively prevents cell transformation induced by trace metals (arsenic, chromium, and nickel). Our results reveal the mechanism of ribosome biogenesis upregulated by m6A in the carcinogenesis of trace metal pollutants. From the perspective of RNA epigenetics, our study improves our understanding of the contradiction between upregulated ribosome biogenesis and decreased ribosomal DNA copy number in the carcinogenesis of environmental carcinogens.

Keywords:  AKT1; Arsenic; Carcinogenicity; Heavy metal contaminants; Ribosomal proteins; m(6)A

DOI:  https://doi.org/10.1016/j.scitotenv.2023.163428
Biosci Rep. 2023 Apr 21. pii: BSR20222100. [Epub ahead of print]

N6-methyladenosine (m6A)-connected lncRNAs is linked to survival and immune infiltration in glioma patients.

Wei Jun Wu, Feng Xiao, Yaping Xiong, Gu Feng Sun, Yun Guo, Xiang Zhou, Guo Wen Hu, Kai Huang, Hua Guo.

   BACKGROUND: The connection between m6A-assiociateed lncRNAs and prognosis has been demonstrated in multiple types of tumors. However, potential roles of m6A-assiociateed lncRNAs in glioma is still rare.  Methods: We implemented consensus cluster analysis to group the downloaded samples into two subtypes. The least absolute shrinkage and selection operator (LASSO) analysis was used to create a risk model. Additionally, the conjunction between m6A-related lncRNAs and immune cells infiltration was explored by conducting the R package. Ultimately, we inspected the underlying downstream pathways of the two subtypes by performing Gene Set Enrichment Analysis (GSEA). The expression level of m6A-connected lncRNAs in glioma were examined by conducting in vitro experiments.</p>  Results: We ascertained two subtypes of glioma in line with the consensus clustering of m6A-associated lncRNAs. We confirmed that age, grade, and IDH are related to the two subtypes. Additionally, the immune cells infiltration and immune checkpoint molecules of the two clusters were discussed. A risk signature including AL359643.3, AL445524.1, AL162231.2, AL117332.1, AP001486.2, POLR2J4, AC120036.4, LINC00641, LINC00900, CRNDE, and AL158212.3, was identified using the Cox regression and LASSO analyses. We also verified the prognostic value and discussed the immune cells infiltration and immune checkpoint molecules of the risk signature. In Vitro experiments verified that the m6A-associated lncRNAs was abnormally expressed in glioma.</p>  Conclusion: We elaborated the significant role of m6A-connected lncRNAs in glioma prognosis and immune infiltration and suggest that these key regulators may serve as underlying therapeutic targets to build up the efficacy of glioma immunotherapy.

Keywords:  Glioma; LncRNA; M6A regulators; immune infiltration; prognosis

DOI:  https://doi.org/10.1042/BSR20222100
Trends Pharmacol Sci. 2023 Apr 15. pii: S0165-6147(23)00062-7. [Epub ahead of print]

The structure and function of YTHDF epitranscriptomic m6A readers.

Vilbert Sikorski, Simona Selberg, Maciej Lalowski, Mati Karelson, Esko Kankuri.

  Specific RNA sequences modified by a methylated adenosine, N6-methyladenosine (m6A), contribute to the post-transcriptional regulation of gene expression. The quantity of m6A in RNA is orchestrated by enzymes that write and erase it, while its effects are mediated by proteins that bind to read this modification. Dysfunction of this post-transcriptional regulatory process has been linked to human disease. Although the initial focus has been on pharmacological targeting of the writer and eraser enzymes, interest in the reader proteins has been challenged by a lack of clear understanding of their functional roles and molecular mechanisms of action. Readers of m6A-modified RNA (m6A-RNA) - the YTH (YT521-B homology) domain-containing protein family paralogs 1-3 (YTHDF1-3, referred to here as DF1-DF3) - are emerging as therapeutic targets as their links to pathological processes such as cancer and inflammation and their roles in regulating m6A-RNA fate become clear. We provide an updated understanding of the modes of action of DF1-DF3 and review their structures to unlock insights into drug design approaches for DF paralog-selective inhibition.

Keywords:  RNA modifications; YTHDF; allosteric regulation; drug development; epitranscriptomics; higher-order cooperativity; liquid–liquid phase separation; m(6)A reader proteins; oligonucleotide therapeutics; post-transcriptional modifications; selective inhibition

DOI:  https://doi.org/10.1016/j.tips.2023.03.004
Cancer Lett. 2023 Apr 14. pii: S0304-3835(23)00129-5. [Epub ahead of print]562 216178

m6A-modification of cyclin D1 and c-myc IRESs in glioblastoma controls ITAF activity and resistance to mTOR inhibition.

Angelica Benavides-Serrato, Jacquelyn T Saunders, Sunil Kumar, Brent Holmes, Kennedy E Benavides, Muhammad T Bashir, Robert N Nishimura, Joseph Gera.

  A major mechanism conferring resistance to mTOR inhibitors is activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated mRNA translation, driving the synthesis of proteins promoting resistance of glioblastoma (GBM). Previously, we found this pathway is stimulated by the requisite IRES-trans-acting factor (ITAF) hnRNP A1, which itself is subject to phosphorylation and methylation events regulating cyclin D1 and c-myc IRES activity. Here we describe the requirement for m6A-modification of IRES RNAs for efficient translation and resistance to mTOR inhibition. DRACH-motifs within these IRES RNAs upon m6A modification resulted in enhanced IRES activity via increased hnRNP A1-binding following mTOR inhibitor exposure. Inhibitor exposure stimulated the expression of m6A-methylosome components resulting in increased activity in GBM. Silencing of METTL3-14 complexes reduced IRES activity upon inhibitor exposure and sensitized resistant GBM lines. YTHDF3 associates with m6A-modified cyclin D1 or c-myc IRESs, regulating IRES activity, and mTOR inhibitor sensitivity in vitro and in xenograft experiments. YTHDF3 interacted directly with hnRNP A1 and together stimulated hnRNP A1-dependent nucleic acid strand annealing activity. These data demonstrate that m6A-methylation of IRES RNAs regulate GBM responses to this class of inhibitors.

Keywords:  Drug resistance; Glioblastoma; IRES; ITAF; N(6)-methyladenosine modification; mTOR inhibitors

DOI:  https://doi.org/10.1016/j.canlet.2023.216178
Transl Oncol. 2023 Apr 13. pii: S1936-5233(23)00056-6. [Epub ahead of print]33 101670

A prognostic signature consisting of N6-methyladenosine modified mRNAs demonstrates clinical potential in prediction of biochemical recurrence and guidance on precision therapy in prostate cancer.

Jianming Lu, Jiahong Chen, Zhuoyuan Lin, Qinwei Liu, Chuanfan Zhong, Zhouda Cai, Zhenyu Jia, Weide Zhong, Yingke Liang, Chao Cai.

  Novel biomarkers are urgently needed to improve the prediction of clinical outcomes and guide personalized treatment for prostate cancer (PCa) patients. However, the role of N6-methyladenosine (m6A) modifications in PCa initiation and progression remains largely elusive. In our study, we collected benign Prostate Hyperplasia (BPH), localized PCa, and metastatic PCa samples from patients and performed methylated RNA immunoprecipitation sequencing (MeRIP-Seq) to map m6A-methylated mRNAs. Furthermore, we developed a prognostic signature based on 239 differentially methylated RNAs and the TCGA-PRAD dataset, which can be used to calculate an m6A-modified mRNA (MMM) score for a PCa patient, validated by independent multi-center cohorts. Our findings revealed that differential m6A modifications were positively correlated with altered expressions of mapped m6A-modified mRNAs. Higher MMM scores were associated with shorter times to biochemical recurrence (BCR) in PCa patients, and the MMM scoring system outperformed three well-established signatures in nine independent validation cohorts, as demonstrated by Kaplan-Meier survival analysis, C-index and ROC. Patients who did not respond to androgen receptor signaling inhibitor (ARSI) therapy and immunotherapy were found to have high MMM scores. Two hub genes, TLE1 and PFKL, were confirmed to have m6A sites through MeRIP-qPCR, and their knockdown promoted PCa cell invasion. Bioinformatics analysis of single-cell databases identified cell types with high transcript abundance levels of these two genes. In summary, our study is the first to perform transcriptome-wide m6A mapping in prostate tissues. The translational potential of a prognostic signature, comprising m6A-methylated mRNAs, in predicting clinical outcomes and therapy responses for PCa patients, is demonstrated.

Keywords:  Androgen receptor signaling inhibitor; Biochemical recurrence; Immunotherapy; N6-methyladenosine modified mRNA; Prognostic signature; Prostate cancer

DOI:  https://doi.org/10.1016/j.tranon.2023.101670
Mol Cell Biochem. 2023 Apr 19.

FTO overexpression expedites wound healing and alleviates depression in burn rats through facilitating keratinocyte migration and angiogenesis via mediating TFPI-2 demethylation.

Zihan Xu, Xiumei Zhu, Shengzhi Mu, Ronghui Fan, Benfeng Wang, Wenjie Gao, Tao Kang.

  Burn injury is a serious traumatic injury that leads to severe physical and psychosocial impairment. Wound healing after burn injury is a substantial challenge in medical community. This study investigated the biological effects of the demethylase fat mass and obesity-associated protein (FTO) on burn injury. FTO protein level in burn skin tissues of patients was measured with Western blot assay. Keratinocytes (HaCaT cells) were given heat stimulation to induce an in vitro burn injury model, and then transfected with overexpression plasmids of FTO (pcDNA-FTO) or small interfering RNA against FTO (si-FTO). Cell proliferation, migration, and angiogenesis in keratinocytes were evaluated with CCK-8, Transwell, and tube formation assays, respectively. Tissue factor pathway inhibitor-2 (TFPI-2) m6A methylation level was detected with MeRIP‑qPCR assay. Then rescue experiments were conducted to explore the effects of FTO/TFPI-2 axis on keratinocyte functions. Lentivirus carrying FTO overexpression plasmids was injected into a burn rat model to detect its effects on wound healing and depressive-like behaviors in burn rats. FTO was downregulated in burn skin and heat-stimulated keratinocytes. FTO prominently augmented proliferation, migration and angiogenesis in heat-stimulated keratinocytes, while FTO knockdown showed the opposite results. FTO inhibited TFPI-2 expression by FTO-mediated m6A methylation modification. TFPI-2 overexpression abrogated FTO mediated enhancement of proliferation, migration and angiogenesis in keratinocytes. Additionally, FTO overexpression accelerated wound healing and improved depressive-like behaviors in burn rat model. FTO prominently augmented proliferation, migration and angiogenesis in heat-stimulated keratinocytes though inhibiting TFPI-2, and then improved wound healing and depressive-like behaviors.

Keywords:  Angiogenesis; Burn injury; FTO; Keratinocytes; TFPI-2

DOI:  https://doi.org/10.1007/s11010-023-04719-x
Cell Prolif. 2023 Apr 17. e13404

YTHDC1 positively regulates PTEN expression and plays a critical role in cisplatin resistance of bladder cancer.

Yinjie Su, Bo Wang, Jian Huang, Ming Huang, Tianxin Lin.

Activation of PI3K/AKT signalling by PTEN loss significantly enhances chemoresistance in bladder cancer. This study aims to evaluate PTEN regulation and identify targets that could be used to relieve chemoresistance. Expression of YTHDC1, γ-H2AX and PTEN were detected by IHC assay. Cell Counting Kit-8 assay, colony formation assay and tumour xenograft experiment evaluated cisplatin response. Flow cytometry and comet assay estimated cell apoptosis, cell cycle distribution and DNA repair capability. Quantitative real-time polymerase chain reaction, Western blot and RIP assay assessed binding properties between PTEN mRNA and YTHDC1. Silencing YTHDC1 in bladder cancer cells reduced PTEN expression and activated PI3K/AKT signalling by destabilizing PTEN mRNA in an m6 A-dependent manner. Low YTHDC1 expression indicated poor cisplatin sensitivity in bladder cancer patients. Reducing YTHDC1 expression promoted drug resistance to cisplatin, while over-expressing YTHDC1 promoted cisplatin sensitivity. Reducing YTHDC1 expression activated DNA damage response, which includes quicker cell cycle recovery, apoptosis evasion and an enhanced DNA repair capability, whereas these effects were attenuated when MK2206, a PI3K/AKT inhibitor was applied. We provide novel evidence that PTEN/PI3K/AKT signalling pathway could be regulated by YTHDC1 in an m6 A-dependent manner and highlight a critical role of YTHDC1 in cisplatin resistance of bladder cancer.

DOI: https://doi.org/10.1111/cpr.13404
BMC Pulm Med. 2023 Apr 18. 23(1): 128

Identification of a m6A-related ferroptosis signature as a potential predictive biomarker for lung adenocarcinoma.

Dongdong Li, Ting Chen, Qiu-Gen Li.

   BACKGROUND: Both N6-methyladenosine (m6A) and ferroptosis-related genes are associated with the prognosis of lung adenocarcinoma. However, the predictive value of m6A-related ferroptosis genes remains unclear. Here, we aimed to identify the prognostic value of m6A-related ferroptosis genes in lung adenocarcinoma.
METHODS: Lung adenocarcinoma sample data were downloaded from the University of California Santa Cruz Xena and Gene Expression Omnibus databases. Spearman's correlation analysis was used to screen for m6A-related ferroptosis genes. Univariate Cox regression, Kaplan-Meier, and Lasso analyses were conducted to identify prognostic m6A-related ferroptosis genes, and stepwise regression was used to construct a prognostic gene signature. The predictive value of the gene signature was assessed using a multivariate Cox analysis. In the validation cohort, survival analysis was performed to verify gene signature stability. The training cohort was divided into high- and low-risk groups according to the median risk score to assess differences between the two groups in terms of gene set variation analysis, somatic mutations, and tumor immune infiltration cells.
RESULTS: Six m6A-related ferroptosis genes were used to construct a gene signature in the training cohort and a multivariate Cox analysis was conducted to determine the independent prognostic value of these genes in lung adenocarcinoma. In the validation cohort, Kaplan-Meier and receiver operating characteristic analyses confirmed the strong predictive power of this signature for the prognosis of lung adenocarcinoma. Gene set variation analysis showed that the low-risk group was mainly related to immunity, and the high-risk group was mainly related to DNA replication. Somatic mutation analysis revealed that the TP53 gene had the highest mutation rate in the high-risk group. Tumor immune infiltration cell analysis showed that the low-risk group had higher levels of resting CD4 memory T cells and lower levels of M0 macrophages.
CONCLUSION: Our study identified a novel m6A-related ferroptosis-associated six-gene signature (comprising SLC2A1, HERPUD1, EIF2S1, ACSL3, NCOA4, and CISD1) for predicting lung adenocarcinoma prognosis, yielding a useful prognostic biomarker and potential therapeutic target.

Keywords:  Bioinformatics; Ferroptosis; Lung adenocarcinoma; Prognosis; m6A

DOI:  https://doi.org/10.1186/s12890-023-02410-x
Cancer Res. 2023 Apr 16. pii: CAN-22-3059. [Epub ahead of print]

Metabolic reprogramming driven by IGF2BP3 promotes acquired resistance to EGFR inhibitors in non-small cell lung cancer.

Ziyou Lin, Jingwei Li, Jian Zhang, Weineng Feng, Jiaye Lu, Xiaofan Ma, Wen Ding, Shumin Ouyang, Jin-Jian Lu, Peibin Yue, Guohui Wan, Peiqing Liu, Xiaolei Zhang.

Acquired resistance represents a bottleneck for effective molecular targeted therapy in lung cancer. Metabolic adaptation is a distinct hallmark of human lung cancer that might contribute to acquired resistance. In this study, we discovered a novel mechanism of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) mediated by IGF2BP3-dependent crosstalk between epigenetic modifications and metabolic reprogramming through the IGF2BP3-COX6B2 axis. IGF2BP3 was upregulated in TKI-resistant non-small cell lung cancer patients, and high IGF2BP3 expression correlated with reduced overall survival. Upregulated expression of the RNA binding protein IGF2BP3 in lung cancer cells reduced sensitivity to TKI treatment and exacerbated the development of drug resistance via promoting oxidative phosphorylation (OXPHOS). COX6B2 mRNA bound IGF2BP3, and COX6B2 was required for increased OXPHOS and acquired EGFR-TKI resistance mediated by IGF2BP3. Mechanistically, IGF2BP3 bound to the 3'-untranslated region of COX6B2 in an m6A-dependent manner to increase COX6B2 mRNA stability. Moreover, the IGF2BP3-COX6B2 axis regulated nicotinamide metabolism, which can alter OXPHOS and promote EGFR-TKI acquired resistance. Inhibition of OXPHOS with IACS-010759, a small-molecule inhibitor, resulted in strong growth suppression in vitro and in vivo in a gefitinib-resistant patient-derived xenograft model. Collectively, these findings suggest that metabolic reprogramming by the IGF2BP3-COX6B2 axis plays a critical role in TKI resistance and confers a targetable metabolic vulnerability to overcome acquired resistance to EGFR-TKIs in lung cancer.

DOI: https://doi.org/10.1158/0008-5472.CAN-22-3059
bioRxiv. 2023 Apr 06. pii: 2023.04.06.535853. [Epub ahead of print]

METTL3 is essential for small intestinal epithelial proliferation via regulation of growth factor signaling including KRAS.

Charles H Danan, Kaitlyn E Naughton, Katharina E Hayer, Sangeevan Vellappan, Emily A McMillan, Yusen Zhou, Rina Matsuda, Shaneice K Nettleford, Kay Katada, Louis R Parham, Xianghui Ma, Afrah Chowdhury, Benjamin J Wilkins, Premal Shah, Matthew D Weitzman, Kathryn E Hamilton.

The METTL3-METTL14 methyltransferase complex adds N6-methyladenosine (m 6 A) to mRNA with profound impacts on cell fate. Studies delete METTL3 or METTL14 interchangeably to define the role of m 6 A in target tissues despite a lack of data confirming that these deletions are equivalent. Intestinal epithelial METTL14 deletion triggers stem cell death in the colon with no overt phenotype in the small intestine. The effect of METTL3 deletion in the same tissues remains unknown. We report that intestinal epithelial METTL3 deletion caused unexpected severe defects in the small intestine, including crypt and villus atrophy associated with cellular senescence and death in the crypt transit amplifying zone. Ribosome profiling and m 6 A-sequencing demonstrated downregulated translation of hundreds of unique methylated transcripts, including genes essential to growth factor signal transduction, such as Kras . Our study suggests that METTL3 is essential for small intestinal homeostasis via enhanced translation of growth factor signaling in crypt transit amplifying cells.
Teaser: METTL3 deletion leads to cell death in proliferating small intestinal epithelium associated with decreased translation of growth factor transducers.

DOI: https://doi.org/10.1101/2023.04.06.535853