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

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N6-Methyladenosine Reader YTHDF1 Promotes ARHGEF2 Translation and RhoA Signaling in Colorectal Cancer
Gastroenterology ( IF 25.7 ) Pub Date : 2021-12-28 , DOI: 10.1053/j.gastro.2021.12.269
Shiyan Wang ₁ , Shanshan Gao ₂ , Yong Zeng ₁ , Lin Zhu ₃ , Yulin Mo ₄ , Chi Chun Wong ₂ , Yi Bao ₂ , Peiran Su ₅ , Jianning Zhai ₂ , Lina Wang ₆ , Fraser Soares ₁ , Xin Xu ₁ , Huarong Chen ₂ , Kebria Hezaveh ₁ , Xinpei Ci ₁ , Aobo He ₇ , Tracy McGaha ₁ , Catherine O'Brien ₅ , Robert Rottapel ₅ , Wei Kang ₈ , Jianfeng Wu ₉ , Gang Zheng ₁₀ , Zongwei Cai ₃ , Jun Yu ₂ , Housheng Hansen He ₅

Affiliation

Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada.
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.
State Key Laboratory of Environmental and Biological Analysis, Department of Chemistry, Hong Kong Baptist University, Hong Kong, China.
Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario, Canada.
Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
Institute of Precision Medicine, The First Affiliated Hospital of Sun Yat-sen University, Guangdong, China.
Princess Margaret Cancer Centre/University Health Network, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
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.
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Fujian, China.
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.

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.

中文翻译：

N6-甲基腺苷阅读器 YTHDF1 促进结直肠癌中的 ARHGEF2 翻译和 RhoA 信号传导

背景与目标

N6-甲基腺苷 (m ^{6 A) 通过 m}⁶ A 阅读器控制 RNA 的命运。结直肠癌 (CRC) 表现出异常的 m ⁶ A 修饰和 m ⁶ A 调节剂的表达。然而，m ⁶ A 读者如何解释致癌 m ⁶ A 甲基化组以促进恶性转化仍有待说明。

方法

生成YTH N6-甲基腺苷 RNA 结合蛋白 1 ( Ythdf1 ) 敲除小鼠以确定Ythdf1在体内 CRC 肿瘤发生中的作用。对 RNA 测序、m ⁶ A 甲基化 RNA 免疫沉淀测序、YTHDF1 RNA 免疫沉淀测序和蛋白质组学进行了多组学分析，以解开 YTHDF1 在 CRC 中的靶标。使用脂质纳米颗粒 (LNP) 包裹的小干扰 RNA (siRNA) 评估靶向 YTHDF1-m ^{6 A-Rho/Rac 鸟嘌呤核苷酸交换因子 2 (ARHGEF2) 的治疗潜力。}

结果

YTHDF1的DNA 拷贝数增加是 CRC 中的常见事件，并导致其过度表达。YTHDF1 的高表达与患者肿瘤中的转移基因特征显着相关。小鼠中的Ythdf1敲除抑制了炎症性 CRC 模型中的肿瘤生长。YTHDF1 促进 CRC 细胞系和原发性类器官的细胞生长以及体内肺和肝转移。综合多组学分析确定 RhoA 激活剂 ARHGEF2 是 YTHDF1 的关键下游目标。YTHDF1 与ARHGEF2信使 RNA 的 m ⁶ A 位点结合，导致ARHGEF2的翻译增强. ARHGEF2 的异位表达恢复了由 YTHDF1 缺失引起的体外和体内受损的 RhoA 信号传导、细胞生长和转移能力，证实了 ARHGEF2 是 YTHDF1 的关键靶点。最后，由 LNP 传递的ARHGEF2 siRNA 在体内显着抑制了肿瘤的生长和转移。