N1-methyladenosine methylations in tRNA drive liver tumorigenesis by PPARδ-mediated cholesterol synthesis

Increased protein translation plays a critical role in cancer development and treatment1,2. However, the molecular mechanism that is involved in this process remains poorly understood. N1-methyladenosine (m1A) methylation in RNA accounts for regulating mRNA translation in a post-transcriptional manner3,4. Here we show that m1A methylation levels are remarkably elevated in hepatocellular carcinoma (HCC) patient tumor tissues, especially in patients with microscopic vascular invasion (MVI). Moreover, m1A methylation signals are increased in liver cancer stem cells (CSCs) and are negatively correlated with HCC patient survival. Consistently, TRMT6 and TRMT61A, forming m1A methyltransferase complex, are highly expressed in advanced HCC tumors and are negatively correlated with HCC survival. TRMT6/TRMT61A-mediated m1A methylations are required for self-renewal of liver CSCs and tumorigenesis. Mechanistically, TRMT6/TRMT61A-dependent m1A in tRNA boost PPARδ expression, which triggers cholesterol synthesis to activate Hedgehog signaling, driving self-renewal of liver CSCs and tumorigenesis. For potential therapeutic benefit, we further identify a specific inhibitor against TRMT6/TRMT61A complex that exerts effective therapeutic effect on liver cancer with high m1A methylations. Our findings provide novel insights into the function and molecular mechanism of m1A modifications underlying liver tumorigenesis and drug target, which will serve as a new biomarker for HCC and pave a new way to develop more effective therapeutic strategies for HCC patients. We find that the overall m1A methylation signal as well as the relevant m1A-methyltransferase TRMT6/61A are of abberrently high level in HCC tumor samples. By performing m1A sequencing for small RNA populations, we discovered several tRNAs with elevated m1A58 methylation stoicheometry. Then we evaluate the influenences on gene expression via Ribo-seq, and proved that TRMT6/TRMT61A-dependent m1A in tRNA boost expression of some metabolic pathway genes, which trigger cholesterol synthesis to activate Hedgehog signaling, driving self-renewal of liver CSCs and tumorigenesis.

蛋白质翻译增强在癌症发生与治疗中发挥关键作用1,2。然而，该过程背后的分子机制仍未被充分阐明。RNA中的N1-甲基腺嘌呤（N1-methyladenosine，m¹A）甲基化可通过转录后方式调控mRNA翻译3,4。本研究发现，肝细胞癌（hepatocellular carcinoma，HCC）患者的肿瘤组织中m¹A甲基化水平显著升高，尤其在存在微观血管侵犯（microscopic vascular invasion，MVI）的患者中更为突出。此外，肝癌干细胞（cancer stem cells，CSCs）内的m¹A甲基化信号增强，且与HCC患者的生存率呈负相关。

与之相符的是，构成m¹A甲基转移酶复合物的TRMT6与TRMT61A在晚期HCC肿瘤中高表达，同样与HCC患者的生存率呈负相关。TRMT6/TRMT61A介导的m¹A甲基化对于肝癌干细胞的自我更新与肿瘤发生是必需的。

从机制层面而言，tRNA上依赖TRMT6/TRMT61A的m¹A修饰可促进PPARδ的表达，进而触发胆固醇合成通路以激活Hedgehog信号通路，最终推动肝癌干细胞的自我更新与肿瘤发生。

为探索潜在的治疗价值，本研究进一步筛选得到一种针对TRMT6/TRMT61A复合物的特异性抑制剂，该抑制剂对高m¹A甲基化水平的肝癌展现出显著的治疗效果。

本研究的发现为m¹A修饰在肝癌发生中的功能与分子机制提供了全新见解，同时确立了其作为HCC新型生物标志物的潜力，为开发更有效的HCC患者治疗策略开辟了新途径。

我们发现，HCC肿瘤样本中整体m¹A甲基化信号以及相关甲基转移酶TRMT6/61A的表达水平均异常升高。通过对小RNA群体进行m¹A测序，我们鉴定出数种m¹A58甲基化化学计量比升高的tRNA。随后，通过核糖体测序（Ribo-seq）评估其对基因表达的影响，证实了tRNA上依赖TRMT6/TRMT61A的m¹A修饰可上调部分代谢通路基因的表达，进而触发胆固醇合成以激活Hedgehog信号通路，最终驱动肝癌干细胞的自我更新与肿瘤发生。