KCTD17-mediated Ras stabilization promotes hepatocellular carcinoma progression

Article information

Clin Mol Hepatol. 2024;30(4):895-913
Publication date (electronic) : 2024 August 5
doi : https://doi.org/10.3350/cmh.2024.0364
1Department of Biomedical Sciences, College of Medicine, Inha University, Incheon, Korea
2Program in Biomedical Science & Engineering, College of Medicine, Inha University, Incheon, Korea
3Research Center for Controlling Intercellular Communication (RCIC), College of Medicine, Inha University, Incheon, Korea
4Department of Biomedical Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Korea
5Department of Medicine, Columbia University, New York, NY, USA
6Institute of Pharmaceutical Sciences, China Pharmaceutical University, Nanjing, China
7Ionis Pharmaceuticals Inc., Carlsbad, CA, USA
8Division of Life Sciences, Jeonbuk National University, Jeonju, Korea
Corresponding author : KyeongJin Kim Department of Biomedical Sciences, College of Medicine, Program in Biomedical Science & Engineering, Research Center for Controlling Intercellular Communication (RCIC), College of Medicine, Inha University, 100 Inha-ro, Michuhol-gu, Incheon 22212, Korea Tel: +82-860-9870, Fax: +82-32-885-8302, E-mail: kimkj@inha.ac.kr
Soon-Sun Hong Department of Biomedical Sciences, College of Medicine, Program in Biomedical Science & Engineering, Research Center for Controlling Intercellular Communication (RCIC), College of Medicine, Inha University, 100 Inha-ro, Michuhol-gu, Incheon 22212, Korea Tel: +82-32-890-3683, Fax: +82-32-890-2462, E-mail: hongs@inha.ac.kr
Sang Bae Lee Division of Life Sciences, Jeonbuk National University, 567, Baekje-daero, Deokjin-gu, Jeonju 54896, Korea Tel: +82-63-270-4856, Fax: +82-63-270-3362, E-mail: leesb@jbnu.ac.kr
*These authors have contributed equally to this work.
Editor: Kin Wah Lee, The Hong Kong Polytechnic University, Hong Kong
Received 2024 May 17; Revised 2024 July 31; Accepted 2024 August 1.

Abstract

Background/Aims

Potassium channel tetramerization domain containing 17 (KCTD17) protein, an adaptor for the cullin3 (Cul3) ubiquitin ligase complex, has been implicated in various human diseases; however, its role in hepatocellular carcinoma (HCC) remains elusive. Here, we aimed to elucidate the clinical features of KCTD17, and investigate the mechanisms by which KCTD17 affects HCC progression.

Methods

We analyzed transcriptomic data from patients with HCC. Hepatocyte-specific KCTD17 deficient mice were treated with diethylnitrosamine (DEN) to assess its effect on HCC progression. Additionally, we tested KCTD17-directed antisense oligonucleotides for their therapeutic potential in vivo.

Results

Our investigation revealed the upregulation of KCTD17 expression in both tumors from patients with HCC and mouse models of HCC, in comparison to non-tumor controls. We identified the leucine zipper-like transcriptional regulator 1 (Lztr1) protein, a previously identified Ras destabilizer, as a substrate for KCTD17-Cul3 complex. KCTD17-mediated Lztr1 degradation led to Ras stabilization, resulting in increased proliferation, migration, and wound healing in liver cancer cells. Hepatocyte-specific KCTD17 deficient mice or liver cancer xenograft models were less susceptible to carcinogenesis or tumor growth. Similarly, treatment with KCTD17-directed antisense oligonucleotides (ASO) in a mouse model of HCC markedly lowered tumor volume as well as Ras protein levels, compared to those in control ASO-treated mice.

Conclusions

KCTD17 induces the stabilization of Ras and downstream signaling pathways and HCC progression and may represent a novel therapeutic target for HCC.

Graphical Abstract

INTRODUCTION

Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is associated with high mortality [1,2]. Most patients with HCC are diagnosed at an advanced stage, leaving limited therapeutic options [3,4]. First-line tyrosine kinase inhibitor (TKI) treatment with sorafenib and lenvatinib offers only a modest extension of the median overall survival of patients with advanced-stage HCC [5-8]. Secondline treatment with angiogenesis inhibitors similarly offers limited improvement in quality of life [9-12]. Thus, identification of reliable biomarkers and the development of novel therapeutic strategies are urgently required for HCC.

Potassium channel tetramerization domain-containing 17 (KCTD17) belongs to a family of 25 proteins named for shared sequence similarities with voltage-gated potassium channels [13]. Unlike its counterparts, KCTD17 is a soluble non-channel protein that serves as a substrate adapter for the Cul3-based ubiquitin-conjugating enzyme E3 ligase to regulate cellular proliferation, transcription, and cytoskeleton organization [13-16]. We recently observed an increase in KCTD17 expression in the livers of obese mice and patients with the recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), which leads to abnormalities in glucose and lipid metabolism [17,18].

MASLD has rapidly become a leading cause of HCC [19-21]. Therefore, we postulated that KCTD17 may additionally regulate HCC initiation and/or progression. Indeed, we observed a significant increase in KCTD17 levels in HCC patient samples, corresponding to disease severity. Similarly, KCTD17 expression was increased in tumor tissues versus non-tumor tissues compared to that in mouse models of HCC, leading to cellular proliferation and migration. These effects are mediated through KCTD17-dependent polyubiquitination and the subsequent degradation of leucine zipper-like transcriptional regulator 1 (Lztr1), known as a Ras destabilizer [22-24]. This degradation process results in stabilization of the Ras oncogene and activation of Ras/Mitogenactivated protein kinase (MAPK) signaling. Thus, hepatocyte-specific KCTD17 deficient mice or liver cancer xenograft models exhibited lower susceptibility to liver carcinogenesis and tumor growth, and treatment with KCTD17-directed antisense oligonucleotide (ASO) in mouse models of HCC markedly reduced tumor growth. These data highlight the mechanism by which KCTD17-mediated regulation of Ras stability may lead to the development of novel therapeutic strategies against HCC.

MATERIALS AND METHODS

Human transcriptomic analyses

All raw transcriptomic data were retrieved from the Gene Expression Omnibus under accession numbers GSE25097, GSE36376, and GSE202853. RNA-seq data and clinical information for the liver hepatocellular carcinoma (LIHC) cohort were downloaded from the Cancer Genome Atlas (TCGA) data portal. All computations were conducted using the R program (RStudio 1.4). Using the R environment for statistical analysis, gene set enrichment analysis was carried out using the clusterProfiler package [25]. All variables and signatures’ normalized enrichment score (NES), P-value, and false discovery rate (FDR) were derived in the R environment using the GTEx database as raw count data from all (371) primary tumor transcriptomes imported from TCGA-LIHC project, with the top and bottom 10% of samples based on KCTD17 expression analyzed after normalization to the log2(TMM+1) value (edgeR package). The findings of the GSEA results were visualized using R packages, including ggplot2, enrichplot, and enhanced volcano.

Animals

C57BL/6J (#664) and Rosa26-LSL-Cas9 knockin (#026175) [26] mice were purchased from the Jackson Labs. Rosa26-LSL-Cas9 KI mice were first treated with 25 mg/kg diethylnitrosamine (DEN; N0259; Sigma-Aldrich, St. Louis, MO, USA) on day 14 and then transduced with 1.5×1011 genome copies per mouse of adeno-associated virus subtype 8 (AAV8) expressing either AAV8-TBG-Cre or AAV8-U6-Kctd17 sgRNA-TBG-Cre, to generate control or L-Kctd17 mice [18]. After transduction, mice were maintained on a choline-deficient, L-amino-acid-defined, high-fat diet (CDAHFD) (60 Kcal% fat and 0.1% methionine by weight, A06071302; Research Diets Inc, New Brunswick, NJ, USA) for 16–18 weeks [27].

To induce the rapid onset of mouse HCC, 10 mg of the plasmids encoding myr-Akt and N-Ras along with sleeping beauty transposase in a ratio of 25:1 was administered by hydrodynamic tail vein injection [28,29]. DEN or carbon tetrachloride (CCl4) with fructose/palmitate/cholesterol (FPC)-enriched NASH diet-feeding to develop mouse HCC has been previously described [30].

For xenograft experiments, cells were subcutaneously injected into the left upper back of BALB/c nude mice (20 mice were randomly allocated to two groups). After 3–4 weeks, the mice were sacrificed and the tumor volume and weight were evaluated. The tumor volume was calculated using the following formula: V=L×W2/2. The mice were housed at 3–5 animals per cage, with a 12 h light/dark cycle, in a temperature-controlled environment. All animal experiments were approved by the Inha University Institutional Animal Care and Utilization Committee (IACUC).

Cell culture, constructs, transfection, and lentivirus infection

The 293T, HL-7702, HepG2, Sk-Hep1, Hep3B, or Hepa1c1c7 cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Plasmids encoding myc-tagged K-Ras or H-Ras were purchased from Sino Biological and transfected using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) to the manufacturer’s protocol. Lentiviruses encoding empty, HA-tagged Kctd17, Kctd17 singleguide RNAs (sgRNAs), or doxycycline-inducible shKCTD17 [31] were generated by co-transfection of LentiX-293T cells (Takara, Kusatsu, Japan) with psPAX2 and pMD2.G. Viral supernatants containing 8 mL/mL of polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) were applied to Hepa1c1c7 or Hep3B cells, which were then selected with puromycin (Thermo Fisher Scientific) before the expansion of single clones.

Cell proliferation, migration, and wound-healing assays

The cell proliferation reagent WST-1 (Roche, Basel, Switzerland) accessed the proliferation of HCC cells. Briefly, cells were cultured in 24- or 96-well plates at 37°C and 5% CO2. The WST-1 solution was added to each well and incubated at 37°C for 4 h prior to analysis using microplate reader (BioTek, Winooski, VT, USA) at 450 nm. For migration assays, cells were seeded into the upper chamber of 24-transwell plates with 8 mm diameter filters for 12 h, and then cells on the bottom of the filter were fixed with 4% paraformaldehyde and stained with crystal violet. For wound healing assays, cells were seeded in 24-well plates and allowed to grow to 70–80% confluence as a monolayer, which was gently scratched across the center of the well. The medium was then removed, and the wells were washed twice with PBS. Images were obtained from the same fields immediately after scratching and after 24 h or 36 h under a microscope.

RNA isolation and quantitative PCR

RNA was isolated using TRIzol (Invitrogen, Waltham, MA, USA) or NucleoSpin RNA (Clontech, Mountain View, CA, USA), and cDNA was synthesized using the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), followed by quantitative RTPCR with TB Green Fast qPCR mix (Takara Bio Inc., Otsu, Japan) or 2X Universal SYBR Green Fast qPCR mix (ABclonal, Woburn, MA, USA) in a CFX Opus 96 Real-Time PCR detection system (Bio-Rad, Hercules, CA, USA).

Antibodies, Western blots and immunoprecipitation

Cells and liver tissues were lysed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 10 mM sodium pyrophosphate, 10 mM b-glycerophosphate, 50 mM sodium fluoride, and EDTA free protease inhibitor cocktail [Roche]). For immunoprecipitation, cell lysates were incubated with primary antibody and protein A/G beads (Santa Cruz, SC-2003), and FLAG M2 or HA affinity gels (Sigma, F2426 or E6779). Protein samples were separated using SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were probed with antibodies against phospho-ERK1/2 (#4370), ERK1/2 (#9102), phospho-MEK1/2 (#9121), MEK1/2 (#4694), Myc-tag (#2276), HA-tag (#3724), and DYKDDDDK-tag (#14793) from Cell Signaling; Ras (05-516) from Merck; and b-actin (A5441) or a-tubulin (T5168) from Sigma.

Ubiquitination assays

Cells were collected, and lysates were prepared in 100 mL of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2% SDS, and then boiled at 100°C for 10 min. The lysates were diluted with 900 mL of Tris-buffered saline containing 1% NP-40. Immunoprecipitation was performed using 1 mg of cellular lysate and an anti-FLAG M2 affinity gel and then analyzed using Western blotting with the indicated antibodies.

ASO studies

Nonspecific control ASO and Kctd17 ASO-2 (5’-AAG GTA ATG ATT GTA G-3’) were synthesized by Ionis Pharmaceuticals [18,32], diluted in saline before injection, and administered by intraperitoneal injection to male C57/BL/6J mice at a dosage of 25 mg per kg body weight once weekly for 6 weeks. One week after the 1st injection of ASO, 10 mg of the plasmids encoding myr-Akt and N-Ras along with sleeping beauty transposase in a ratio of 25:1 were further administered via hydrodynamic tail vein injection for 6 weeks before the mice were euthanized [28]. C57/BL/6J mice treated with DEN and CDAHFD feeding were injected with control or Kctd17 ASO once weekly for the indicated times prior to sacrifice.

Immunohistochemistry

Following sacrifice and rapid excision of mouse livers, tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 7 mm thickness slides, which were then deparaffinized, rehydrated and stained for H&E or Ki-67 (MA5-14520 from Invitrogen).

Statistical analysis

All data are shown as mean±standard error of the mean. Differences between the two groups were calculated using a two-tailed Student’s t test. Analyses involving multiple groups were performed using one-way ANOVA or the Kruskal–Wallis’s test. P<0.05 was considered statistically significant.

RESULTS

KCTD17 is upregulated in patients with HCC and associated with poor prognosis

We observed higher expression of KCTD17 in both human and mouse HCC and adenocarcinoma cell lines (Huh7, HepG2, Sk-Hep1, Hep3B, and Hepa1c1c7) than that in normal cells (HL7702 and AML12) (Supplementary Fig. 1A, B). We assessed patients with HCC databases, which revealed higher KCTD17 levels in HCC tissues than that in adjacent non-tumor tissues (Fig. 1A). Next, we investigated the association with clinicopathological features in patients with HCC classified and analyzed according to tumornode-metastasis (TNM) stage; in this cohort, patients with advanced stage (III/IV) disease showed higher KCTD17 as compared to those with early-stage disease (Fig. 1B). These data suggested that KCTD17 expression may predict survival. To test this possibility, we conducted a Kaplan-Meier estimation analysis. Patients were stratified into high- and low-KCTD17 expression groups using optimal expression levels determined by multiple receiver operating characteristic curve analysis. Patients with HCC and high KCTD17 expression showed a poorer prognosis (Fig. 1C). Consistently, across the TNM stages, higher expression of KCTD17 was consistently associated with decreased survival (Fig. 1D). These findings underscore the potential of Kctd17 as a prognostic marker of advanced HCC and poor survival.

Figure 1.

Increased KCTD17 is associated with poor prognosis in hepatocellular carcinoma (HCC). (A) Box and whiskers plots showing expression levels of KCTD17 in non-tumor and tumor from HCC in two independent cohorts (GSE25097, non-tumor=249, tumor=268; GSE36376, non-tumor=193, tumor=240). (B) Box plots showing expression levels of KCTD17 in each stage (stage I=171, II=86, and III+IV=88) in the liver hepatocellular carcinoma (LIHC) from the Cancer Genome Atlas (TCGA) database. Significance is determined using the Wilcoxon rank sum test. (C) Survival probabilities (Kaplan–Meier curves) (left) or cumulative hazard curve (right) based on hepatic KCTD17 expression. The R package, multipleROC, is divided into two groups (i.e., high vs. low KCTD17 expression) according to the optimal gene expression level of KCTD17. (D) Kaplan–Meier curves comparing hepatic KCTD17 expression and overall survival (top) or cumulative hazard curve (bottom) in different stages of HCC. ***P<0.001, ****P<0.0001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±standard error of the mean.

KCTD17 promotes proliferation, migration, and drug resistance in HCC

To investigate the implications of increased KCTD17 expression in HCC, we generated Kctd17 knockout (KO) or doxycycline (Dox)-inducible KCTD17 knockdown (KD) cells using sgRNA or shRNA against Kctd17 in Hepa1c1c7 or Hep3B cell lines (Supplementary Fig. 2A, B), which had moderate or high expression of KCTD17. In both experiments, cells lacking KCTD17 showed lower proliferation than that in the control cells (Fig. 2A, B). To test if observed growth inhibition may affect metastatic potential of cancer cells, we evaluated the effect of KCTD17 on tumor migration using trans-well and wound healing assays. Indeed, KCTD17 depletion impeded the migration of HCC cells (Fig. 2CE). Consistent with the altered cellular behavior, spheroid formation assays revealed significantly smaller spheroids in cells lacking KCTD17 than those in control cells (Fig. 2F).

Figure 2.

KCTD17 deficiency attenuates hepatocellular carcinoma (HCC) cell growth and migration. (A, B) Cell proliferation of control or Kctd17 KO Hepa1c1c7 cells (A), or doxycycline (Dox)-inducible KCTD17 knockdown Hep3B cells (B). (C, D) Representative pictures and quantitation of transwell migration assay in Kctd17 KO Hepa1c1c7 cells (C), or (D) KCTD17 knockdown Hep3B cells. (E) Representative pictures and quantitation of wound width in KCTD17 knockdown Hep3B cells. (F) Representative pictures and quantitation of three-dimensional spheroid cluster assay in KCTD17 knockdown Hep3B cells. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

We next established a gain-of-function model by overexpressing Kctd17 in Hepa1c1c7 cells (Supplementary Fig. 2C). As anticipated, Kctd17 overexpression showed the opposite results in loss-of-function experiments—increased cellular proliferation, migration, wound healing, and spheroid formation (Fig. 3AD)—associated with increased expression of oncogenic genes such as Myc and B-Cell-specific Moloney murine leukemia virus integration site 1 (Bmi1) (Fig. 3E). These findings suggested that Kctd17 affects key HCC properties. As resistance to chemotherapy poses a major obstacle in patients with advanced HCC, given the tumorigenic effects seen above, we next investigated whether Kctd17 influences the response to two commonly used chemotherapeutic approaches in HCC, TKI (sorafenib) [33] and anti-metabolite (5-FU) [34] drug treatment. In fact, forced Kctd17 inhibited the sensitivity of HCC cells to both drugs in a dose-dependent manner (Fig. 3F and Supplementary Fig. 2D), including the effects of these agents on MAPK and Akt pathways and spheroid formation (Fig. 3G, H). Collectively, these data demonstrate that increased Kctd17 expression is likely associated with chemoresistance in HCC.

Figure 3.

Kctd17 promotes hepatocellular carcinoma (HCC) proliferation, migration and drug resistance. (A–D) Cell proliferation (A), cell migration (B), would healing ability (C), three-dimensional spheroid cluster assay (D) and gene expression of Myc and Bmi1 (E) in control or Kctd17 overexpressing Hepa1c1c7 cells. (F–H) Control or Kctd17-overexpressing cells were treated with different dosages of sorafenib for 48 h, followed by cell proliferation assay (F), Western blot (G) and three-dimensional spheroid cluster assay (H). *P<0.05, **P<0.01 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

KCTD17 drives Ras signaling

To understand the mechanism of KCTD17-induced HCC, we performed functional annotation of differential expression using human datasets, which revealed significant enrichment of Ras-related signaling features (Fig. 4AD). We observed enrichment in KRAS and ERK1/2 (extracellular signal-regulated kinase 1/2) signaling, which are features indicative of malignant characteristics, including high proliferative activity. Similar results were observed in the GSE202853 dataset (Supplementary Fig. 3AD), further supporting the association between KCTD17 expression and activation of the Ras-related signaling pathway in HCC. We confirmed activation of Ras/MAPK (mitogen-activated protein kinase) signaling by Kctd17 upon serum stimulation (Fig. 4E). Conversely, KCTD17 depletion attenuated this activation (Fig. 4F), suggesting that KCTD17 enhances growth factor-induced Ras activation.

Figure 4.

KCTD17 drives Ras signaling. (A) Violin plot based on the expression of KCTD17 (red: 37 top expressed samples, blue: 37 bottom expressed samples). (B, C) Volcano (B) and bubble plots (C) of normalized enrichment score (NES) distribution with RAS signalingrelated gene sets marked. (D) RNA-seq datasets of low-KCTD17 enriched and high-KCTD17 enriched as compared with KRAS and ERK signature in LIHC from TCGA. (E, F) Hepa1c1c7 cells expressing control or HA/Kctd17 (E) or Hep3B cells expressing doxycycline-inducible shKCTD17 (F) were cultured in serum-free medium and then treated with serum for the indicated time, followed by evaluation of Ras/MAPK signaling using Western blot.

Kctd17 mediates Ras stabilization by Lztr1 degradation

Interestingly, we observed increased Ras protein levels with Kctd17 overexpression (Fig. 5A), without a change in Ras mRNA expression (Supplementary Fig. 4A). Kctd17 overexpression also attenuated the degradation rates of endogenous and exogenous Ras, as demonstrated by measurement in the presence of the de novo protein synthesis inhibitor cycloheximide (CHX) (Fig. 5B and Supplementary Fig. 4B). These results suggest that Kctd17 promotes MAPK signaling through Ras stabilization.

Figure 5.

Kctd17 prompts Lztr1 degradation to mediate Ras stabilization. (A) Western blots from 293T cells transfected with H/K-Ras with or without Kctd17. (B) Hepa1c1c7 cells transfected with control or Kctd17 were treated with 50 mg/mL cycloheximide (CHX) and then harvested at the indicated time points after treatment prior to Western blotting and quantitation of endogenous Ras. (C, D) Co-immunoprecipitation of Ras and Lztr1 or Kctd17 from Hepa1c1c7 cells (C) or Western blots in lysate from 293T cells (D). (E) Western blots from Hepa1c1c7 cells transfected with Kctd17 and Lztr1 with or without MG-132. (F) Co-immunoprecipitation of Lztr1 by Kctd17 from Hepa1c1c7 cells. (G) Polyubiquitination of Lztr1 by Kctd17 Myc/Ub-transfected 293T cells with MG-132. (H) Ras ubiquitination in Flag/Ub-transfected 293T cells with or without Kctd17 and Lztr1. (I) Cell proliferation in control or Kctd17 overexpressing cells with or without Lztr1/Flag from Hepa1c1c7 cells. **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

We next sought to understand the mechanism by which Kctd17 regulates Ras stability. Previous studies have reported that leucine zipper-like transcription regulator 1 (Lztr1) promotes polyubiquitination and degradation of Ras family members by recruiting a Cul3 ubiquitin ligase complex, thereby inhibiting Ras/MAPK signaling [22-24]. Germ-line mutations in Lztr1 are associated with Noonan syndrome, a RASopathy [35]. Furthermore, Lztr1 is mutated in HCC [36] and its expression is significantly lower in normal cells than those in HCC cells [37]. Given these data, we hypothesized that Kctd17 affects Ras stability by interfering with the Lztr1-Ras interaction. Intriguingly, we observed that Kctd17 reduced Lztr1 protein levels which disrupted the interaction between Lztr1 and Ras (Fig. 5C), without altering Lztr1 mRNA expression (Supplementary Fig. 4C). Similarly, the Lztr1-mediated loss of Ras was prevented by Kctd17 overexpression (Fig. 5D), whereas the Kctd17-mediated reduction of Lztr1 was rescued by concomitant MG-132 treatment to prevent proteasomal degradation (Fig. 5E). Consistent with these data, we observed that Kctd17 interacted with Lztr1 (Fig. 5F) and increased its ubiquitination (Fig. 5G). As such, Lztr1-mediated Ras polyubiquitination was inhibited by Kctd17 under denaturing conditions (Fig. 5H). Finally, Kctd17 reversed the anti-proliferative effects of Lztr1 on hepatoma cells (Fig. 5I). Taken together, our results indicate that Kctd17 induces Lztr1 proteasomal degradation, which provokes Ras stabilization and cellular proliferation.

Kctd17 is required for HCC progression in vivo

To test repercussion of altered Kctd17 expression in HCC, we first conducted in vivo tumorigenicity assays using a xenograft mouse model. Both Kctd17 KO (Fig. 6AD) or doxycycline-inducible KCTD17 knockdown (Fig. 6EH) resulted in a significant decrease in tumor growth, leading to lower tumor size and weight, without significant changes in body weight (Supplementary Fig. 5A, B).

Figure 6.

Kctd17 is required for hepatocellular carcinoma (HCC) progression in vivo. (A) Experimental schematic for tumor xenograft assay using control or Kctd17 KO Hepa1c1c7 cells. (B–D) Representative pictures (B), tumor size (C) and weight (D) of control or Kctd17 KO xenografts (n=10 per group). (E) Experimental schematic for tumor xenograft assay using doxycycline-inducible KCTD17 knockdown Hep3B cells. (F–H) Representative pictures (F), tumor size (G) and weight (H) of control or doxycycline-inducible shKCTD17 xenografts (n=10 per group). (I) Experimental schematic for hepatocyte-specific Kctd17 KO mice (L-Kctd17). (J–N) Kctd17 expression in tumors and adjacent non-tumors (J), representative images of whole livers (K) and tumor volume (L), Representative images of livers stained with H&E or Ki-67 (M), Western blots from liver tumor lysates (N) of control or L-Kctd17 mice (n=7 to 9 per group), as indicated. Scale bar, 100 or 200 mm. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

We assessed Kctd17 expression in several mouse HCC models, including carcinogen-induced (CCl4 or DEN), dietinduced (CDAHFD feeding or fructose/palmitate/cholesterol (FPC)-enriched NASH diet-feeding) [27,30], and oncogene induction (myr-Akt/N-Ras) [38,39]. All showed higher Kctd17 mRNA expression in tumors than in non-tumors (Supplementary Fig. 6AD). To ascertain the role of Kctd17 in HCC pathogenesis, we treated hepatocyte-specific Kctd17 (L-Kctd17) knockout mice [18] with DEN, and then fed a CDAHFD to accelerate HCC [27] (Fig. 6I, J). L-Kctd17 mice showed reduced tumor numbers, Ki-67-positive cells, lower Ras, and increased Lztr1 protein levels, compared to those in control mice, without significant changes in body weight (Fig. 6KN, Supplementary Fig. 6E). These data demonstrate that Kctd17 functions as a tumor promoter in HCC.

Kctd17 inhibition ameliorates HCC progression

Next, to assess the potential therapeutic applications of these data, we tested the effects of liver-directed Kctd17 ASO [18] in mouse HCC. We leveraged the rapid onset of HCC development and the large number of tumors in the oncogene models (Fig. 7A). Treatment with Kctd17 ASO reduced Kctd17 expression in both tumor and adjacent non-tumor liver tissues, decreased tumor size, lowered Ras levels, and increased Lztr1 protein levels (Fig. 7BE).

Figure 7.

Kctd17-specific ASO decreases hepatocellular carcinoma (HCC) progression. (A) Experimental schematic for oncogene-induced HCC models. After hydrodynamic injection of myr-Akt and N-Ras in WT male mice, control or Kctd17-directed ASO was administered weekly by intraperitoneal injection. (B–E) Kctd17 expression in tumors and adjacent non-tumors (B), representative images of whole liver (C), tumor size (D) and Western blots from liver tumor lysate (E) in control or Kctd17 ASO-treated mice (n=5 or 7 per group). (F) Experimental schematic for DEN/CDAHFD-induced HCC models. Mice were injected with DEN at 2 weeks of life, then started on CDAHFD diet-feeding with concurrent weekly intraperitoneal injections of control or Kctd17 ASO. (G–K) Kctd17 expression in tumors and adjacent non-tumors (G), representative images of whole mouse liver (H), tumor number (I), liver H&E and Ki-67 staining (J), and Western blots in lysate from liver tumor lysate (K), in control or Kctd17 ASO-treated mice (n=6 per group). Scale bar, 100 or 200 mm. (L) Model of Kctd17-mediated HCC progression. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

We confirmed these data in DEN/CDAHFD-induced mouse HCC (Fig. 7F). Similar to above, Kctd17 ASO reduced Kctd17 mRNA expression, tumor number, Ki-67-positive cells, and Ras protein levels, whereas it increased Lztr1 protein levels, without significant changes in body weight (Fig. 7GK, Supplementary Fig. 6F). These data suggest that the liver-selective inhibition of Kctd17 can attenuate HCC progression in vivo, indicating its potential as a therapeutic target for the treatment of liver tumors.

DISCUSSION

Liver cancer is a significant global health concern, ranking as the sixth most commonly diagnosed cancer and the third leading cause of cancer-related deaths [40]. Despite significant progress, challenges persist in the treatment of HCC, and the identification of novel therapeutic targets is crucial for improving patient outcomes. Although KCTD17 expression has been associated with poor prognosis of HCC in co-expression network analysis [41], the underlying mechanisms have not been elucidated.

In this study, we observed that KCTD17 is highly expressed in HCC and is correlated with tumor grade, predicting poor patient survival. Previous studies have shown that KCTD17 expression is increased in the livers of obese mice and humans, as well as in obese adipose tissues [17,18,31]. Interestingly, KCTD17 expression was higher in tumors than in non-tumors, independent of obesity, suggesting that its expression might be regulated by other factors involved in cancer initiation and/or progression. Importantly, genetic or pharmacological ablation of KCTD17 in human cell lines and mouse models led to reduced tumor cell proliferation in vitro and in vivo, suggesting that KCTD17 could represent a novel treatment target for HCC. Nevertheless, despite consistent results in mouse models showing a reduction in tumor growth, there are inherent limitations associated with relying solely on results derived from cell lines and rodent models. The intricate heterogeneity observed in human HCC necessitates further studies to validate the efficacy of KCTD17 inhibitors and explore their therapeutic applications in clinical settings.

These results may also have further application beyond HCC. Ras proteins, small GTPases that play a crucial role in linking extracellular signaling pathways to intracellular effector signals, are essential for fundamental cellular processes such as proliferation, survival, differentiation, motility, and transcription [42,43]. The activity of Ras proteins is finely regulated by the interplay between GDP and GTP binding states, as well as their membrane localization [44]. While oncogenic mutations in Ras, including constitutive GTP binding forms, are frequently implicated in human cancer [45,46], elevated Ras levels are also commonly observed in HCC patients [46-50]. This observation prompts the consideration that manipulation of the regulation of Ras stability could serve as an additional therapeutic strategy for the treatment of HCC. Our study unveils Lztr1, a Golgi protein [51,52] known as a tumor suppressor by means of regulation of degradation of various Ras proteins [22], as a novel substrate of the KCTD17-Cul3 ubiquitin ligase complex, ultimately amplifying Ras/MAPK signaling (Fig. 7L). Interestingly, while KCTD17 overexpression significantly upregulated MEK1/2 activation, KCTD17 knockdown had only a marginal effect on ERK and MEK regulation (Fig. 4E, F), suggesting the possibility that KCTD17 might also influence Ras signaling through alternative pathways. This intricate regulatory axis holds profound implications in the context of HCC, but likely also in other cancers dependent on Ras signaling.

In conclusion, our study uncovered a novel mechanism for the regulation of Ras stability orchestrated by KCTD17 and positioned the KCTD17-Lztr1-Ras axis as a promising strategic approach for overcoming the multifaceted challenges associated with HCC. These findings provide a solid foundation for further investigations of KCTD17 modulation during HCC treatment.

Notes

Authors’ contribution

Y.H.J. and K.K designed the whole project. Y.H.J., Y.J.L., T.D., J.Y., A.R.O., Y.J., H.G., Y.U.K., D.R., M.C., S.B.L. and K.K. performed the experiments. Y.H.J., Y.J.L., T.D., K.H.J., D.R., U.B.P., S.B.L., S.S.H. and K.K. performed data analysis and interpretation. Y.H.J., U.B.P. and K.K. drafted the main manuscript. A.R.O., D.R., U.B.P., D.R., S.B.L., S.S.H. and K.K. obtained the funding.

Conflicts of Interest

The authors have no conflicts to disclose.

Acknowledgements

This work was supported by an INHA UNIVERSITY Research Grant (K.K), National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) [No. RS-2023-00208008 to K.K, 2021R1A5A2031612 to S.S.H and K.K, 2020R1C1C1014281 and 2021R1A5A80 29876 to S.B.L, 2023R1A2C3006220 to D.R, and 2022R1I1A1A01069078 to A.R.O] and NIH DK103818 and DK119767 (U.B.P). We thank the members of the Kim laboratories and Dr. Kook Hwan Kim for the insightful discussion.

SUPPLEMENTAL MATERIAL

Supplementary material is available at Clinical and Molecular Hepatology website (http://www.e-cmh.org).

Supplementary Figure 1.

KCTD17 is upregulated in liver cancer cell lines. (A, B) Western blot of KCTD17 in lysates from normal liver cell lines and multiple liver cancer cell lines.

cmh-2024-0364-Supplementary-Fig-1.pdf
Supplementary Figure 2.

KCTD17 promotes HCC proliferation, migration and drug resistance. (A, B) Kctd17 expression in control or Kctd17 knockout Hep1c1c7 cells (A), or doxycycline (Dox)-inducible KCTD17 knockdown Hep3B cells (B). (C) Western blot in lysates from control or Kctd17 overexpressing Hepa1c1c7 cells. (D) The effect of chemoresistance by Kctd17 overexpression. Control or Kctd17-over-expressing cells were treated with different dosages of 5-FU for 48 h, followed by cell proliferation assay. **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the means±s.e.m.

cmh-2024-0364-Supplementary-Fig-2.pdf
Supplementary Figure 3.

KCTD17 drives Ras signaling. (A) Violin plot based on expression of KCTD17 (red: 4 top expressed samples, blue: 4 bottom expressed samples). (B, C) Volcano plot (B) and bubble plots (C) of normalized enrichment score (NES) distribution with RAS signaling-related gene sets marked. (D) RNA-seq datasets of low-KCTD17 enriched and high-KCTD17 enriched as compared with MAPK and ERK1/2 cascade signature from GSE202853 data.

cmh-2024-0364-Supplementary-Fig-3.pdf
Supplementary Figure 4.

Kctd17 prompts Lztr1 degradation to mediate Ras stabilization. (A) Kctd17, H-Ras or K-Ras expression from 293T cells transfected with H/K-Ras with or without Kctd17. (B) 293T cells transfected with H-Ras with or without Kctd17 were treated with 50 μg/mL cycloheximide (CHX) and then harvested at the indicated time points after treatment prior to Western blot. (C) Lztr1 mRNA expression from Hepa1c1c7 cells transfected with or without Kctd17. ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the means±s.e.m.

cmh-2024-0364-Supplementary-Fig-4.pdf
Supplementary Figure 5.

Kctd17 is required for HCC progression in vivo. (A, B) Body weight curves of control or Kctd17 KO xenograft (A) or dox-inducible shKCTD17 xenografts (B).

cmh-2024-0364-Supplementary-Fig-5.pdf
Supplementary Figure 6.

Kctd17 is required for HCC progression in vivo. (A–D) mRNA expression levels of Kctd17 in non-tumor and tumor of primary liver cancer induced by DEN with CDAHFD (A), NASH diet (B), CCl4 with NASH diet (C), hydrodynamic tail vein injection of myr-Akt/N-Ras (D). (E, F) Body weight curves of control or L-Kctd17 mice (E) and control ASO or Kctd17 ASO (F) injected with DEN with CDAHFD feeding. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the means±s.e.m.

cmh-2024-0364-Supplementary-Fig-6.pdf

Abbreviations

AAV8

adeno-associated virus subtype 8

CDAHFD

choline-deficient

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

FDR

false discovery rate

HCC

hepatocellular carcinoma

LIHC

liver hepatocellular carcinoma

MASLD

metabolic dysfunction-associated steatotic liver disease

NES

normalized enrichment score

PVDF

polyvinylidene fluoride

TCGA

The Cancer Genome Atlas

TKI

tyrosine kinase inhibitor

TNM

tumor-node-metastasis

References

1. Niu ZS, Niu XJ, Wang WH. Genetic alterations in hepatocellular carcinoma: an update. World J Gastroenterol 2016;22:9069–9095.
2. Zhou J, Sun HC, Wang Z, Cong WM, Wang JH, Zeng MS, et al. Guidelines for diagnosis and treatment of primary liver cancer in China (2017 edition). Liver Cancer 2018;7:235–260.
3. Lepage C, Capocaccia R, Hackl M, Lemmens V, Molina E, Pierannunzio D, et al, ; EUROCARE-5 Working Group. Survival in patients with primary liver cancer, gallbladder and extrahepatic biliary tract cancer and pancreatic cancer in Europe 1999-2007: results of EUROCARE-5. Eur J Cancer 2015;51:2169–2178.
4. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin 2018;68:7–30.
5. Zhu AX, Kudo M, Assenat E, Cattan S, Kang YK, Lim HY, et al. Effect of everolimus on survival in advanced hepatocellular carcinoma after failure of sorafenib: the EVOLVE-1 randomized clinical trial. JAMA 2014;312:57–67.
6. Llovet JM, Montal R, Sia D, Finn RS. Molecular therapies and precision medicine for hepatocellular carcinoma. Nat Rev Clin Oncol 2018;15:599–616.
7. Marisi G, Cucchetti A, Ulivi P, Canale M, Cabibbo G, Solaini L, et al. Ten years of sorafenib in hepatocellular carcinoma: are there any predictive and/or prognostic markers? World J Gastroenterol 2018;24:4152–4163.
8. Personeni N, Pressiani T, Rimassa L. Lenvatinib for the treatment of unresectable hepatocellular carcinoma: evidence to date. J Hepatocell Carcinoma 2019;6:31–39.
9. Vogel A, Cervantes A, Chau I, Daniele B, Llovet JM, Meyer T, et al. Correction to: “Hepatocellular carcinoma: ESMO Clinical Practice Guidelines for diagnosis, treatment and followup”. Ann Oncol 2019;30:871–873. Erratum for: Ann Oncol 2018;29(Suppl 4):iv238-iv255.
10. Abou-Alfa GK, Meyer T, Cheng AL, El-Khoueiry AB, Rimassa L, Ryoo BY, et al. Cabozantinib in patients with advanced and progressing hepatocellular carcinoma. N Engl J Med 2018;379:54–63.
11. Bruix J, Qin S, Merle P, Granito A, Huang YH, Bodoky G, et al, ; RESORCE Investigators. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017;389:56–66. Erratum in: Lancet 2017;389:36.
12. De Luca E, Marino D, Di Maio M. Ramucirumab, a second-line option for patients with hepatocellular carcinoma: a review of the evidence. Cancer Manag Res 2020;12:3721–3729.
13. Teng X, Aouacheria A, Lionnard L, Metz KA, Soane L, Kamiya A, et al. KCTD: a new gene family involved in neurodevelopmental and neuropsychiatric disorders. CNS Neurosci Ther 2019;25:887–902.
14. Ji AX, Chu A, Nielsen TK, Benlekbir S, Rubinstein JL, Privé GG. Structural insights into KCTD protein assembly and Cullin3 recognition. J Mol Biol 2016;428:92–107.
15. Pinkas DM, Sanvitale CE, Bufton JC, Sorrell FJ, Solcan N, Chalk R, et al. Structural complexity in the KCTD family of Cullin3-dependent E3 ubiquitin ligases. Biochem J 2017;474:3747–3761.
16. Skoblov M, Marakhonov A, Marakasova E, Guskova A, Chandhoke V, Birerdinc A, et al. Protein partners of KCTD proteins provide insights about their functional roles in cell differentiation and vertebrate development. Bioessays 2013;35:586–596.
17. Kim K, Ryu D, Dongiovanni P, Ozcan L, Nayak S, Ueberheide B, et al. Degradation of PHLPP2 by KCTD17, via a glucagondependent pathway, promotes hepatic steatosis. Gastroenterology 2017;153:1568–1580.e10.
18. Oh AR, Jeong Y, Yu J, Minh Tam DT, Kang JK, Jung YH, et al. Hepatocyte Kctd17 inhibition ameliorates glucose intolerance and hepatic steatosis caused by obesity-induced Chrebp stabilization. Gastroenterology 2023;164:439–453.
19. Sanyal A, Poklepovic A, Moyneur E, Barghout V. Populationbased risk factors and resource utilization for HCC: US perspective. Curr Med Res Opin 2010;26:2183–2191.
20. Baffy G, Brunt EM, Caldwell SH. Hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace. J Hepatol 2012;56:1384–1391.
21. Wong RJ, Cheung R, Ahmed A. Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S. Hepatology 2014;59:2188–2195.
22. Abe T, Umeki I, Kanno SI, Inoue SI, Niihori T, Aoki Y. LZTR1 facilitates polyubiquitination and degradation of RAS-GTPases. Cell Death Differ 2020;27:1023–1035.
23. Bigenzahn JW, Collu GM, Kartnig F, Pieraks M, Vladimer GI, Heinz LX, et al. LZTR1 is a regulator of RAS ubiquitination and signaling. Science 2018;362:1171–1177.
24. Steklov M, Pandolfi S, Baietti MF, Batiuk A, Carai P, Najm P, et al. Mutations in LZTR1 drive human disease by dysregulating RAS ubiquitination. Science 2018;362:1177–1182.
25. Yu G, Wang LG, Han Y, He QY. clusterProfiler: an R package for comparing biological themes among gene clusters. OMICS 2012;16:284–287.
26. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell 2014;159:440–455.
27. Li S, Ghoshal S, Sojoodi M, Arora G, Masia R, Erstad DJ, et al. Pioglitazone reduces hepatocellular carcinoma development in two rodent models of cirrhosis. J Gastrointest Surg 2019;23:101–111.
28. Ho C, Wang C, Mattu S, Destefanis G, Ladu S, Delogu S, et al. AKT (v-akt murine thymoma viral oncogene homolog 1) and N-Ras (neuroblastoma ras viral oncogene homolog) coactivation in the mouse liver promotes rapid carcinogenesis by way of mTOR (mammalian target of rapamycin complex 1), FOXM1 (forkhead box M1)/SKP2, and c-Myc pathways. Hepatology 2012;55:833–845.
29. Stauffer JK, Scarzello AJ, Andersen JB, De Kluyver RL, Back TC, Weiss JM, et al. Coactivation of AKT and β-catenin in mice rapidly induces formation of lipogenic liver tumors. Cancer Res 2011;71:2718–2727.
30. Zhu C, Ho YJ, Salomao MA, Dapito DH, Bartolome A, Schwabe RF, et al. Notch activity characterizes a common hepatocellular carcinoma subtype with unique molecular and clinicopathologic features. J Hepatol 2021;74:613–626.
31. Shin MC, Jung YH, Jeong Y, Oh AR, Lee SB, Kim K. Kctd17-mediated Chop degradation promotes adipogenic differentiation. Biochem Biophys Res Commun 2023;653:126–132.
32. Vasquez G, Freestone GC, Wan WB, Low A, De Hoyos CL, Yu J, et al. Site-specific incorporation of 5’-methyl DNA enhances the therapeutic profile of gapmer ASOs. Nucleic Acids Res 2021;49:1828–1839.
33. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al, ; SHARP Investigators Study Group. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378–390.
34. Kalyan A, Nimeiri H, Kulik L. Systemic therapy of hepatocellular carcinoma: current and promising. Clin Liver Dis 2015;19:421–432.
35. Zenker M, Edouard T, Blair JC, Cappa M. Noonan syndrome: improving recognition and diagnosis. Arch Dis Child 2022;107:1073–1078.
36. The Cancer Genome Atlas Research Network, Wheeler DA, Roberts LR. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell 2017;169:1327–1341.e23.
37. Yi T, Luo H, Qin F, Jiang Q, He S, Wang T, et al. LncRNA LL22NC03-N14H11.1 promoted hepatocellular carcinoma progression through activating MAPK pathway to induce mitochondrial fission. Cell Death Dis 2020;11:832.
38. Liu YT, Tseng TC, Soong RS, Peng CY, Cheng YH, Huang SF, et al. A novel spontaneous hepatocellular carcinoma mouse model for studying T-cell exhaustion in the tumor microenvironment. J Immunother Cancer 2018;6:144.
39. Chen X, Calvisi DF. Hydrodynamic transfection for generation of novel mouse models for liver cancer research. Am J Pathol 2014;184:912–923.
40. Rumgay H, Arnold M, Ferlay J, Lesi O, Cabasag CJ, Vignat J, et al. Global burden of primary liver cancer in 2020 and predictions to 2040. J Hepatol 2022;77:1598–1606.
41. Fang Y, Yang Y, Zhang X, Li N, Yuan B, Jin L, et al. A co-expression network reveals the potential regulatory mechanism of lncRNAs in relapsed hepatocellular carcinoma. Front Oncol 2021;11:745166.
42. Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol 2008;9:517–531.
43. Pylayeva-Gupta Y, Grabocka E, Bar-Sagi D. RAS oncogenes: weaving a tumorigenic web. Nat Rev Cancer 2011;11:761–774.
44. Mor A, Philips MR. Compartmentalized Ras/MAPK signaling. Annu Rev Immunol 2006;24:771–800.
45. Cha PH, Cho YH, Lee SK, Lee J, Jeong WJ, Moon BS, et al. Small-molecule binding of the axin RGS domain promotes β-catenin and Ras degradation. Nat Chem Biol 2016;12:593–600.
46. Moon BS, Jeong WJ, Park J, Kim TI, Min do S, Choi KY. Role of oncogenic K-Ras in cancer stem cell activation by aberrant Wnt/β-catenin signaling. J Natl Cancer Inst 2014;106:djt373.
47. Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, et al. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006;130:1117–1128.
48. Chen L, Shi Y, Jiang CY, Wei LX, Wang YL, Dai GH. Expression and prognostic role of pan-Ras, Raf-1, pMEK1 and pERK1/2 in patients with hepatocellular carcinoma. Eur J Surg Oncol 2011;37:513–520.
49. Jura N, Scotto-Lavino E, Sobczyk A, Bar-Sagi D. Differential modification of Ras proteins by ubiquitination. Mol Cell 2006;21:679–687.
50. Sasaki AT, Carracedo A, Locasale JW, Anastasiou D, Takeuchi K, Kahoud ER, et al. Ubiquitination of K-Ras enhances activation and facilitates binding to select downstream effectors. Sci Signal 2011;4:ra13.
51. Nacak TG, Leptien K, Fellner D, Augustin HG, Kroll J. The BTB-kelch protein LZTR-1 is a novel Golgi protein that is degraded upon induction of apoptosis. J Biol Chem 2006;281:5065–5071.
52. Piotrowski A, Xie J, Liu YF, Poplawski AB, Gomes AR, Madanecki P, et al. Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas. Nat Genet 2014;46:182–187.

Article information Continued

Notes

Study Highlights

• Transcriptomic data from HCC patients showed a significant increase in KCTD17 expression, correlating with disease severity and progression.

• KCTD17 promotes cellular proliferation and migration via polyubiquitination and degradation of Lztr1, stabilization of the Ras oncogene, and activation of Ras/MAPK signaling.

• Hepatocyte-specific Kctd17 knockout mice or liver cancer xenograft models exhibits lower susceptibility of liver carcinogenesis and tumor growth.

• Treatment with Kctd17-directed ASO in mouse models of HCC markedly reduced tumor growth.

Figure 1.

Increased KCTD17 is associated with poor prognosis in hepatocellular carcinoma (HCC). (A) Box and whiskers plots showing expression levels of KCTD17 in non-tumor and tumor from HCC in two independent cohorts (GSE25097, non-tumor=249, tumor=268; GSE36376, non-tumor=193, tumor=240). (B) Box plots showing expression levels of KCTD17 in each stage (stage I=171, II=86, and III+IV=88) in the liver hepatocellular carcinoma (LIHC) from the Cancer Genome Atlas (TCGA) database. Significance is determined using the Wilcoxon rank sum test. (C) Survival probabilities (Kaplan–Meier curves) (left) or cumulative hazard curve (right) based on hepatic KCTD17 expression. The R package, multipleROC, is divided into two groups (i.e., high vs. low KCTD17 expression) according to the optimal gene expression level of KCTD17. (D) Kaplan–Meier curves comparing hepatic KCTD17 expression and overall survival (top) or cumulative hazard curve (bottom) in different stages of HCC. ***P<0.001, ****P<0.0001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±standard error of the mean.

Figure 2.

KCTD17 deficiency attenuates hepatocellular carcinoma (HCC) cell growth and migration. (A, B) Cell proliferation of control or Kctd17 KO Hepa1c1c7 cells (A), or doxycycline (Dox)-inducible KCTD17 knockdown Hep3B cells (B). (C, D) Representative pictures and quantitation of transwell migration assay in Kctd17 KO Hepa1c1c7 cells (C), or (D) KCTD17 knockdown Hep3B cells. (E) Representative pictures and quantitation of wound width in KCTD17 knockdown Hep3B cells. (F) Representative pictures and quantitation of three-dimensional spheroid cluster assay in KCTD17 knockdown Hep3B cells. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

Figure 3.

Kctd17 promotes hepatocellular carcinoma (HCC) proliferation, migration and drug resistance. (A–D) Cell proliferation (A), cell migration (B), would healing ability (C), three-dimensional spheroid cluster assay (D) and gene expression of Myc and Bmi1 (E) in control or Kctd17 overexpressing Hepa1c1c7 cells. (F–H) Control or Kctd17-overexpressing cells were treated with different dosages of sorafenib for 48 h, followed by cell proliferation assay (F), Western blot (G) and three-dimensional spheroid cluster assay (H). *P<0.05, **P<0.01 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

Figure 4.

KCTD17 drives Ras signaling. (A) Violin plot based on the expression of KCTD17 (red: 37 top expressed samples, blue: 37 bottom expressed samples). (B, C) Volcano (B) and bubble plots (C) of normalized enrichment score (NES) distribution with RAS signalingrelated gene sets marked. (D) RNA-seq datasets of low-KCTD17 enriched and high-KCTD17 enriched as compared with KRAS and ERK signature in LIHC from TCGA. (E, F) Hepa1c1c7 cells expressing control or HA/Kctd17 (E) or Hep3B cells expressing doxycycline-inducible shKCTD17 (F) were cultured in serum-free medium and then treated with serum for the indicated time, followed by evaluation of Ras/MAPK signaling using Western blot.

Figure 5.

Kctd17 prompts Lztr1 degradation to mediate Ras stabilization. (A) Western blots from 293T cells transfected with H/K-Ras with or without Kctd17. (B) Hepa1c1c7 cells transfected with control or Kctd17 were treated with 50 mg/mL cycloheximide (CHX) and then harvested at the indicated time points after treatment prior to Western blotting and quantitation of endogenous Ras. (C, D) Co-immunoprecipitation of Ras and Lztr1 or Kctd17 from Hepa1c1c7 cells (C) or Western blots in lysate from 293T cells (D). (E) Western blots from Hepa1c1c7 cells transfected with Kctd17 and Lztr1 with or without MG-132. (F) Co-immunoprecipitation of Lztr1 by Kctd17 from Hepa1c1c7 cells. (G) Polyubiquitination of Lztr1 by Kctd17 Myc/Ub-transfected 293T cells with MG-132. (H) Ras ubiquitination in Flag/Ub-transfected 293T cells with or without Kctd17 and Lztr1. (I) Cell proliferation in control or Kctd17 overexpressing cells with or without Lztr1/Flag from Hepa1c1c7 cells. **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

Figure 6.

Kctd17 is required for hepatocellular carcinoma (HCC) progression in vivo. (A) Experimental schematic for tumor xenograft assay using control or Kctd17 KO Hepa1c1c7 cells. (B–D) Representative pictures (B), tumor size (C) and weight (D) of control or Kctd17 KO xenografts (n=10 per group). (E) Experimental schematic for tumor xenograft assay using doxycycline-inducible KCTD17 knockdown Hep3B cells. (F–H) Representative pictures (F), tumor size (G) and weight (H) of control or doxycycline-inducible shKCTD17 xenografts (n=10 per group). (I) Experimental schematic for hepatocyte-specific Kctd17 KO mice (L-Kctd17). (J–N) Kctd17 expression in tumors and adjacent non-tumors (J), representative images of whole livers (K) and tumor volume (L), Representative images of livers stained with H&E or Ki-67 (M), Western blots from liver tumor lysates (N) of control or L-Kctd17 mice (n=7 to 9 per group), as indicated. Scale bar, 100 or 200 mm. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.

Figure 7.

Kctd17-specific ASO decreases hepatocellular carcinoma (HCC) progression. (A) Experimental schematic for oncogene-induced HCC models. After hydrodynamic injection of myr-Akt and N-Ras in WT male mice, control or Kctd17-directed ASO was administered weekly by intraperitoneal injection. (B–E) Kctd17 expression in tumors and adjacent non-tumors (B), representative images of whole liver (C), tumor size (D) and Western blots from liver tumor lysate (E) in control or Kctd17 ASO-treated mice (n=5 or 7 per group). (F) Experimental schematic for DEN/CDAHFD-induced HCC models. Mice were injected with DEN at 2 weeks of life, then started on CDAHFD diet-feeding with concurrent weekly intraperitoneal injections of control or Kctd17 ASO. (G–K) Kctd17 expression in tumors and adjacent non-tumors (G), representative images of whole mouse liver (H), tumor number (I), liver H&E and Ki-67 staining (J), and Western blots in lysate from liver tumor lysate (K), in control or Kctd17 ASO-treated mice (n=6 per group). Scale bar, 100 or 200 mm. (L) Model of Kctd17-mediated HCC progression. *P<0.05, **P<0.01, ***P<0.001 as compared to the indicated control by two-way ANOVA. All data are shown as the mean±s.e.m.