N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer

Shiyan Wang; Shanshan Gao; Yong Zeng; Lin Zhu; Yulin Mo; Chi Chun Wong; Yi Bao; Peiran Su; Jianning Zhai; Lina Wang; Fraser Soares; Xin Xu; Huarong Chen; Kebria Hezaveh; Xinpei Ci; Aobo He; Tracy McGaha; Catherine O'Brien; Robert Rottapel; Wei Kang; Jianfeng Wu; Gang Zheng; Zongwei Cai; Jun Yu; Housheng Hansen He

doi:10.1053/j.gastro.2021.12.269

N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer

Gastroenterology. 2022 Apr;162(4):1183-1196. doi: 10.1053/j.gastro.2021.12.269. Epub 2021 Dec 28.

Authors

Shiyan Wang¹, Shanshan Gao², Yong Zeng¹, Lin Zhu³, Yulin Mo⁴, Chi Chun Wong², Yi Bao², Peiran Su⁵, Jianning Zhai², Lina Wang⁶, Fraser Soares¹, Xin Xu¹, Huarong Chen², Kebria Hezaveh¹, Xinpei Ci¹, Aobo He⁷, Tracy McGaha¹, Catherine O'Brien⁵, Robert Rottapel⁵, Wei Kang⁸, Jianfeng Wu⁹, Gang Zheng¹⁰, Zongwei Cai³, Jun Yu¹¹, Housheng Hansen He¹²

Affiliations

¹ Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada.
² Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China.
³ State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
⁴ Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.
⁵ Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
⁶ Institute of Precision Medicine, The First Affiliated Hospital of Sun Yat-sen University, Guangdong, China.
⁷ Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
⁸ Department of Anatomical and Cellular Pathology, State Key Laboratory of Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong, China.
⁹ State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Fujian, China.
¹⁰ Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
¹¹ Institute of Digestive Disease and Department of Medicine and Therapeutics, State Key Laboratory of Digestive Disease, Li Ka Shing Institute of Health Sciences, CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Hong Kong, China. Electronic address: junyu@cuhk.edu.hk.
¹² Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. Electronic address: hansenhe@uhnresearch.ca.

PMID: 34968454
DOI: 10.1053/j.gastro.2021.12.269

Abstract

Background & aims: N6-methyladenosine (m⁶A) governs the fate of RNAs through m⁶A readers. Colorectal cancer (CRC) exhibits aberrant m⁶A modifications and expression of m⁶A regulators. However, how m⁶A readers interpret oncogenic m⁶A methylome to promote malignant transformation remains to be illustrated.

Methods: YTH N6-methyladenosine RNA binding protein 1 (Ythdf1) knockout mouse was generated to determine the effect of Ythdf1 in CRC tumorigenesis in vivo. Multiomic analysis of RNA-sequencing, m⁶A methylated RNA immunoprecipitation sequencing, YTHDF1 RNA immunoprecipitation sequencing, and proteomics were performed to unravel targets of YTHDF1 in CRC. The therapeutic potential of targeting YTHDF1-m⁶A-Rho/Rac guanine nucleotide exchange factor 2 (ARHGEF2) was evaluated using small interfering RNA (siRNA) encapsulated by lipid nanoparticles (LNP).

Results: DNA copy number gain of YTHDF1 is a frequent event in CRC and contributes to its overexpression. High expression of YTHDF1 is significantly associated with metastatic gene signature in patient tumors. Ythdf1 knockout in mice dampened tumor growth in an inflammatory CRC model. YTHDF1 promotes cell growth in CRC cell lines and primary organoids and lung and liver metastasis in vivo. Integrative multiomics analysis identified RhoA activator ARHGEF2 as a key downstream target of YTHDF1. YTHDF1 binds to m⁶A sites of ARHGEF2 messenger RNA, resulting in enhanced translation of ARHGEF2. Ectopic expression of ARHGEF2 restored impaired RhoA signaling, cell growth, and metastatic ability both in vitro and in vivo caused by YTHDF1 loss, verifying that ARHGEF2 is a key target of YTHDF1. Finally, ARHGEF2 siRNA delivered by LNP significantly suppressed tumor growth and metastasis in vivo.

Conclusions: We identify a novel oncogenic epitranscriptome axis of YTHDF1-m⁶A-ARHGEF2, which regulates CRC tumorigenesis and metastasis. siRNA-delivering LNP drug validated the therapeutic potential of targeting this axis in CRC.

Keywords: ARHGEF2; Colorectal Cancer; N6-Methyladenosine; Nanoparticle; YTHDF1.

MeSH terms

Adenosine / analogs & derivatives
Adenosine / metabolism
Animals
Carcinogenesis / genetics
Colorectal Neoplasms* / pathology
Gene Expression Regulation, Neoplastic*
Humans
Liposomes
Mice
Nanoparticles
RNA, Small Interfering
RNA-Binding Proteins / genetics
RNA-Binding Proteins / metabolism
Rho Guanine Nucleotide Exchange Factors / genetics
Rho Guanine Nucleotide Exchange Factors / metabolism
rhoA GTP-Binding Protein / genetics
rhoA GTP-Binding Protein / metabolism

Substances

ARHGEF2 protein, human
Lipid Nanoparticles
Liposomes
RNA, Small Interfering
RNA-Binding Proteins
Rho Guanine Nucleotide Exchange Factors
YTHDF1 protein, human
RHOA protein, human
N-methyladenosine
rhoA GTP-Binding Protein
Adenosine