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. 2023 Dec 20;10(1):68.
doi: 10.1186/s40779-023-00501-8.

Elevated FBXL6 activates both wild-type KRAS and mutant KRASG12D and drives HCC tumorigenesis via the ERK/mTOR/PRELID2/ROS axis in mice

Hao-Jun Xiong  1 , Hong-Qiang Yu  1 , Jie Zhang  1 , Lei Fang  1 , Di Wu  1 , Xiao-Tong Lin  1 , Chuan-Ming Xie  2
Affiliations

Elevated FBXL6 activates both wild-type KRAS and mutant KRASG12D and drives HCC tumorigenesis via the ERK/mTOR/PRELID2/ROS axis in mice

Hao-Jun Xiong et al. Mil Med Res. .

Abstract

Background: Kirsten rat sarcoma (KRAS) and mutant KRASG12D 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 KRASG12D activity in hepatocellular carcinoma (HCC).

Methods: We constructed transgenic mouse strains LC (LSL-Fbxl6KI/+;Alb-Cre, n = 13), KC (LSL-KrasG12D/+;Alb-Cre, n = 10) and KLC (LSL-KrasG12D/+;LSL-Fbxl6KI/+;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 KRASG12D at lysine 128, leading to the activation of both KRAS and KRASG12D 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 KRASG12D 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 KRASG12D 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 KRASG12D-induced tumorigenesis, providing a potential therapeutic strategy to treat this aggressive subtype of liver cancer.

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

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
FBXL6 activates KRAS and KRASG12D by K63-linked polyubiquitination at the site K128. a Analysis of FBXL6 expression across cancers in the TCGA database shows that FBXL6 is upregulated in various types of human cancers, including liver hepatocellular carcinoma (LIHC), namely, HCC. b Alb-Cre and LSL-Fbxl6KI/+;Alb-Cre (LC) male mice were monitored for 320 d and then euthanized. The livers were imaged. c Diagram showing the number of proteins with increased ubiquitinated sites and upregulated expression in HCC tumors. Ub-C/A > 4 indicates proteins with a more than fourfold increase in ubiquitinated sites in tumors compared with normal tissues from male mice; Ub-B/A > 3 indicates proteins with a more than threefold increase in ubiquitinated sites in adjacent tissues compared with normal tissues. Ub-C/B > 1.3 indicates proteins with a more than 1.3-fold increase in ubiquitinated sites in tumors compared with adjacent tissues. TP-C/B > 1.4 and TP-C/A > 1.5 indicate that the protein levels were upregulated 1.4- or 1.5-fold in tumors compared with adjacent tissues or normal tissues, respectively. d Evolutionary conservation of the site K128 on KRAS from different species. A red star (*) indicates a conserved site in KRAS in different species. e A co-IP assay was utilized to measure the interaction between FBXL6 and KRAS. Huh7 and Hep3B cells were transfected with Flag-FBXL6 plasmids for 48 h and then lysed. The indicated antibodies and protein A/G PLUS-Agarose were added to the cell lysates. f HEK293T cells were transfected with the indicated plasmids for 72 h and then lysed with a 6 mol/L guanidine solution, followed by pull-down using Ni–NTA beads or direct Western blotting with the indicated antibodies. g Huh7 cells were transfected with the indicated plasmids for 72 h and lysed with lysis buffer. Activated KRAS was pulled down with an anti-active RAS monoclonal antibody (RBD peptide), followed by Western blotting. Band intensity was quantified by ImageJ software. h After serum deprivation for 12 h, Huh7 cells were transfected with the indicated plasmids for 72 h, followed by Western blotting. ns non‑significant; ***P < 0.001. BLCA bladder urothelial carcinoma, BRCA breast invasive carcinoma, CESC cervical squamous cell carcinoma and endocervical adenocarcinoma, CHOL cholangiocarcinoma, COAD colon adenocarcinoma, ESCA esophageal carcinoma, GBM glioblastoma multiforme, HNSC head and neck squamous cell carcinoma, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LUAD lung adenocarcinoma, LUSC lung squamous cell carcinoma, PAAD pancreatic adenocarcinoma, PRAD prostate adenocarcinoma, PCPG pheochromocytoma and paraganglioma, READ rectum adenocarcinoma, SARC sarcoma, SKCM skin cutaneous melanoma, THCA thyroid carcinoma, THYM thymoma, STAD stomach adenocarcinoma, UCEC uterine corpus endometrial carcinoma, FBXL6 F-box and leucine-rich repeat 6, TCGA The Cancer Genome Atlas, WCE whole-cell extract, HA hemagglutinin, KRAS kirsten rat sarcoma, KRASG12D glycine to aspartic acid mutation of KRAS, Ub ubiquitin, HA-KRAS (DM) double mutation of KRAS at the sites K128 and G12, A normal liver tissue, B adjacent tumor tissue, C cancer or tumor tissue, TP total protein, K128 lysine 128, Co-IP coimmunoprecipitation, RBD RAS-binding domain
Fig. 2
Fig. 2
FBXL6 facilitates KRASG12D-driven hepatocarcinogenesis and lung metastasis. LSL-Fbxl6KI/+;Alb-Cre (LC), LSL-KrasG12D/+;Alb-Cre (KC) and LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre (KLC) mice were monitored for 320 d and then sacrificed. a Representative images of tumorigenesis in LC, KC and KLC mice. b Quantification of the tumor number, tumor size, and liver/body weight ratio of LC (n = 13), KC (n = 10), and KLC mice (n = 12). qPCR was utilized to measure the expression of HCC markers (Cd44, Afp, Gpc3 and Ly6d) (c), proliferation markers (Ki67, Pcna, Ccnb1, and Ccnb2) (d), and metastasis-related markers (Icam1, Vcam1, Mmp9 and Ccl2) (e), in WT liver tissues, LC tumors, KC tumors and KLC tumors. Lung tissues were collected from LC, KC and KLC mice for HE and IHC staining. Representative images of HE and IHC staining for lipase C (LIPC) showing distinct lung metastatic foci expressing LIPC (f). Scale bars = 200 or 50 μm. The ratio of lung metastasis in each cohort was calculated (g). Data are represented as the mean ± SEM. One-way ANOVA was used in (b–e). ns non‑significant; *P < 0.05; **P < 0.01; ***P < 0.001. Fbxl6 F-box and leucine-rich repeat 6, Kras kirsten rat sarcoma, Afp alpha fetoprotein, Gpc3 glypican 3, Ly6d lymphocyte antigen 6 family member D, Ki67 marker of proliferation Ki-67, Pcna proliferating cell nuclear antigen, Ccnb1 cyclin B1, Ccnb2 cyclin B2, Icam1 intercellular adhesion molecule 1, Vcam1 vascular cell adhesion molecule 1, Mmp9 matrix metallopeptidase 9, Ccl2 C–C motif chemokine ligand 2, IHC immunohistochemistry, SEM standard error of the mean
Fig. 3
Fig. 3
FBXL6 activates the oncogenic KRAS/MEK/ERK axis and promotes mTOR activation. a HE staining was utilized to determine the cancer tissue structure, and IHC was used to measure the p-ERK, p-mTOR, and p-S6 protein signals in WT, LC, KC and KLC mice. Representative consecutive IHC staining images are presented. Scale bars = 200 or 50 μm. b Western blotting was utilized to determine the protein levels of p-mTOR, p-4EBP1, p-70S6K, p-S6, p-Akt and p-ERK in WT (X112, 115 and 116), LC (X136, 178 and 182), KC (X151, 154 and 156) and KLC (X148, 171 and 190) mice. c After treatment with pharmacological inhibitors of PI3K (GDC-0326, 1 μmol/L), MEK (trametinib, 100 nmol/L), or mTOR (everolimus, 100 nmol/L); a PI3K inhibitor combined with an mTOR inhibitor; or an MEK inhibitor combined with an mTOR inhibitor for 48 h, KLC primary cells were lysed to extract total proteins. Thereafter, Western blotting was employed to measure the protein levels of p-mTOR, p-S6, p-4EBP1, p-70S6K, p-Akt and p-ERK. d After treatment with pharmacological inhibitors of PI3K (GDC-0326, 1 μmol/L), MEK (trametinib, 100 nmol/L), and mTOR (everolimus, 100 nmol/L) alone; a PI3K inhibitor combined with an mTOR inhibitor; or an MEK inhibitor combined with an mTOR inhibitor for 24, 48, and 72 h, the proliferation of KLC primary cells was analyzed by CCK-8 assay. Two-way ANOVA with the Bonferroni correction for multiple comparisons was used. ***P < 0.001. Fbxl6 F-box and leucine-rich repeat 6, Kras kirsten rat sarcoma, MEK mitogen-activated protein kinase kinase, ERK extracellular signal-regulated kinase, IHC immunohistochemistry, WT wild-type, LC LSL-Fbxl6KI/+;Alb-Cre, KC LSL-KrasG12D/+;Alb-Cre, KLC LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre, PI3K phosphatidylinositol 3‑kinase, mTOR mammalian target of rapamycin, S6 ribosomal protein S6, 70S6K ribosomal protein S6 kinase B1, 4EBP1 eukaryotic translation initiation factor 4E binding protein 1, PI3K-i inhibitor of PI3K, MEK-i inhibitor of MEK, mTOR-i inhibitor of mTOR
Fig. 4
Fig. 4
Fbxl6 elevation synergizes with KrasG12D to drive HCC by upregulating Prelid2. a Venn diagram showing genes that were significantly differentially expressed at the mRNA level between liver tissues from KC and KLC mice and those from LC and KLC mice. b Heatmap showing the top 21 gene signatures representing different groups. c qPCR was used to detect the mRNA levels of Prelid2, Slc41a3 and Gldn in WT, LC, KC and KLC mice. d HCC tissues were collected from LC, KC and KLC mice for HE and IHC staining. Representative consecutive IHC staining images for Prelid2, Slc41a3 and Gldn are presented. Scale bars = 200 or 50 μm. e Cells that were positive for Prelid2, Slc41a3, or Gldn were counted among a total of 500 cells on average from 3 independent tumors derived from 3 mice per group. f The Prelid2 knockdown efficiency was determined by qPCR. Knockdown of Prelid2 inhibited cell proliferation (g), migration (h), and ROS generation (i) in KLC primary cells. j Blockade of ROS with NAC (1 or 10 mmol/L) inhibited cell migration in KLC primary cells. Scale bars = 250 (h), 200 (i), or 100 (j) μm. Unpaired Student’s t test was used to analyze the data in (i). One-way ANOVA with Tukey’s multiple comparisons test was used to analyze the data in (c, e, f, j). *P < 0.05; **P < 0.01; ***P < 0.001. Fbxl6 F-box and leucine-rich repeat 6, Kras kirsten rat sarcoma, Prelid2 the proteins of relevant evolutionary and lymphoid interest (PRELI) domain 2, WT wild-type, LC LSL-Fbxl6KI/+;Alb-Cre, KC LSL-KrasG12D/+;Alb-Cre, KLC LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre, Slc41a3 solute carrier family 41 member 3, Gldn gliomedin, IHC immunohistochemistry, HCC hepatocellular carcinoma, ROS reactive oxygen species, NAC N-acetylcysteine
Fig. 5
Fig. 5
mTOR and MEK inhibition significantly blocks hepatocarcinogenesis and lung metastasis triggered by Fbxl6 elevation and Kras mutation. a Nude mice bearing KLC orthotopic HCC tumors were randomized into three groups: the negative control (NC) group, everolimus (E) group and everolimus combined with trametinib (E + T) group. The NC group was treated with vehicle, the E group was treated with everolimus (diluted in DMSO and coil oil, 4 mg/kg, intraperitoneal injection) twice per week for six rounds, and the E + T group was treated with both everolimus (the everolimus dose was as in the E group) and trametinib (diluted in DMSO and coil oil, 0.5 mg/kg, intraperitoneal injection) twice per week for six rounds. The mice were sacrificed after the last injection, and representative images showing tumorigenesis are presented. n = 7. b Tumor weight, the tumor/liver weight ratio, and tumor volume were analyzed (n = 7). c Representative images showing the effect of mTOR and MEK/ERK inhibitors on lung metastases. The lung metastasis rate (d) and number of lung distant lung metastatic foci (e) were quantified. f Representative HE and IHC staining images for lipase C (LIPC) showing distinct lung metastatic foci expressing LIPC. Scale bars = 200 or 50 μm. g Representative images of HE and IHC staining for Prelid2, p-mTOR, p-4EBP1, and p-ERK in the NC, E and E + T groups. Representative consecutive IHC staining images are presented. Scale bars = 200 or 50 μm. One-way ANOVA with Tukey’s multiple comparisons test was used in (b, e). *P < 0.05; **P < 0.01; ***P < 0.001. KLC LSL-KrasG12D/+;LSL-Fbxl6KI/+;Alb-Cre, mTOR mammalian target of rapamycin, MEK mitogen-activated protein kinase kinase, Fbxl6 F-box and leucine-rich repeat 6, Kras kirsten rat sarcoma, NC negative control, E everolimus, T trametinib, DMSO dimethyl sulfoxide, ERK extracellular signal-regulated kinase, IHC immunohistochemistry
Fig. 6
Fig. 6
Elevated PRELID2 is positively correlated with the FBXL6/p-ERK/p-mTOR pathway and poor prognosis of HCC. a Representative images of IHC staining for FBXL6, PRELID2, p-ERK, and p-mTOR in human HCC tumors. Scale bar = 50 µm. b The association between PRELID2 and FBXL6, p-ERK, or p-mTOR in 129 paired HCC tumors and adjacent normal tissues was analyzed by χ2 test. c The prognostic significance of PRELID2 in HCC patients was evaluated by Kaplan–Meier analysis. High expression of PRELID2 predicted a shorter overall survival (OS) time. d The association between FBXL6 and p-ERK/p-mTOR protein levels in 129 HCC tissues was evaluated by the χ2 test. e The prognostic significance of the coexpression of FBXL6 and p-ERK in HCC patients was evaluated by Kaplan–Meier analysis. f Kaplan–Meier survival curves showing the overall survival of FBXL6high/p-ERKhigh HCC patients with high or low PRELID2 expression. PRELID2 the proteins of relevant evolutionary and lymphoid interest (PRELI) domain 2, FBXL6 F-box and leucine-rich repeat 6, ERK extracellular signal-regulated kinase, mTOR mammalian target of rapamycin, IHC immunohistochemistry, HCC hepatocellular carcinoma
Fig. 7
Fig. 7
Working model of KRAS/KRASG12D-triggered HCC in response to high FBXL6 expression. FBXL6 elevation facilitates the KRAS/KRASG12D mutation-mediated activation of MEK/ERK/mTOR signaling by promoting K63-linked KRAS/KRASG12D polyubiquitination at the site K128. Hyperactive mTOR increases the expression of PRELID2, leading to HCC tumorigenesis and metastasis in mice. This picture was created by BioRender. FBXL6 F-box and leucine-rich repeat 6, KRAS kirsten rat sarcoma, PRELID2 the proteins of relevant evolutionary and lymphoid interest (PRELI) domain 2, HCC hepatocellular carcinoma, MEK mitogen-activated protein kinase kinase, ERK extracellular signal-regulated kinase, mTOR mammalian target of rapamycin, WT wild-type, Ub ubiquitin, ROS reactive oxygen species
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