Key Laboratory of Hepatobiliary and Pancreatic Surgery, Institute of Hepatobiliary Surgery, Southwest Hospital, Army Medical University, Chongqing, 400038, China.
Mil Med Res. 2023 Dec 20;10(1):68. doi: 10.1186/s40779-023-00501-8.
Kirsten rat sarcoma (KRAS) and mutant KRAS have been implicated in human cancers, but it remains unclear whether their activation requires ubiquitination. This study aimed to investigate whether and how F-box and leucine-rich repeat 6 (FBXL6) regulates KRAS and KRAS activity in hepatocellular carcinoma (HCC).
We constructed transgenic mouse strains LC (LSL-Fbxl6;Alb-Cre, n = 13), KC (LSL-Kras;Alb-Cre, n = 10) and KLC (LSL-Kras;LSL-Fbxl6;Alb-Cre, n = 12) mice, and then monitored HCC for 320 d. Multiomics approaches and pharmacological inhibitors were used to determine oncogenic signaling in the context of elevated FBXL6 and KRAS activation. Co‑immunoprecipitation (Co-IP), Western blotting, ubiquitination assay and RAS activity detection assay were employed to investigate the underlying molecular mechanism by which FBXL6 activates KRAS. The pathological relevance of the FBXL6/KRAS/extracellular signal-regulated kinase (ERK)/mammalian target of rapamycin (mTOR)/proteins of relevant evolutionary and lymphoid interest domain 2 (PRELID2) axis was evaluated in 129 paired samples from HCC patients.
FBXL6 is highly expressed in HCC as well as other human cancers (P < 0.001). Interestingly, FBXL6 drives HCC in transgenic mice. Mechanistically, elevated FBXL6 promotes the polyubiquitination of both wild-type KRAS and KRAS at lysine 128, leading to the activation of both KRAS and KRAS and promoting their binding to the serine/threonine-protein kinase RAF, which is followed by the activation of mitogen-activated protein kinase kinase (MEK)/ERK/mTOR signaling. The oncogenic activity of the MEK/ERK/mTOR axis relies on PRELID2, which induces reactive oxygen species (ROS) generation. Furthermore, hepatic FBXL6 upregulation facilitates KRAS to induce more severe hepatocarcinogenesis and lung metastasis via the MEK/ERK/mTOR/PRELID2/ROS axis. Dual inhibition of MEK and mTOR effectively suppresses tumor growth and metastasis in this subtype of cancer in vivo. In clinical samples, FBXL6 expression positively correlates with p-ERK (χ = 85.067, P < 0.001), p-mTOR (χ = 66.919, P < 0.001) and PRELID2 (χ = 20.891, P < 0.001). The Kaplan-Meier survival analyses suggested that HCC patients with high FBXL6/p-ERK levels predicted worse overall survival (log‑rank P < 0.001).
FBXL6 activates KRAS or KRAS via ubiquitination at the site K128, leading to activation of the ERK/mTOR/PRELID2/ROS axis and tumorigenesis. Dual inhibition of MEK and mTOR effectively protects against FBXL6- and KRAS-induced tumorigenesis, providing a potential therapeutic strategy to treat this aggressive subtype of liver cancer.
Kirsten 大鼠肉瘤（KRAS）和突变型 KRAS 被认为与人类癌症有关，但它们的激活是否需要泛素化仍不清楚。本研究旨在探讨 F-box 和富含亮氨酸重复 6（FBXL6）是否以及如何调节肝癌（HCC）中的 KRAS 和 KRAS 活性。
我们构建了转基因小鼠品系 LC（LSL-Fbxl6；Alb-Cre，n = 13）、KC（LSL-Kras；Alb-Cre，n = 10）和 KLC（LSL-Kras；LSL-Fbxl6；Alb-Cre，n = 12）小鼠，并监测 HCC 长达 320 天。采用多组学方法和药理学抑制剂，确定在 FBXL6 升高和 KRAS 激活的情况下致癌信号。采用免疫共沉淀（Co-IP）、Western blot、泛素化测定和 RAS 活性检测试验，研究 FBXL6 激活 KRAS 的潜在分子机制。评估了 129 对 HCC 患者的配对样本中 FBXL6/KRAS/细胞外信号调节激酶（ERK）/哺乳动物雷帕霉素靶蛋白（mTOR）/相关进化和淋巴细胞感兴趣域 2（PRELID2）轴的病理相关性。
FBXL6 在 HCC 以及其他人类癌症中高度表达（P < 0.001）。有趣的是，FBXL6 驱动转基因小鼠中的 HCC。在机制上，升高的 FBXL6 促进野生型 KRAS 和 KRAS 在赖氨酸 128 处的多泛素化，导致 KRAS 和 KRAS 的激活，并促进它们与丝氨酸/苏氨酸蛋白激酶 RAF 结合，随后激活丝裂原活化蛋白激酶激酶（MEK）/ERK/mTOR 信号。MEK/ERK/mTOR 轴的致癌活性依赖于 PRELID2，它诱导活性氧（ROS）的产生。此外，肝 FBXL6 的上调通过 MEK/ERK/mTOR/PRELID2/ROS 轴促进 KRAS 诱导更严重的肝癌发生和肺转移。体内双重抑制 MEK 和 mTOR 可有效抑制该亚型癌症的肿瘤生长和转移。在临床样本中，FBXL6 表达与 p-ERK（χ = 85.067，P < 0.001）、p-mTOR（χ = 66.919，P < 0.001）和 PRELID2（χ = 20.891，P < 0.001）呈正相关。Kaplan-Meier 生存分析表明，HCC 患者中 FBXL6/p-ERK 水平较高预示着总体生存率较差（对数秩 P < 0.001）。
FBXL6 通过泛素化 KRAS 或 KRAS 的赖氨酸 128 残基激活 KRAS，从而激活 ERK/mTOR/PRELID2/ROS 轴并促进肿瘤发生。MEK 和 mTOR 的双重抑制可有效预防 FBXL6 和 KRAS 诱导的肿瘤发生，为治疗这种侵袭性肝癌亚型提供了一种潜在的治疗策略。
