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...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(χ^(2)=85.067,P<0.001),p-mTOR(χ^(2)=66.919,P<0.001)and PRELID2(χ^(2)=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.展开更多
Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultu...Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.展开更多
基金supported by the National Natural Science Foundation of China(82370631)the Talent Foundations from Army Medical University(4174C6),the Chongqing Government(CQYC20220303727)to Xie CMthe National Natural Science Foundation of China(31900449)to Xiong HJ.
文摘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(χ^(2)=85.067,P<0.001),p-mTOR(χ^(2)=66.919,P<0.001)and PRELID2(χ^(2)=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.
基金in part by Natural Sciences Foundation of China (No. 39870239)by the Sasagawa Fellowship,Japan.
文摘Objective: To explore the effects of γ-irradiation on mitogen-activated protein kinases (MAPKs) and role of intracellular calcium in this event in intestinal epithelial cell line 6 (IEC-6 cells). Methods: After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca2+ chelator were exposed to Y-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry. Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting. Results: In response to γ-irradiation, phosphorylation of ERK was not significantly observed, while the levels of phosphorylated c-Jun NH2-terminal kinase (JNK) and p38 MAPK were increased in 30 min and reached the peak 2 h after exposure to 6 Gy γ-irradiation, though the cell viability was significantly lowered 12 h. On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK. Chelation of intracellular Ca2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradia-tion, but removal of external Ca2+ had no such effect. Activation of p38 MAPK, but not of ERK, was seen to have a correlation with γ-irradiation induced apoptosis. Conclusion: The results suggest that γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.
文摘目的观察口虾蛄乙酸乙酯提取物(ESO)对人肺腺癌耐药细胞株A549/DDP增殖、耐药的影响,并探讨其机制。方法选取人肺腺癌耐药细胞株A549/DDP,用不同浓度(0、200、400、800μg/m L)的ESO干预细胞,24 h后,采用MTT法测算细胞增殖抑制率,流式细胞技术测算细胞周期百分率。采用流式细胞技术观察400μg/m L ESO干预0、30、60 min细胞内荧光染料罗丹明123(Rh123)蓄积和外排情况。采用Western blotting法检测不同浓度ESO干预24 h后细胞多药耐药蛋白1(MDR1),并检测400μg/m L ESO干预2、4、8 h后细胞ERK1/2、p-ERK1/2蛋白。结果 200、400、800μg/m L的ESO干预细胞增殖抑制率分别为19.940%±0.041%、37.360%±0.024%、42.510%±0.008%,两两相比P均<0.01。0、200、400、800μg/m L ESO干预细胞G2/M期分别为24.06%±2.22%、31.10%±0.79%、35.64%±1.15%、43.19%±0.43%,两两相比P均<0.01。Rh123在ESO干预细胞内积累增多、外排减少,且60 min时较30 min时明显(P均<0.01)。0、200、400、800μg/m L ESO干预细胞MDR1蛋白相对表达量分别为1.22±0.23、0.83±0.16、0.37±0.09、0.31±0.04,两两相比P均<0.01。ESO干预不同时间A549/DDP细胞ERK1/2蛋白表达无变化,p-ERK1/2蛋白表达上调(P均<0.01)。结论 ESO通过使A549/DDP细胞发生周期阻滞,从而抑制其增殖;通过调节MAPK-ERK1/2信号通路,使其多药耐药发生逆转。
