1. High expression of RBCK1 is correlated with poor outcomes in patients with HCC
TCGA (n = 424) cohort analyses revealed the upregulation of RBCK1 in HCC, and high expression of RBCK1 in HCC patients was positively correlated with stage and distant invasion (Fig. 1a, b and c). To further determine RBCK1 expression in HCC, qRT-PCR analyses were performed on HCC tissues and their corresponding adjacent tissues. Results showed that the average fold change of RBCK1 mRNA expression in HCC tissues compared with adjacent non-tumour-bearing tissues (Fig. 1d and e). Moreover, analysis of the results obtained from western blotting (Fig. 1f and g) and IHC staining (Fig. 1h) revealed that the RBCK1 protein was upregulated in HCC tissues, compared with adjacent non-tumour-bearing tissues. These results indicated that RBCK1 expression is significantly upregulated in HCC tissues.
Evaluations of the correlations between RBCK1 overexpression and HCC clinicopathologic parameters revealed no significant association between RBCK1 expression and tumour size, age or histological type, but a significant correlation with TNM (p < 0.002), distant metastasis (p = 0.013) and clinical stage (p = 0.006) (Table 1). Additionally, the 216 HCC patients were divided into two groups based on the results of the immunohistochemically analysis: high RBCK1 expression group (n = 146) and low RBCK1 expression group (n = 70). Kaplan-Meier survival analysis showed that HCC patients with high RBCK1 expression levels exhibited poor overall survival (OS) (p = 0.025), poor disease-free survival (DFS) (p = 0.0034), compared to patients with low RBCK1 levels (Fig. 1i and j). This prognosis effect was also observed in the TCGA (n = 425) cohorts (Fig. S1). It should be noted that the results of multivariate Cox regression analysis indicate that RBCK1 overexpression was one of the independent predictive factors for detecting poor outcome in HCC patients (Table 2). Collectively, these data suggest that RBCK1 was upregulated in HCC tissues and associated with an unfavourable prognosis in HCC patients.
Table 2
Univariate and multivariate analyses of overall survival in HCC patients
Parameters | Univariate analysis | | | Multivariate analysis | | |
| HR | 95%CI | P value | HR | 95%CI | P value |
Age (≥ 65 vs ༜65) | 1.437 | 0.731–2.826 | 0.736 | — | — | — |
Sex (Female vs Male) | 1.851 | 0.584–2.927 | 0.724 | — | — | — |
Tumor size (༜5 vs ≥ 6) | 1.637 | 1.153–5.176 | 0.226 | — | — | — |
Differentiation (Well vs Moderate/Poor) | 1.724 | 1.652–4.16 | 0.278 | — | — | — |
TNM stage (T1-T2 vs T3-T4) | 2.607 | 1.415–5.542 | 0.012* | 1.673 | 1.441–4.534 | 0.031* |
Distant metastasis (No vs Yes) | 1.381 | 0.716–3.765 | 0.031* | 1.721 | 1.419–2.917 | 0.089 |
Stage ( I-II vs III-IV) | 0.749 | 0.654–1.867 | 0.017* | 1.432 | 0.983–3.837 | 0.037* |
RBCK1 expression (High vs Low) | 3.847 | 2.546–5.629 | 0.001* | 2.231 | 1.736–4.841 | 0.019* |
- RBCK1 accelerates the metastasis of HCC cells in vitro and in vivo
To investigate the potential biological function of RBCK1 in HCC development, we first determined RBCK1 expression in HCC cell lines. qRT-PCR and western blot results showed that RBCK1 was significantly upregulated in HCC cells, compared to that in normal HL-7702 cell line (Fig. 2a and b). Based on the RBCK1 expression levels in HCC cell lines, we next established stable models of RBCK1 knockdown in HCCLM3 cell lines, as well as stable models of RBCK1 overexpression in Hep3B cell lines (Fig. 2c). Migration and invasion assays revealed that the mobility and invasiveness of HCC cells was markedly inhibited by RBCK1 knockdown, but significantly promoted by RBCK1 overexpression, compared with control cells (Fig. 2d and e). Similarly, RTCA assay results also showed that RBCK1 knockdown notably suppressed the metastatic ability of HCCLM3 cells, and RBCK1 overexpression promoted the metastatic ability of Hep3B cells (Fig. S2). As EMT is significantly associated with the metastatic abilities of cancer cells, we examined the effects of RBCK1 expression on the EMT phenotype of HCC cells. As shown in Fig. 2f and g, immunofluorescence assays indicate that RBCK1 knockdown increased the epithelial marker, but decreased the mesenchymal marker in HCCLM3 cells. The stable RBCK1 knockdown can therefore inhibit HCC invasion and metastasis.
We further examined the effects of RBCK1 on HCC metastasis by establishing an orthotopic liver tumour model in nude mice. The experiment included shNC and shRBCK1 groups. Histological analysis showed the development of intrahepatic metastasis in five cases from the shNC group, compared to only one case in the HCCLM3-shRBCK1 group (Fig. 2h and j). In addition, H&E-stained serial lung sections revealed that the number of HCC lung micrometastases significantly decreased in the shRBCK1 group (Fig. 2i). In contrast, overexpression of RBCK1 increased the number of intrahepatic and lung metastatic nodules (Fig. 2k). Collectively, these results indicate that the stable knockdown of RBCK1 can inhibit HCC invasion and metastasis both in vitro and in vivo, while also acting as a tumour oncogene candidate during HCC progression and metastasis.
3. RBCK1 promotes HCC progression by enhancing the Warburg effect
E3 ubiquitin ligase contributes to reprogrammed metabolism in the progression of several types of cancer. As the Warburg effect is a well-characterised metabolic shift that ubiquitously occurs in tumour cells, including HCC, we explored the role of RBCK1 in HCC glucose metabolism. RBCK1 knockdown also dramatically decreased the cellular levels of glucose-6-phosphate (G6P), glucose consumption, lactate production, and ATP in HCCLM3 cells (Fig. 3a), while RBCK1 overexpression generated opposite trends in Hep3B cells (Fig. 3f). To further validate the impact of RBCK1 on glycolysis in HCC, ECAR, which reflects overall glycolytic flux, was measured. RBCK1 knockdown was shown to significantly decrease the glycolytic rate and capacity of HCCLM3 cells (Fig. 3b and c), whereas RBCK1 overexpression increased ECAR in Hep3B cells (Fig. 3g and h). Results obtained from the measurement of OCR, an indicator of mitochondrial respiration, revealed an increase in HCCLM3/shRBCK1 cells (Fig. 3d and e), whereas RBCK1 overexpression produced a decrease in Hep3B cells (Fig. 3i and j). Moreover, these responses were also exhibited in SMCC7721/shRBCK1 and SK-Hep-1/p-RBCK1 cells (Fig. S3).
To investigate whether the Warburg effect was responsible for the progression of HCC cells, HCCLM3/shRBCK1 and Hep3B/p-RBCK1 cells were treated with 2-DG at different concentrations (0, 4, or 8 mM) for 24 h. Results showed that, in HCCLM3/shRBCK1 and Hep3B/p-RBCK1 cells, 2-DG significantly inhibited glycolysis in a dose-dependent manner (Fig. 3k and l). The migratory and invasive ability of HCCLM3/shRBCK1 and Hep3B/p-RBCK1 cells was also decreased in a dose-dependent manner (Fig. 3m and n). In order to demonstrate that glycolysis modulates HCC migration and invasion, cells were cultured in medium containing galactose instead of glucose, thereby reducing the glycolytic flux and forcing the cells to rely on oxidative phosphorylation. We observed that this reduced glycolytic flux greatly abrogated the increased migratory and invasive ability of Hep3B cells induced by RBCK1 overexpression (Fig. 3o). These findings indicate that RBCK1 suppresses oxidative phosphorylation while promoting aerobic glycolysis (Warburg effect) in HCC cells, but promotes migration and invasion by enhancing the Warburg effect in HCC cell lines in vitro.
4. RBCK1 promotes the Warburg effect via GLUT1 in HCC cells
Previous studies have demonstrated that GLUT1 plays an important role in glycolysis [6]. We explored whether RBCK1 regulated GLUT1 expression, by initially observing the expression of GLUT1 in RBCK1-knockdown or -overexpressing HCC cells. Western blotting results showed that RBCK1 knockdown significantly decreased GLUT1 expression in HCCLM3 cells (Fig. 4a). Conversely, RBCK1 overexpression markedly increased GLUT1 expression in Hep3B cells (Fig. 4b). Further, the upregulation of GLUT1 attenuated the loss of GLUT1 expression in HCCLM3/shRBCK1 cells (Fig. 4c). Meanwhile, the rescue experiments indicate that restoration of GLUT1 expression abolished the reduced metastasis ability of HCC cells induced by RBCK1 silence (Fig. 4d and e). Importantly, the in vivo tumour metastatic assay revealed that overexpression of GLUT1 rescued the decreased incidence of intrahepatic and lung metastasis of HCCLM3/shRBCK1 cells (Fig. 4f). Moreover, investigations into whether RBCK1 increased glycolysis via GLUT1 expression revealed that an upregulation of GLUT1 expression rescued the RBCK1-mediated reduction in cellular G6P, glucose consumption, lactate production, and ATP levels in HCC cells (Fig. 4g). Meanwhile, RBCK1 knockdown decreased ECAR and OCR in HCC cells, whereas a simultaneous overexpression of GLUT1 attenuated the decrease in glycolytic rate and capacity (Fig. 4h-k).
Next, we assessed the effect of decreased GLUT1 expression on RBCK1 and GLUT1 protein levels, as well as on cell migration and invasion abilities, in RBCK1-overexpressing Hep3B cells. Western blotting analyses showed that RBCK1 overexpression significantly increased GLUT1 expression, whereas the knockdown of GLUT1 expression dramatically inhibited the RBCK1-induced increase in GLUT1 expression in Hep3B cells (Fig. 4l). Meanwhile, GLUT1 downregulation significantly reduced RBCK1-enhanced cell migration and invasion (Fig. 4m and n). Furthermore, in vivo metastatic assay results showed that GLUT1 downregulation decreased the incidence of intrahepatic and lung metastasis in the Hep3B-RBCK1 group (Fig. 4o). Moreover, investigations into whether RBCK1 increased glycolysis via GLUT1 expression revealed that knockdown of GLUT1 expression rescued the RBCK1-mediated increase in cellular G6P, glucose consumption, lactate production, and ATP levels in HCC cells (Fig. 4p). Meanwhile, RBCK1 overexpression increased ECAR and OCR in HCC cells, whereas a simultaneous knockdown of GLUT1 attenuated the increase in glycolytic rate and capacity (Fig. 4q-t). Collectively, these findings indicated that GLUT1 is a functional downstream target of RBCK1 in the regulation of aerobic glycolysis, and that it is critical in RBCK1-mediated tumour progression.
5. RBCK1-induced activation of GLUT1 is mediated by WNT/β-catenin signalling
To further clarify the mechanism by which RBCK1 regulates GLUT1 in HCC, the effect of RBCK1 on the global gene expression patterns of HCCLM3 cells was assessed at the transcriptome level via RNA sequencing (RNA-seq) (Fig. 5a). Gene set enrichment analysis (GSEA) was performed to determine the effects of transcriptomic changes on biological functions and pathways. The WNT signalling pathway was significantly positively correlated with RBCK1 in HCCLM3 cells (Fig. 5a). As GLUT1 is a target gene of WNT/β-catenin, we speculated that RBCK1 regulated GLUT1 via the WNT/β-catenin signalling pathway. To test this hypothesis, we measured changes in β-catenin expression in RBCK1-knockdown HCCLM3 cells. Western blotting analyses showed that total and nuclear β-catenin protein expression decreased with decreasing RBCK1 expression in HCCLM3 cells (Fig. 5b). The TOP-Flash reporter luciferase assay showed that RBCK1 knockdown decreased the transcriptional activity of TCF4 in MHCC97H cells, compared with the shNC control (Fig. 5c). In contrast, RBCK1 overexpression generated opposite effects in Hep3B cells (Fig. 5d and e). We further determined that β-catenin upregulation rescued the decrease in GLUT1 expression, cell migration, and cell invasion induced by RBCK1 knockdown (Fig. 5f-i).
To verify that RBCK1 regulates GLUT1 expression through the WNT/β-catenin signalling pathway, we measured the levels of GLUT1 and β-catenin in the presence of the WNT/β-catenin pathway inhibitors XAV-939. Consistently, XAV-939 inhibited the mRNA and protein levels of GLUT1 in HCCLM3 cells (Fig. 5j and k). Transwell assay showed that XAV-939 markedly decreased RBCK1-induced cell migration and invasion (Fig. 5l). XAV-939 leads to decrease in cellular G6P, glucose consumption, lactate production, and ATP levels in HCC cells (Fig. 5m). Meanwhile, XAV-939 inhibited ECAR in HCC cells (Fig. 5n). Meanwhile, the rescue experiments indicate that XAV-939 attenuated the enhanced metastasis ability and Warburg effect of HCC cells induced by RBCK1 overexpression (Fig. 5o-t). Consistently, β-catenin silence abolished the increased metastasis ability and Warburg effect of HCC cells induced by RBCK1 overexpression (Fig. S4). Taken together, RBCK1 promotes the metastasis of HCC cells via GLUT1-mediated Warburg effect through activation of WNT/β-catenin signalling.
6. RBCK1 destroyed the PPARγ/PGC1α complex to activate the WNT/β-catenin pathway and Warburg effect in HCC cells
To clarify the mechanism through which RBCK1 regulates the WNT/β-catenin signalling pathway in HCC cells, we first determined whether there was a direct interaction between the RBCK1 and β-catenin proteins. Co-IP showed that no direct interaction existed between these proteins (Fig. 6a). The PPARγ/PGC1α complex induces the inhibition of the canonical WNT/β-catenin pathway and contributes to glucose homeostasis. Therefore, we speculated that RBCK1 regulated WNT/β-catenin via the destroyed PPARγ/PGC1α complex. To test this hypothesis, we first determined whether RBCK1 influenced the activation of the β-catenin/GLUT1 pathway via the destroyed PPARγ/PGC1α complex. Changes in β-catenin, GLUT1, PGC1α, and PPARγ expression, as well as in the PPARγ/PGC1α complex, were measured in RBCK1-knockdown HCC cells. Results showed that RBCK1 knockdown in HCCLM3 cells significantly increased the levels of PPARγ expression and PPARγ/PGC1α complex, decreased β-catenin and GLUT1 expression, but produced no change in PGC1α protein levels (Fig. 6b and c). By contrast, RBCK1 overexpression in Hep3B cells significantly decreased the levels of PPARγ protein expression and PPARγ/PGC1α complex, increased β-catenin and GLUT1 expression, while also producing no change in PGC1α protein levels (Fig. 6d and e). In addition, the PPARγ mRNA level in HCC cells remained unaffected by changes in RBCK1 expression (Fig. 6f). The PPARγ/PGC1α complex is therefore involved in RBCK1-mediated regulation of the β-catenin/GLUT1 pathway.
To verify that RBCK1 regulates the β-catenin/GLUT1 pathway through the destroyed PPARγ/PGC1α complex, PPARγ was knocked down in RBCK1-downregulated HCC cells. PPARγ downregulation inhibited the decrease in β-catenin and GLUT1 expression, cell migration, and aerobic glycolysis observed in RBCK1-knockdown HCCLM3 cells (Fig. 6g-i). By contrast, PPARγ upregulation enhanced the increase in β-catenin and GLUT1 expression, cell migration, and aerobic glycolysis observed in RBCK1-upregulated HepG2 cells (Fig. 6j-m). To determine whether the RBCK1-induced anti-Warburg effect was dependent on the PPARγ/PGC1α complex, we treated HCC cells with the PPARγ inhibitor GW9662. As expected, GW9662 reversed the decreased ECAR levels induced by RBCK1 knockdown (Fig. 6n). Importantly, IHC staining (Fig. 6o) revealed that compared with adjacent non-tumour-bearing tissues, the RBCK1, Ki67, β-catenin, GLUT1 and Vimentin protein were upregulated, whereas PPARγ and PGC1α were downregulated in HCC tissues. These results indicated that the RBCK1-mediated regulation of β-catenin/GLUT1 pathway-induced HCC cell migration and aerobic glycolysis is therefore dependent on the destroyed PPARγ/PGC1α complex.
7. RBCK1 destroyed the PPARγ/PGC1α complex by promoting the ubiquitination and degradation of PPARγ
RBCK1 has the ability to interact with different substrates in order to exert its effects. We observed whether RBCK1, PPARγ, and PGC1α directly interacted in HCC cells. Co-IP analysis detected endogenous RBCK1 and PPARγ in the immunoprecipitate, indicating an interaction between RBCK1 and PPARγ, but no direct interaction between RBCK1 and PGC1α (Fig. 7a and b). Moreover, GST-pull down assay indicate that RBCK1 could bind to PPARγ in vitro system (Fig. 7c and d). These findings confirmed that RBCK1 directly bind with PPARγ in HCC cells, and that RBCK1 destroyed the PPARγ/PGC1α complex by regulating PPARγ expression.
We then assessed the mechanisms by which RBCK1 regulates PGC1α. Consistent with findings from a previous study, which showed PPARγ degradation via the UPS, treatment with the proteasome inhibitor MG132 led to significant accumulation of endogenous PPARγ protein in HCC cells (Fig. 7e and f). Moreover, the data indicate that efficient ubiquitination of His-PPARγ was detected in the presence of E1, E2 (UBCH5c), FBXO9 (an E3 ubiquitin ligase for PPARγ), and Ub (Fig. 7g). PPARγ is therefore also degraded by the UPS in HCC cells. Next, we determined whether RBCK1 could directly mediate PPARγ ubiquitination. Interestingly, PPARγ poly-ubiquitination was substantially increased by ectopic RBCK1 expression, but decreased by RBCK1 knockdown (Fig. 7h and i). The data also showed that mutations in all the Lys sites of PPARγ abolished its poly-ubiquitination (Fig. 7j). As expected, mutation of the Lys48 site on ubiquitin (Ub) almost completely abolished RBCK1-mediated PPARγ ubiquitination, whereas the K63R mutation produced no effect (Fig. 7k). Consistent with the ubiquitination results, a degradation dynamics assay showed that the half-life of exogenously expressed PPARγ was significantly increased in RBCK1-overexpressing HCC cells, compared with that in control cells (Fig. 7l and m). These data showed that RBCK1 mediates Lys48-linked poly-ubiquitination of PPARγ, which leads to PPARγ degradation in the proteasome. Collectively, these results indicate that RBCK1 destroyed the PPARγ/PGC1α complex by promoting PPARγ ubiquitination and degradation (Fig. 8).