Background:Kirsten rat sarcoma (KRAS) and mutant KRAS^G12D 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^G12D activity in hepatocellular carcinoma (HCC).Methods:We constructed transgenic mouse strains LC (LSL-Fbxl6^KI/+; Alb-Cre, n=13), KC (LSL-Kras^G12D/+; Alb-Cre, n=10) and KLC (LSL-Kras^G12D/+; LSL-Fbxl6^KI/+; 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.Results: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^G12D at lysine 128, leading to the activation of both KRAS and KRAS^G12D 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^G12D 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 (χ²=85.067, P<0.001), p-mTOR (χ²=66.919, P<0.001) and PRELID2 (χ²=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).Conclusions:FBXL6 activates KRAS or KRAS^G12D 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^G12D-induced tumorigenesis, providing a potential therapeutic strategy to treat this aggressive subtype of liver cancer.

Ubiquitination;Kirsten rat sarcoma (KRAS);F-box and leucine-rich repeat 6 (FBXL6);PRELID2;Reactive oxygen species;Extracellular signal-regulated kinase (ERK);Mammalian target of rapamycin