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METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability

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Abstract

Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3′-untranslated Region (3′-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.

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Availability of data and materials

The tumor data of BALB/C NUDE mice have been deposited in the NCBI BioProject database (www.ncbi.nlm.nih.gov/bioproject) under BioProject accession no. PRJNA669425, PRJNA669419. The RNA-Seq data in gastric cancer in the TCGA database was downloaded from https://cptac-data-portal.georgetown.edu/study-summary/S025. Obtain single-cell sequencing data of early gastric cancer tissue from the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE134520). All relevant data could be obtained from the corresponding author.

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Acknowledgements

The authors would like to thank Professor Antonio Tedeschi (Adult Stem Cell Laboratory, The Francis Crick Institute) for valuable comments on this article.

Funding

This study was supported by the National Natural Science Foundation of China (grant No. 82073148), the Sanming Project of Medicine in Shenzhen (SZSM201911010), the Shenzhen Key Medical Discipline Construction Fund (SZXK016), the Shenzhen Sustainable Project (KCXFZ202002011010593), Shenzhen-Hong Kong-Macau Technology Research Programme (Type C) (Grant No. SGDX2020110309260100) and the Guangdong Provincial Key Laboratory of Digestive Cancer Research (No. 2021B1212040006).

Author information

Author notes

  1. Huafu Li, Chunming Wang, Linxiang Lan and Leping Yan are joint first authors.

Authors and Affiliations

  1. Guangdong Provincial Key Laboratory of Digestive Cancer Research, The Seventh Affiliated Hospital of Sun Yat-Sen University, No. 628, Zhenyuan Rd, Guangming Dist., Shenzhen, 518107, China

    Huafu Li, Chunming Wang, Leping Yan, Wenhui Wu, Wei Chen & Changhua Zhang

  2. The Institute of Cancer Research, 123 Old Brompton Road, London, SW7 3RP, UK

    Huafu Li, Linxiang Lan, Ian Evans, E. Josue Ruiz & Axel Behrens

  3. Adult Stem Cell Laboratory, The Francis Crick Institute, 1 Midland Road, London, NW1 1AT, UK

    Huafu Li, Linxiang Lan, Ian Evans, E. Josue Ruiz & Axel Behrens

  4. Digestive Diseases Center, The Seventh Affiliated Hospital of Sun Yat-Sen University, Shenzhen, 518107, China

    Huafu Li, Chunming Wang, Wenhui Wu, Haiyong Zhang, Wei Chen & Changhua Zhang

  5. Department of Gastrointestinal Surgery, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, China

    Chunming Wang & Haiyong Zhang

  6. Animal Experiment Center, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China

    Chunming Wang, Wuguo Li, Qiao Su & Guangying Zhao

  7. Scientific Research Center, The Seventh Affiliated Hospital Sun Yat-Sen University, Shenzhen, Guangdong, China

    Leping Yan & Zhenran Hu

  8. Department of Medicine, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA

    Zhijun Zhou

  9. 3B’s Research Group, I3Bs-Research Institute On Biomaterials, Biodegradables and Biomimetics of University of Minho, Headquarters of the European Institute of Excellence On Tissue Engineering and Regenerative Medicine, Avepark, Parque de Ciência E Tecnologia, Zona Industrial da Gandra, Barco, 4805-017, Guimarães, Portugal

    Joaquim M. Oliveira & Rui L. Reis

  10. ICVS/3B’s-PT Government Associate Laboratory, Braga/Guimarães, Portugal

    Joaquim M. Oliveira & Rui L. Reis

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  1. Huafu Li
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Contributions

All authors participated in the writing of the manuscript. AB, YH, CZ, HL and RLR designed the study. HL, CW and LL worked on the methodologies. HL, CW, LL, IE, WL, QS and ZZ performed data collection (providing animals, collection of patients samples, providing facilities, etc.). HL, CW, LL, ER conducted data analysis and interpretation (for example, statistical analysis, biostatistics, computational analysis). HL, CW, WC, ZH, LY were responsible for cultivation and development of organoids. LY, JMO and RLR supervised the study and revised the manuscript.

Corresponding authors

Correspondence to Axel Behrens, Rui L. Reis or Changhua Zhang.

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Conflict of interest

No potential conflicts of interest were disclosed.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (Ethical Review [2018] No. 087). And the clinical research and animal experiment ethics committee of the First Affiliated Hospital of Sun Yat-sen University (Ethical Review [2017] No. 208).

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18_2022_4129_MOESM1_ESM.pdf

Figure S1. Verifying the drug resistance level in GC resistant strains. (A) Representative images of colony formation of SNU719, MKN74 and AGS resistant strains and their corresponding wild-type cell lines in the same dose of oxaliplatin. (B) Comparison of the number of colonies in (A). (C-E) Indicate the comparison of cell viability between AGS, SNU719 and MNK74 Oxaliplatin resistance strains and wild-type cell strains under the effects of oxaliplatin respectively. OXA: Oxaliplatin. OLP: Olaparib. CON: Control group. AGSRE: AGS oxaliplatin resistance. SNU719RE: SNU719 oxaliplatin resistance. MKN74RE: MKN74 oxaliplatin resistance. * p < 0.05, ** p < 0.01, *** p < 0.001 (PDF 1686 KB)

18_2022_4129_MOESM2_ESM.pdf

Figure S2. Oxaliplatin-resistant human gastric cancer cell lines enrich for CD133+ cells. (A) Representative image of the comparison of the spheroidizing ability of SNU719, MKN74, AGS-resistant strains and their wild-type cell lines. (B) Proportion of spheroids in (A). (C-E) RT-qPCR was used to compare the mRNA expression levels of SNU719, MKN74 and AGS resistant strains and their wild-type cell lines LGR5, CD133, and CD44 respectively. (F) Flow cytometric analysis and comparison of CD133+ cells in SNU719, AGS and MKN74 resistant strains and their corresponding wild-type cell lines. Isotype is a control without antibody. The abscissa represents CD133, and the ordinate represents cells. * p < 0.05, ** p < 0.01, *** p < 0.001. (PDF 1870 KB)

18_2022_4129_MOESM3_ESM.pdf

Figure S3. CD133+ cell show highly stem-like properties. (A) A representative image of the spheroidization experiment based on the cells processed in flow cytometry. (B) The proportion of spheroids in (A). (C, D) RT-qPCR was performed on the cells sorted in H and their mRNA differences were analyzed. OXA: Oxaliplatin. OLP: Olaparib. CON: Control group. AGSRE: AGS oxaliplatin resistance. SNU719RE: SNU719 oxaliplatin resistance. MKN74RE: MKN74 oxaliplatin resistance. * p < 0.05, ** p < 0.01, *** p < 0.001 (PDF 4920 KB)

18_2022_4129_MOESM4_ESM.pdf

Figure S4. CD133 and PARP1 are elevated in CSCs. (A) Volcano plot for the statistical significance (y-axis, log2 transformation) versus the fold change of gene expression (x-axis, log2 transformation) between populations with high and low stemness indices determined by RNA-seq (n = 3). FC, fold change; padj, false-discovery-rate adjusted p value. The red dots denote significantly differentially expressed genes with padj < 0.05 of low stemness. The green dots denote significantly differentially expressed genes with padj < 0.05 of high stemness. (B-E) Stemness index is highly enriched in JAK STAT signaling pathway, MAPK Signaling pathway, TGF BETA signaling pathway, WNT Signaling pathway (PDF 3013 KB)

18_2022_4129_MOESM5_ESM.pdf

Figure S5. CD133+ cells are a subgroup of cells capable of self-renewal and DNA repair. (A, B) Nonlinear dimension reduction analysis of pathway activity score using t-SNE and UMAP method. (C) The heatmap of cell-type-specific activation pathways. (D) The ratio of other groups of CD133+ cells. (E) The first eight sites of C6 and C5 enrichment pathways (PDF 3788 KB)

18_2022_4129_MOESM6_ESM.pdf

Figure S6. The expression of PARP1 and CD133 in PT2, PT4, CD133+, CD133-, pLKO and SH-mettl3 were verified by WB. (A) comparison of PARP1 expression between PT2 and PT4. (B) comparison of expression of CD133+ and CD133-organoidPARP1. (C) CD133+ and CD133- PARP1 expression in subcutaneous tumors. (D) comparison of PARP1 expression in pLKO and SH-MettL3 organs and subcutaneous tumors (PDF 973 KB)

18_2022_4129_MOESM7_ESM.pdf

Figure S7. PARP1 and METTL3 were elevated in CSCs and oxaliplatin resistance cells. (A) RT-qPCR was used to compare the expression of PARP1 mRNA in SNU719, MKN74 and AGS-resistant CD133+ and CD133- cells. (B) RT-qPCR was used to compare PARP1 mRNA expression in SNU719, MKN74 and AGS resistant strains and their wild-type cell lines. (C) RT-qPCR was used to compare the expression of METTL3 mRNA in SNU719, MKN74 and AGS-resistant CD133+ and CD133- cells. (D) RT-qPCR was used to compare METTL3 mRNA expression in SNU719, MKN74 and AGS resistant strains and their wild-type cell lines. (E) By comparing the expression levels of PARP1 of SNU719, MKN74 and AGS resistant strains and their wild-type cell lines. (F) By comparing the expression levels of METTL3 of SNU719, MKN74 and AGS resistant strains and their wild-type cell lines. AGSRE: AGS oxaliplatin resistance. SNU719RE: SNU719 oxaliplatin resistance. MKN74RE: MKN74 oxaliplatin resistance. * p < 0.05, ** p < 0.01, *** p < 0.001. (PDF 1137 KB)

18_2022_4129_MOESM8_ESM.pdf

Figure S8. PARP1 knockdown and overexpression were verified by WB. (A) Comparing the expression levels of PARP1 of PT1 pLKO, PT1 sh1, PT1 sh2, PT1 sh3. (B) Comparing the expression levels of PARP1 of PT2 pLKO, PT2 sh1, PT2 sh2, PT2 sh3. (C) Comparing the expression levels of PARP1 of PT3 CON, PT3 PARP1, PT4 CON, PT4 PARP1 (PDF 1305 KB)

18_2022_4129_MOESM9_ESM.pdf

Figure S9. CD133+ cells were enriched in tumor stem cells and DNA damage repair related pathways and RNA modification. (A, B) CD133+ CSCs were enriched in tumor stem cells and DNA damage repair related pathways and RNA modification. (C, D) WB analysis of METTL3 and YTHDF1 in successfully transfected stable strains. (E, F) WB analysis of PT3 and PT4 organoids following METTL3 knockdown and PARP1 overexpression, PARP1 knockdown and METTL3 overexpression, METTL3 overexpression, PARP1 overexpression, METTL3 overexpression and PARP1 overexpression (PDF 631 KB)

18_2022_4129_MOESM10_ESM.pdf

Figure S10. m6A positive and negative controls and Dual Luciferase plasmid system validation. (A, B, C) MerIP-QPCR positive and negative controls indicated that the experimental results were reliable. (D) RNA decay rate positive and negative controls indicated that the experimental results were reliable. (E, F) RNA decay rate followed by Dual luciferase plasmid system assay demonstrated the PARP1 mRNA half-lives upon the METTL3 knockdown and YTHDF1 knockdown. Data were detected at indicated timepoint with actinomycin D (Act D, 5 μg/mL) treatment (PDF 488 KB)

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Li, H., Wang, C., Lan, L. et al. METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability. Cell. Mol. Life Sci. 79, 135 (2022). https://doi.org/10.1007/s00018-022-04129-0

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  • DOI: https://doi.org/10.1007/s00018-022-04129-0

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