Elevated FBXL18 promotes RPS15A ubiquitination and SMAD3 activation to drive HCC

Background: F-box and leucine-rich repeat protein 18 (FBXL18) is an E3 ubiquitin ligase that is reported to be involved in the tumorigenesis of various types of cancer. However, it remains unknown whether FBXL18 is correlated with hepatocarcinogenesis. Methods and results: In the current study, we found that FBXL18 was highly expressed in HCC tissues and positively associated with poor overall survival of HCC patients. FBXL18 was an independent risk factor for HCC patients. We observed that FBXL18 drove HCC in FBXL18 transgenic mice. Mechanistically, FBXL18 promoted the K63-linked ubiquitination of small-subunit ribosomal protein S15A (RPS15A) and enhanced its stability, increasing SMAD family member 3 (SMAD3) levels and translocation to the nucleus and promoting HCC cell proliferation. Moreover, the knockdown of RPS15A or SMAD3 significantly suppressed FBXL18-mediated HCC proliferation. In clinical samples, elevated FBXL18 expression was positively associated with RPS15A expression. Conclusion: FBXL18 promotes RPS15A ubiquitination and upregulates SMAD3 expression, leading to hepatocellular carcinogenesis, and this study provides a novel therapeutic strategy for HCC treatment by targeting the FBXL18/RPS15A/SMAD3 pathway.


INTRODUCTION
Hepatocellular carcinoma (HCC), which is the fourth leading cause of cancer-related death worldwide, is the most common primary liver malignancy, accounting for 90% of liver cancer cases [1] . Despite various treatment strategies, including surgical resection, adjuvant chemotherapy and radiotherapy, high-intensity focused ultrasound, and especially the application of molecular-targeted drugs in recent years, the mortality and recurrence rates of HCC remain high. Possible reasons that explain these high mortality and recurrence rates are the robust proliferation and invasive behavior of cancer cells and immune system disorders. Therefore, it is important to identify the precise molecular mechanisms underlying HCC pathogenesis to facilitate the development of novel therapies.
Ubiquitination refers to the attachment of ubiquitin molecules to target proteins through covalent bonds to regulate the stability, activity, and localization of these proteins. E3 ubiquitin-protein ligases, which are the critical components of the ubiquitin-proteasome system, mediate the recognition of specific target proteins [2] . F-box proteins are indispensable substrate-recognition subunits of the SKP1-Cullin1-F-box protein E3 ubiquitin ligase complex and are associated with tumorigenesis and progression [3][4][5][6] . F-box proteins can be divided into 3 families, namely, the FBXW, FBXL, and FBXO families, according to their substrate-recognition domain [7] . FBXL family members have an F-box domain and a leucine-rich repeat domain, and they have been reported to be related to the occurrence and development of HCC. As a member of the FBXL subgroup, F-box and leucine-rich repeat protein 18 (FBXL18) regulates apoptosis through the proteasomal degradation of FBXL7 in HeLa cells [8] and promotes glioma progression by promoting the ubiquitination of AKT [9] . However, the biological function of FBXL18 in HCC and the molecular mechanism have not yet been elucidated.
Small-subunit ribosomal protein S15A (RPS15A) is a member of the ribosomal protein family and is a 40S ribosomal subunit, and it is important for the synthesis of proteins that are related to the expression and transmission of genetic information in humans; abnormal RPS15A expression is associated with various types of cancers [10][11][12] , including HCC [13] . RPS15A is located at chromosome 16p13.1 and has a full length of 7369 bp, including 5 exons and a 393 bp-long coding domain that encodes a protein consisting of 130 amino acids (MW 14.8 kDa) [13] .
In this study, we found that FBXL18 was upregulated and correlated with poor prognosis in HCC patients. FBXL18 promoted HCC cell proliferation and drove tumorigenesis in FBXL18-overexpressing (OE-FBXL18) mice. Furthermore, FBXL18 interacted with RPS15A and enhanced its stability by ubiquitination; then, SMAD3 was upregulated to promote HCC cell proliferation. Our study suggests that the FBXL18/RPS15A/ SMAD3 axis is a novel target for HCC treatment.

Patient subjects
All the human HCC tissue and paired adjacent normal tissue paraffin specimens were obtained from the Department of Hepatobiliary Surgery, Southwest Hospital, Chongqing, PR China, from 2011 to 2013. Samples of 4% formaldehyde-fixed tissues were used for immunohistochemistry (IHC) and hematoxylin & eosin analysis. This study was approved by the Ethics Committee of Southwest Hospital (KY2020127), and all of the patients provided informed consent. Pathological data and detailed clinical data were obtained from each patient (Table S3, http://links.lww.com/HC9/A333). In addition, the clinical relevance of the mRNA expression of FBXL18 was assessed using a TCGA dataset (downloaded from https://xenabrowser.net) and a GEO dataset (GSE36376).
Animal studies and the DEN/CCl4-induced animal model of HCC FBXL18 allele-overexpressing (OE-FBXL18) mice were generated by Cyagen Biosciences (Guangzhou, China). To generate this mouse strain, fertilized mouse zygotes were co-injected with a mixture of Cas9 mRNA, gRNA targeting the mouse ROSA 26 gene, and a construct carrying mouse FBXL18 complementary DNA (cDNA), and then the zygotes were transferred into pseudopregnant C57BL/6N female mice. PCR and gene sequencing were used to identify mice harboring these constructs. To further enhance liver tumorigenesis, OE-FBXL18 or wild-type (WT) mice were injected with 25 mg/kg DEN (N0756, Sigma-Aldrich, USA) in the abdomen at the age of 2 weeks, followed by 14 weekly injections with 0.5 mL/kg CCl 4 dissolved in corn oil. At the age of 24 weeks, the mice were sacrificed by isoflurane. The number of tumors was counted, and the largest tumor diameter was measured. Livers were isolated from the mice and photographed. The liver and body weights were measured [14] . Mouse breeding was performed in the specific pathogen-free facility of the Animal Center of Army Medical University. All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Army Medical University and followed all the relevant ethical regulations (AMUWEC2020936).

Cell culture studies
The human HCC Huh7 cell line was obtained from the Fudan Cell Bank (Shanghai, China), and the HepG2 cell line was obtained from ATCC. All the cell lines tested were negative for mycoplasma contamination. All the experiments were carried out in DMEM supplemented with 10% fetal bovine serum (Gibco, New Zealand), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, USA) at 37°C, 95% humidity, and 5% CO 2 . For the half-life experiments, Huh7 cells overexpressing FBXL18 were treated with 100 µg/mL cycloheximide (MedChemExpress, USA) at different times.

CCK-8 and colony formation assays
HCC cells were seeded in 96-well plates at 3000 cells per well and incubated for 24 hours; then, the cells were transiently transfected with the indicated plasmids or siRNAs. After 48 hours of incubation, cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8; Dojindo, Japan) assay. Cell proliferation is presented as relative cell growth compared with the control group (%). For colony formation assays, 1000 treated cells were seeded in 6-well plates in 1.5 mL DMEM supplemented with 1% penicillin and streptomycin and 10% fetal bovine serum. Colonies with >50 cells were counted 14 days later after staining with 0.1% crystal violet staining at room temperature for 30 minutes.

Cell cycle assay
Huh7 cells were transfected with siRNA control or siRNA targeting FBXL18 for 48 hours. Next, the cells were digested with 0.25% trypsin and then fixed in 70% ethanol overnight. After fixation, the cells were washed twice in a wash buffer. The cells were stained in the dark with 10 μg/mL PI (Beyotime, China) and 200 μg/mL RNase A(Beyotime, China) at final concentrations in PBS for 30 minutes at room temperature and analyzed on a flow cytometer [15] .

Immunohistochemical staining assay
Tissue specimens were fixed in 10% formalin for 72 hours, dehydrated for 5 hours, and embedded in paraffin (Leica, USA). Paraffin-embedded mouse tissue samples were cut into 4 μm-thick tissue sections, and then, the tissue sections were mechanically deparaffinized and incubated in a 2% sodium citrate antigen retrieval solution (Solarbio, USA) at a high temperature and pressure for 2.5 minutes; then, the samples were washed 3 times for 5 minutes each with PBS. Endogenous peroxidase activity was blocked with methanol containing 5% hydrogen peroxide for 10 minutes, and the samples were washed 3 times for 5 minutes each with PBS and incubated with goat serum for 30 minutes at room temperature. Then, the tissue sections were incubated with primary antibodies at 4°C overnight, followed by incubation with a secondary antibody at room temperature for 30 minutes. Next, the tissue sections were incubated with peroxidase at room temperature for 30 minutes. Then, the sections were stained with DAB (ZSGB-BIO, China). Subsequently, the sections were counterstained with hematoxylin (Biosharp, China) for 30 seconds and incubated in warm water for 20 minutes. The tissue sections were dehydrated and preserved using neutral balsam (Biosharp, China) at room temperature. The primary antibodies used were as follows: anti-FBXL18 (Santa Cruz, USA), anti-RPS15A (Abclonal, China), and anti-Ki-67 (Millipore, USA) antibodies. The staining was evaluated by different specialized pathologists who were blinded to the patient characteristics. The staining intensity was determined from 3 different areas for each patient.

Immunofluorescence assay
Huh7 cells were cultured in 6-well plates with coverslips overnight and then transfected with control and Flag-FBXL18 plasmids for 48 hours. The cells were subsequently fixed with 4% paraformaldehyde for 40 minutes and permeabilized with Triton X-100 for 15 minutes. After blocking for 30 minutes, the cells were incubated with primary antibodies at 4°C overnight, and secondary antibodies were conjugated to fluorophores at room temperature for 1 hour. Finally, the nuclei of the cells were stained with Hoechst 33342. Images of the cells were captured using an Olympus microscope.

qRT-PCR
Total RNA was isolated from cultured Huh7 and HepG2 cells using RNAiso Plus reagent (TaKaRa, Japan). cDNA was synthesized using the PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan) according to the manufacturer's instructions. Then, the mRNA levels of the target genes were determined by qRT-PCR using TB Green Premix Ex Taq II (TaKaRa, Japan) with the CFX96 Touch Realtime PCR Detection System. The primers were listed in Table S5, http://links.lww.com/HC9/A333. The results were calculated based on the threshold cycle (Ct), and the relative fold change was determined using the 2 −ΔΔCt method.

Co-immunoprecipitation assay
To examine the interaction between FBXL18 and RPS15A, Huh7 and HepG2 cells were transfected with Flag-FBXL18 plasmids and incubated for 48 hours. The cells were lysed using immunoprecipitation lysate buffer (100 mM NaCl, pH 8.0, 20 mM Tris/HCl, 1% NP-40, and a protease inhibitor cocktail tablet) for 25 min on ice. The lysates were centrifuged at 10,000 × g for 15 minutes at 4°C. After centrifugation, the supernatants were transferred to new Eppendorf tubes, and 1-10 µL (0.2-2 µg) of primary antibody (the optimal antibody concentration was determined by titration) was added and incubated for 1 hour at 4°C. After incubation, 50 µL Protein A/G PLUS-Agarose (Santa Cruz, USA) was added to the protein-antibody complexes and incubated at 4°C on a rotating device overnight. The immunoprecipitates were washed 3 times with immunoprecipitation buffer, and a 5 × sample loading buffer was added to the beads before boiling for 5 minutes. The supernatants were collected and used in a Western blot assay.

In vivo ubiquitination assay
To examine the ubiquitination of RPS15A and the linked form of the polyubiquitin chains by FBXL18, Huh7 cells were transfected with HA-Ub and Flag-FBXL18 plasmids. After 48 hours of transfection, the cells were lysed with immunoprecipitation lysate buffer and denatured by boiling for 5 minutes. The detailed procedures in immunoprecipitation and IB analysis were conducted as described previously. The eluted proteins were analyzed by Western blotting analysis with an anti-RPS15A antibody.

Proteome analysis
Cell samples were sonicated 3 times on ice using a high-intensity ultrasonic processor in lysis buffer (8 M urea and 1% Protease Inhibitor Cocktail). The remaining debris was removed by centrifugation at 12,000 × g at 4°C for 15 minutes. Then, the supernatants were collected, and the protein concentrations were determined with a BCA Kit according to the manufacturer's instructions. For digestion, the proteins were reduced by incubation with 5 mM dithiothreitol at 56°C for 30 minutes, followed by alkylation with 11 mM iodoacetamide at room temperature in the dark for 15 minutes. The protein samples were then diluted by adding 100 mM TEAB to achieve final urea concentrations of less than 2 M. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio and incubated overnight for the first round of digestion, and a 1:100 trypsinto-protein mass ratio was incubated 4 hours for the second round of digestion. The samples were subsequently subjected to LC-MS/MS analysis. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient comprised an increase in solvent B (0.1% formic acid in 98% acetonitrile) from 6% to 23% over 26 minutes, an increase from 23% to 35% in 8 minutes, and a further increase to 80% in 3 minutes; then, the samples were maintained at 80% for the last 3 min. All of these steps were performed all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350-1800 for the full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were then selected for MS/MS using an NCE setting of 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. The data-dependent procedure alternated between 1 MS scan followed by 20 MS/MS scans with 15 sc dynamic exclusion. Automatic gain control was set to 5E4. The fixed first mass was set to 100 m/z. A 1.5-fold change and p value < 0.05 were defined as the criteria that indicated statistically significantly different expression in the proteome.

Data and code availability
All the detailed data in this paper are available upon request. This paper does not report the original code.

Statistics
Prism 8.0 (GraphPad, USA), SPSS20 (Statistical Product Service Solutions, IBM), R (R 4.1.2), and R-Studio (v7.2 Build 153957) software were used to analyze the data, and the data are presented as the mean ± SEM. Comparisons between 2 groups were analyzed with a 2-tailed unpaired t-test. Comparisons among more than 2 groups were analyzed with 1-way ANOVA with Tukey's post hoc test when the data passed the Shapiro-Wilk normality test. When the data did not pass the normality test, the nonparametric Kruskal-Wallis test was used, followed by Dunn's multiple range test for post hoc comparisons. Area determination was performed using an imaging system (Olympus, Germany) and FIJI software (ImageJ, National Institutes of Health). The correlations between the genetic expression and clinicopathological factors were performed by Pearson's chi-square test. OS was analyzed using a log-rank test and the Kaplan-Meier method. The correlation between FBXL18 expression and RPS15A expression was determined by Pearson's correlation coefficient. p ≤ 0.05 were considered statistically significant.

FBXL18 is positively correlated with poor survival in HCC patients
To determine the role of FBXL18 in human HCC, a volcano plot was generated and showed that the mRNA levels of FBXL18, CCNF, FBXO43, and FBXW10 were significantly highly expressed in HCC tissues according to The Cancer Genome Atlas (TCGA) dataset (Fig. 1A). CCNF, FBXO43 and FBXW10 were reported to be associated with HCC [16][17][18][19] , but whether FBXL18 is correlated with HCC remains unknown. The mRNA levels of FBXL18 were significantly higher in HCC tissues than in adjacent normal tissues in the TCGA dataset (Fig. 1B) and in the GSE36376 dataset (Fig. 1C). The protein expression of FBXL18 in a cohort of 92 paired human HCC and adjacent normal liver tissue specimens was measured by IHC staining. The results demonstrated that the expression of FBXL18 was higher in 57.6% (53/92) of the HCC samples than in adjacent normal liver tissues. (Fig. 1D). The relationships between FBXL18 protein expression and clinicopathological parameters were investigated. FBXL18 was positively associated with TNM stage, histological grade, tumor size, vascular thrombosis, and metastasis in HCC patients (Fig. 1E, F and Table 1), indicating that FBXL18 expression is positively correlated with poor prognosis in HCC patients. To extend this analysis, the association between overall survival (OS) and various risk factors in 92 HCC patient tissue samples was analyzed. Kaplan-Meier analysis showed that FBXL18 expression in 92 HCC patients was positively associated with poor survival in HCC patients (p < 0.001; Fig. 1G). Consistently, FBXL18 expression in a cohort of 354 HCC patients was positively associated with poor survival in the TCGA dataset (p = 0.003; Fig. 1H). Univariate and multivariate analyses revealed that FBXL18 expression (p = 0.048), histological grade (p = 0.037), and recurrence (p = 0.013) were negatively associated with OS in HCC patients, indicating that FBXL18 is an independent risk factor in HCC patients (

FBXL18 promotes inflammation and hepatocarcinogenesis in mice
Subsequently, the role of FBXL18 in liver tumorigenesis in vivo was addressed. In this study, WT mice and FBXL18-overexpressing (OE-FBXL18) mice were used and i.p. injected with 25 mg/kg DEN and 2.5 mL/kg 20% CCl 4 to shorten the observation period. Mice with high FBXL18 expression (OE-FBXL18 mice; n = 14) developed more liver tumors, larger tumor sizes, and increased liver and body ratios compared with WT mice (n = 16) up until 24 weeks, demonstrating that FBXL18 overexpression drives hepatocarcinogenesis in mice ( Fig. 2A-C). Consistently, compared with WT mice, hematoxylin & eosin and IHC staining showed that the expression of FBXL18 and the proliferation biomarker Ki-67 was significantly increased in OE-FBXL18 mice, indicating that FBXL18 overexpression promoted liver tumor growth (Fig. 2D, E). Furthermore, OE-FBXL18 mice tolerated regenerative hepatocytes and were more sensitive to necrosis and ballooning generation than wild-type mice under conditions of DEN/CCl 4 -induced injury (Fig. 2F). In addition, FBXL18 induced more severe mild lobular, periportal and pericentral inflammation in OE-FBXL18 mice than in wild-type mice (Fig. 2G). Taken together, these results indicate that FBXL18 participates in inflammation during the process of HCC tumorigenesis.

FBXL18 promotes the proliferation of HCC cells
First, we determined the FBXL18 levels in L02, Huh7, and HepG2 cells. The expression of FBXL18 in Huh7 and HepG2 cells was higher than that in L02 cells (Fig. 3A). Next, to evaluate the effect of FBXL18 on HCC cell proliferation, Huh7 and HepG2 cells were transfected with empty vector or Flag-FBXL18 plasmid, and cell proliferation and colony formation assays were then performed. We found that the overexpression of FBXL18 significantly promoted cell proliferation and colony formation in both Huh7 and HepG2 cells (Fig. 3B-D). Reciprocally, silencing of FBXL18 significantly inhibited cell proliferation in Huh7 and HepG2 cells (Fig. 3E, F). Moreover, the cell cycle progression of Huh7 cells transfected with the FBXL18 siRNAs was significantly arrested at the G1 phase, as determined by flow cytometry (Fig. 3G). Taken together, FBXL18 promotes HCC cell proliferation in vitro.  FBXL18 promotes K63-linked RPS15A polyubiquitination and enhances its stability To identify the direct substrates of FBXL18, Huh7 cells were transfected with Flag-tagged FBXL18, and then pull-down assays were performed with an anti-FBXL18 antibody. The proteins were analyzed by mass spectrometry. A total of 162 proteins were identified among those that were immunoprecipitated with FBXL18. We analyzed all changed proteins and FBXL18interacting proteins after FBXL18 overexpression using proteome analysis. A total of 106 proteins exhibited a greater than 1.5-fold change after FBXL18 overexpression. Furthermore, 162 proteins were identified as candidates for interaction with FBXL18 in HCC through a pull-down assay using an anti-Flag-FBXL18 antibody. We generated a chart showing the overlapping proteins to identify potential FBXL18 substrates. There were 2 overlapping proteins, PLEC and RPS15A, that exhibited high degrees of change (Fig. 4A). PLEC and RPS15A were reported to be oncogenes in cancers. RPS15A was upregulated and PLEC was downregulated upon FBXL18 expression. RPS15A, but not PLEC, was reported to be associated with HCC [20,21] . These findings indicated that RPS15A may be a key substrate of FBXL18 for HCC tumorigenesis. However, it remains unknown whether the role of RPS15A in HCC depends on its ubiquitination.
To examine the interaction between FBXL18 and RPS15A, Huh7 and HepG2 cells were transfected with Flag-FBXL18, and then, pull-down assays were conducted with an anti-Flag antibody. The results showed that exogenous RPS15A was pulled down by ectopically expressed FBXL18 (Fig. 4B). This finding was further confirmed by FBXL18 LRRs domain binding with RPS15A ( Figure S1A, B, http://links.lww.com/HC9/A334). Next, whether FBXL18 promoted RPS15A protein ubiquitination was evaluated. An in vivo ubiquitination assay indicated that the overexpression of FBXL18 significantly promoted the polyubiquitination of RPS15A (Fig. 4C). K63-linked ubiquitination promotes protein activation or enhances protein stability, whereas K48-linked ubiquitination mediates protein degradation [6,22,23] . To analyze the type of ubiquitin chains involved in FBXL18-mediated RPS15A polyubiquitination, 2 ubiquitin mutants (K63R and K48R) were used along with wild-type ubiquitin. The ubiquitination assay showed that a K63R ubiquitin mutant significantly reduced the formation of the polyubiquitin chain on RPS15A compared with wild-type ubiquitin and its K48R mutant (Fig. 4D), suggesting that FBXL18 may promote RPS15A stability or activation. We found that FBXL18 upregulated RPS15A expression in a dosedependent manner (Fig. 4E). Furthermore, FBXL18 prolonged the half-life of RPS15A in a cycloheximide chase assay (Fig. 4F). Taken together, FBXL18 promotes the K63-linked polyubiquitination of RPS15A and enhances its stability.

FBXL18 promotes cell proliferation via RPS15A-mediated SMAD3 upregulation
To validate the effect of RPS15A on FBXL18-mediated HCC cell proliferation, Huh7 and HepG2 cells were transfected with FBXL18 plasmids with or without siRNA targeting RPS15A. The results showed that silencing of RPS15A significantly inhibited HCC cell proliferation, and this effect could significantly block FBXL18-mediated cell proliferation (Fig. 5A-D). The KEGG pathway analysis showed that the SMAD pathway was enriched in FBXL18-overexpressed Huh7 cells ( Figure S2, http:// links.lww.com/HC9/A334). The SMAD signaling pathway plays a critical role in regulating multiple steps of carcinogenesis. The DNA-binding protein (consisting of SMAD2, SMAD3, and SMAD4) is an important transcription protein that regulates multiple target genes in cooperation with coactivators and corepressors in carcinogenesis. We wanted to determine whether FBXL18-mediated cell proliferation is correlated with SMAD. We found that FBXL18 significantly upregulated the protein expression of SMAD3 but not SMAD2 and SMAD4, and the knockdown of RPS15A blocked this effect (Fig. 5E). Furthermore, knockdown of SMAD3 significantly attenuated FBXL18-mediated cell proliferation in both Huh7 and HepG2 cells (Fig. 5F-I). To validate the effect of SMAD3 on transcription, we determined the SMAD3 levels in Huh7 cells transfected with control or Flag-FBXL18 plasmids by immunofluorescence assay.

Correlation between FBXL18 expression and RPS15A expression in HCC tissues
We next evaluated the significance of RPS15A in HCC using patient tissues. First, we used IHC to measure RPS15A expression in 90 paired human HCC tissues and adjacent normal liver tissues. We found that RPS15A was highly expressed in 55.6% (50/90) of HCC tissues (Fig. 6A). Subsequently, the relationship between RPS15A expression and clinicopathological characteristics was evaluated. The expression of RPS15A was positively associated with TNM tumor stage, recurrence, vascular thrombosis, and metastasis (Fig. 6B Fig. 6D). To evaluate the association between FBXL18 and RPS15A, we used IHC to measure FBXL18 expression in HCC tissues and correlated it with RPS15A staining in the same 90 HCC tissues (Fig. 6A). In total, 42.2% (38/90) of HCC tissues had high FBXL18 and high RPS15A staining intensity, which was statistically significant compared to the staining intensity in the normal tissues, suggesting the possibility that FBXL18 stabilized RPS15A in HCC (Fig. 6E). Moreover, the protein levels of RPS15A and SMAD3 were markedly increased in OE-FBXL18 mice ( Figure S4, http://links.lww.com/HC9/A334). In conclusion, the increased expression of FBXL18 in HCC patients promoted hepatocarcinogenesis through the induction of RPS15A ubiquitination and upregulation of SMAD3 expression (Fig. 6F).