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. 2019 Jun 27;42(5):705–715. doi: 10.1007/s13402-019-00458-8

Predictors of ribociclib-mediated antitumour effects in native and sorafenib-resistant human hepatocellular carcinoma cells

Florian P Reiter 1, Gerald Denk 1, Andreas Ziesch 1, Andrea Ofner 1, Ralf Wimmer 1, Simon Hohenester 1, Tobias S Schiergens 2, Matilde Spampatti 1, Liangtao Ye 1, Timo Itzel 3, Stefan Munker 1, Andreas Teufel 3, Alexander L Gerbes 1, Julia Mayerle 1, Enrico N De Toni 1,✉
PMCID: PMC12994347  PMID: 31250364

Abstract

Purpose

The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC.

Methods

The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA).

Results

We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated.

Conclusions

From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.

Electronic supplementary material

The online version of this article (10.1007/s13402-019-00458-8) contains supplementary material, which is available to authorized users.

Keywords: Hepatocellular carcinoma, Ribociclib, Sorafenib, CDK inhibition, Targeted tumour therapy, Retinoblastoma protein

Introduction

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide [1]. Unfortunately, HCC is diagnosed most often in advanced stages at which surgical or local treatment options are not effective [2, 3]. Over the past 10 years sorafenib has been the gold standard treatment for patients with advanced HCC [3–5]. Lately, however, a number of additional compounds targeting tumour-specific signalling pathways, such as regorafenib, lenvatinib, cabozantinib and ramucirumab, have shown positive results in phase 3 trials [6–9], indicating that therapy via different lines of treatment may be feasible in HCC patients. In addition, the approval of nivolumab and pembrolizumab as second-line treatment options has highlighted an important role of the immune system in determining HCC development, and has set the stage for extensive research of these checkpoint inhibitors [10–12]. With the plethora of newly available therapeutic agents, biomarkers are needed to direct the choice of these agents. As yet, however, no reliable response biomarker for any of these new agents has been defined, with the notable exception of alpha-fetoprotein (AFP), which is a biomarker of response to ramucirumab [9]. This lack of suitable biomarker identification is likely due to both the molecular heterogeneity of HCC and the multiplicity of targets typical of kinase inhibitors. The cyclin D-cyclin-dependent kinase (CDK) 4/6-retinoblastoma protein (Rb) pathway plays a crucial role in the regulation of cell cycle progression [13] and is known to be altered in many tumours [13, 14]. This knowledge provided a rationale for the clinical development of CDK4/6 inhibitors such as palbociclib and ribociclib, which have recently been approved for the treatment of oestrogen receptor-positive, HER2-negative advanced breast cancer [15–17]. In HCC, immunohistochemical studies have revealed that CDK4 overexpression, which is found in 73% of cases, is associated with a poor survival [18]. Moreover, 81% of HCC cases carry at least one inactivation in phospho-Rb (pRb) or p16 [19], a negative regulator of the cell cycle that prevents interactions between CDK4/6 and cyclin D1 and, thereby, their downstream effect on pRb and the transcription factor E2F [19]. Consistent with these findings, preclinical evidence from a previous study [20] has recently shown that palbociclib, a CDK4/6 inhibitor, exerts strong antineoplastic effects in HCCs with high levels of Rb expression [20], suggesting that Rb may serve as a marker for the clinical response when agents such as palbociclib are used for the treatment of HCC. CDK4/6-targeting agents may thus represent a therapeutic resource for the treatment of HCC. Here, we aimed to assess the potential antineoplastic activity of ribociclib on native and sorafenib-resistant HCC cells.

Materials and methods

Cell lines, culture conditions and chemicals

The human hepatocellular carcinoma (HCC) cell lines HuH7, HepG2 and PLCPRF5 were maintained in DMEM (Sigma, Germany). Hep3B cells were maintained in MEM (Sigma, Germany). Primary hepatocytes were cultured in William’s E medium (Thermo Fisher Scientific, USA). All media were supplemented with 10% foetal bovine serum (Biochrom, Germany) and antibiotics (penicillin, streptomycin-Sigma, Germany) and the cells were kept in a humidified atmosphere with 5% CO2 and 21% O2 at 37 °C. All cell lines used were analysed by an external independent institution (Leibniz-Institut; DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) by genetic fingerprinting to confirm authenticity and to exclude contamination. Ribociclib (LEE011) was kindly provided by Novartis (Switzerland). Sorafenib was purchased from Selleck Chemicals (Germany). Sorafenib-resistant cells were generated from native HepG2 cells that underwent continuous incubation with sorafenib at increasing concentrations for 8 months to a final sorafenib concentration of 4 μM. Resistant cells were maintained in medium containing 4 μM sorafenib.

Isolation of primary human hepatocytes

Double-coded tissues and the corresponding data used in this study were provided by the Biobank of the Department of General, Visceral and Transplantation Surgery, Ludwig Maximilian University, Munich, Germany, under the administration of the Human Tissue and Cell Research Foundation. The charter of the Human Tissue and Cell Research Foundation [21], which includes obtaining written informed consent from all donors, has been approved by the ethics commission of the Faculty of Medicine at LMU (approval number 025–12) as well as the Bavarian State Medical Association (approval number 11142), Germany. Primary human hepatocytes were isolated by the Cell Isolation Core Facility of the Biobank using a two-step collagenase perfusion technique with modifications, as described earlier [22]. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The use of all human material was approved by the ethics commission of the Faculty of Medicine at the University of Munich.

Cell viability assay

For assessment of cell viability, SYBR Green assays (Lonza, Germany) were performed. Densitometric values were expressed as values normalized to the controls, as previously described [23].

Fluorescence-activated cell sorting

Cells were harvested from 6-well plates. After propidium iodide staining (Sigma, Germany), fluorescence-activated cell sorting (FACS Accuri C6 flow cytometer-BD Biosciences, San Jose, CA USA) was performed. Apoptosis was quantified by calculating the fraction of cells with a sub-diploid DNA content (sub-G1) and assessed morphologically using Hoechst staining and fluorescence microscopy (Zeiss, Jena, Germany).

Western blotting

Proteins were loaded in equal amounts, separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Germany). The membranes were incubated with monoclonal antibodies directed against Cyclin D1 (Cell Signalling Technology, USA), Rb (Biolegend, Germany), CDK4 (Cell Signaling Technology, USA), CDK6 (Cell Signaling Technology, USA), p16 (Abcam, UK), pRb (Cell Signaling Technology, USA), LC3B (Cell Signaling Technology, USA), AMP-activated protein kinase 1/2 (AMPK1/2-Santa Cruz Biotechnology, USA), pAMPK 1/2 (Cell Signaling Technology, USA), protein phosphatase 5 (PP5-Santa Cruz Biotechnology, USA) or β-Actin (Sigma, Germany), which was used as the loading control, followed by HRP-conjugated anti-mouse or anti-rabbit antibodies (GE Healthcare, Germany). Immunoreactions were visualized using a SuperSignal West Pico substrate (Thermo Fischer Scientific, USA) and detected with a ChemoCam (INTAS, Germany).

Colony formation assay

Cells were seeded in 6-well plates and incubated with ribociclib for 2 weeks. Next, colony formation was evaluated after fixation in 10% paraformaldehyde and staining with crystal violet for 15 min (Sigma Aldrich, Germany). Quantification of the colony formation assay results was performed using ImageJ software (Bethesda, National Institutes of Health, USA).

SiRNA-mediated gene silencing

HuH7 cells were grown to 30%–50% confluence and subsequently incubated with a siRNA directed against Rb (Qiagen, Netherlands) or with a non-coding sequence of β-galactosidase (β-GAL, Dharmacon, USA), which served as the control. Cells were harvested 48 h after transfection for proliferation assays or Western blot analyses.

The Cancer genome atlas data analysis

Open-access RNA sequencing data for HCC samples (n = 361) were downloaded from the Cancer Genome Atlas (http://cancergenome.nih.gov/). The expression levels of genes in HCC patient samples were measured in fragments per kilobase of transcript per million fragments, as previously described [24]. For survival analysis, patients were stratified according to the expression of CDK4 or CDKN2A (encoding p16) RNA by using the median fragments per kilobase of transcript per million fragments value as the cut-off.

Synergism analysis

Synergism analyses were performed according to the method proposed by Chou [25]. In brief, the median-effect method (CompuSyn software; Biosoft, Ferguson, USA) was used to determine the drug concentrations resulting in 50% growth inhibition (IC50) and to calculate isobolograms indicating the Chou-Talalay combination index (CI) for different levels of growth inhibition. CI values of <1, 1, and > 1 indicated synergistic, additive and antagonistic effects, respectively.

Graphics

GraphPad Prism 7 (GraphPad Software, USA), Adobe Illustrator CC 2019 (Adobe, USA) and Adobe Photoshop CC 2019 were used to generate the figures and graphics.

Statistical analysis

Statistical calculations were performed with the SPSS 25 software package (IBM, USA) using analysis of variance (ANOVA), Spearman correlation, log-rank tests, Mann-Whitney U tests or t-tests. When a relevant influence of an experiment was observed, univariate ANOVA was performed, after which the degrees of freedom (df), means of squares and F-values were calculated. The model of total variance reflects the variance of cell preparation, treatment and residual error. P values < 0.05 were considered statistically significant. Data are presented as the means ± standard errors of the mean.

Results

Ribociclib exerts a potent anti-proliferative effect in HCC cells

Using a cell viability assay, we found that ribociclib inhibited HCC cell growth in vitro in a dose-dependent manner, showing higher effectiveness in Huh7 and HepG2 cells (IC50 = 64.3 nM and 80.2 nM, respectively) than in PLCPRF5 cells (IC50 = 43,985.1 nM). In particular, we found that upon incubation with 2000 nM ribociclib, the cell viabilities decreased by 96% and 91% compared to those of the corresponding control Huh7 and HepG2 cells, respectively, but only by 27% in PLCPRF5 cells (Fig. 1a). In contrast, we found that ribociclib had no effect on the viability of Hep3B cells (IC50 = 54,336.1 nM; Fig. 1a). These results were confirmed by colony formation assays, which showed that ribociclib caused a reduction in colony size of Huh7 cells (control vs. 1000 nM ribociclib; 17.05% ± 1.70% vs. 2.94% ± 1.20%) and HepG2 cells (control vs. 1000 nM ribociclib; 16.91% ± 1.87% vs. 5.12% ± 1.48%), but not of Hep3B cells (control vs. 1000 nM ribociclib; 34.59% ± 8.09% vs. 37.48% ± 14.61%; Fig. 1b-c). We found that ribociclib marginally, but significantly, affected the colony size of PLCPRF5 cells (control vs. 1000 nM ribociclib; 14.73% ± 2.16% vs. 9.29% ± 2.29%; Fig. 1b-c).

Figure 1.

Figure 1

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Ribociclib exerts an antiproliferative effect on HCC cells with a high Rb expression and a low p16 expression. The indicated HCC cell lines were incubated with increasing concentrations of ribociclib, after which the basal expression levels of proteins in the CDK4/6–Rb pathway were measured using Western blotting. a Proliferation was assessed using a SYBR Green assay; the plotted mean and SEM values are expressed as the ratios of the control/treated cell values for each cell line and are representative of three experiments (n = 3; HuH7 control vs. ribociclib: ** p < 0.01; HepG2 control vs. ribociclib: ##p < 0.01; PLCPRF5 control vs. ribociclib: † p < 0.05 and †† p < 0.01; ANOVA). b Quantification of positive areas in colony formation assays assessed after staining with crystal violet. The mean and SEM values of the controls were compared with the values for cells treated with increasing doses of ribociclib (n = 3; **, p < 0.01; ANOVA). c Representative images of colony formation. d Baseline protein expression levels of Rb, cyclin D1, CDK4, CDK6 and p16. e Protein expression levels were quantified via densitometry in four different HCC cell lines (HuH7, HepG2, PLCPRF5 and Hep3B), and expression was normalized to that in HuH7 cells (n = 6–9; *, p < 0.05, **, p < 0.01; ANOVA)

Subsequent assessment of Rb expression by Western blotting showed that it was high in cells highly responsive to the antineoplastic activity of ribociclib (Huh7 and HepG2), but lower in cells less sensitive (PLCPRF5) or insensitive (Hep3B) to ribociclib (Fig. 1d-e). Conversely, we found that the basal levels of p16 were higher in Hep3B cells than in Huh7 and HepG2 cells (Fig. 1d-e). In contrast, no apparent association was observed between the reduction in cell viability caused by ribociclib and the expression of Cyclin D1, CDK4 or CDK6 (Fig. 1d and suppl. Fig. 1). We also assessed the expression levels of Rb and p16 in HCC cell lines compared to those in primary non-tumour hepatocytes isolated from surgical specimens of three different patients and found that the expression of Rb and p16 was almost undetectable in the primary normal cells (suppl. Fig. 2). Collectively, these results suggest a possible role of Rb as a marker of addiction to CDK4/6 signalling activation in HCC cells.

Fig. 2.

Fig. 2

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Ribociclib induces cell cycle arrest in sensitive HCC cell lines without inducing apoptosis. Different HCC cell lines (HuH7, HepG2, PLCPRF5 and Hep3B) were incubated with up to 1000 nM Ribociclib for up to 48 h. a Cell cycle analysis was performed using fluorescence-activated cell sorting, and the results were compared to those of controls (n = 3–4; * p < 0.05, ** p < 0.01; ANOVA). b Differences in cell cycle distribution between ribociclib-sensitive HuH7 and ribociclib-resistant Hep3B cells treated with ribociclib. c-d Apoptosis was assessed using Hoechst staining and FACS-based analysis of sub-G1 phase events. Hoechst staining did not show nuclear changes indicating apoptosis under ribociclib treatment, consistent with the lack of alterations in sub-G1-phase distribution observed after FACS analysis (n = 3–4; not significant; ANOVA)

Ribociclib causes cell cycle arrest but negligible apoptosis in HuH7, HepG2 and PLCPRF5 cells

Based on to the known role of CDK4/6 in the regulation of cell proliferation, a FACS-based analysis of the cell cycle and sub-G1 cell fraction after PI staining was conducted to assess the respective contributions of cell cycle arrest and apoptosis to the antineoplastic effect of ribociclib at different time points and concentrations. We found that administration of ribociclib induced G1-phase cell cycle arrest and a concomitant reduction in the S- and G2-phase populations in Huh7, HepG2 and PLCPRF5 cells, but not in Hep3B cells (Fig. 2a-b). In contrast, analysis of DNA fragmentation by assessment of sub-G1 events revealed that ribociclib did not cause apoptosis in any of the cell lines tested. This result was confirmed by the lack of typical features of apoptosis, such as nuclear fragmentation or chromatin condensation, after Hoechst staining (Fig. 2c-d). Consistent with the role of CDK4/6 as a crucial cell cycle regulator, these data indicate that the antineoplastic effect of ribociclib results from cell cycle arrest induction.

Ribociclib-mediated downregulation of Rb is not essential for its antineoplastic effect

To assess the mechanisms underlying the action of ribociclib, the expression of Rb and other members of the CDK4/6 signalling pathway, along with other molecular targets potentially involved in cell cycle regulation [26, 27], was investigated. Interestingly, we found that the levels of Rb and its phosphorylated form (pRb) were decreased in highly sensitive HuH7 and HepG2 cells, but not in PLCPRF5 and Hep3B cells (Fig. 3). However, no consistent pattern of change in the expression of other potential targets of ribociclib, including CDK4/6, LC3B, p-AMPK, AMPK and PP5, was seen. To evaluate the possible functional relevance of Rb downregulation in the effect of ribociclib on HCC cells, siRNA-based silencing experiments targeting Rb were conducted (Fig. 4a). We found that, although Rb silencing caused a significant reduction in viability compared to that of control cells (Fig. 4b), it did not alter the sensitivity of Huh7 cells to the action of ribociclib (Fig. 4c), suggesting that Rb downregulation mediated by ribociclib is not essential for its antineoplastic effect.

Fig. 3.

Fig. 3

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Ribociclib reduces Rb and pRb protein levels in highly ribociclib-sensitive HuH7 and HepG2 cells. The effects of ribociclib treatment on the levels of proteins in the cyclin D-CDK4/6-Rb pathway and on the levels of LC3B, AMPK1/2 and PP5 were assessed using Western blotting. Representative blots are shown

Fig. 4.

Fig. 4

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siRNA-based knockdown of Rb reduces proliferation in HuH7 cells without affecting their sensitivity to ribociclib. a Effect of siRNA-based Rb silencing; the greatest effect was seen after 48 h (n = 4; * p < 0.05; t-test). b siRNA-based Rb silencing significantly reduced the proliferation of HuH7 cells (n = 4; * p < 0.05; t-test). c siRNA-based Rb silencing did not alter sensitivity to ribociclib (n = 4; control group: ** p < 0.01; siRb group: ##p < 0.01; ANOVA)

Ribociclib synergizes with sorafenib and causes loss of viability in sorafenib-resistant cells

To assess the significance of ribociclib as a potential treatment for HCC, its interaction with sorafenib in inducing loss of cell viability was assessed by co-incubating HuH7 and HepG2 cells with increasing concentrations of both agents. We found that co-incubation with ribociclib and sorafenib resulted in a synergistic loss of cell viability (Fig. 5a-f). We also used sorafenib-resistant HepG2 (srHepG2) cells with an acquired resistance to this agent after long-term exposure. As expected, srHepG2 cells were found to be approximately 4.5-fold less sensitive to sorafenib than non-resistant control cells (Fig. 6a), but retained sensitivity to ribociclib (Fig. 6b). Consistent with these results, we found that the expression levels of Rb in srHepG2 cells did not differ from those in sorafenib-sensitive wild-type HepG2 cells (Fig. 6c and suppl. Fig. 4). These data indicate that the mechanisms of action of ribociclib are not affected by the mechanisms of resistance to sorafenib, supporting a combined use of these agents for HCC treatment.

Fig. 5.

Fig. 5

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Combination of ribociclib and sorafenib leads to synergistic anti-proliferative effects. Ribociclib-sensitive hepatoma cell lines (HuH7 and HepG2) were incubated with increasing concentrations of sorafenib for 44 h. a-f Dose-effect relationship of ribociclib, sorafenib, and their combination (constant ratio of 1:10) on growth inhibition of Huh7 (a) and HepG2 (d) cells. The combination index (CI) values and fraction affected (Fa) for each dose were used to generate the Fa-CI plots (b, HuH7; e, HepG2) and isobolograms (c, HuH7; f, HepG2) using CompuSyn software

Fig. 6.

Fig. 6

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Ribociclib shows effects in sorafenib-resistant cells expressing normal basal Rb protein levels. HepG2 cells were continuously incubated with sorafenib for 8 months to generate a sorafenib-resistant cell line (srHepG2). a Sorafenib resistance in HepG2 cells was confirmed by a proliferation assay; srHepG2 cells showed a significantly lower sensitivity to 4 μM sorafenib than non-resistant control cells (HepG2) when normalized to that of the sorafenib-untreated controls for each group (n = 3; * p < 0.05; t-test;). b Native and srHepG2 cells were exposed to increasing doses of ribociclib; a significant reduction in proliferation was seen in both groups (n = 3; native HepG2 control vs. ribociclib: * p < 0.05, ** p < 0.01; srHepG2 control vs. ribociclib: #p < 0.05, ##p < 0.01; ANOVA), without significant differences between the groups (n = 3; n.s.; t-test;). (c) Representative baseline protein expression levels of Rb and β-Actin

CDK4 expression is associated with a poor clinical outcome and Rb expression is inversely correlated with CDKN2A expression

To assess the relevance of the cyclin D-CDK 4/6-Rb pathway in primary human specimens, we analysed the expression and prognostic significance of CDK4, CDK6, Rb and a counter-regulator of their effect on the cell cycle, i.e., CDKN2A (which encodes p16), in a cohort of 361 patients using publicly available data from The Cancer Genome Atlas. Survival analysis revealed that HCC patients with a high CDK4 or a high CDKN2A expression had a poorer median overall survival than patients with a low CDK4 or a low CDK2NA expression (Fig. 7a-b). In contrast, no correlation was found between the CDK6 or Rb expression and survival (data not shown). These data underscore the hypothesis that CDK4/6 activation plays an important role in the pathogenesis of HCC and confirm an inverse correlation between Rb and CDKN2A expression (Fig. 7c). This inverse correlation was also observed after Western blot analysis, showing that high p16 protein expression levels were associated with low Rb expression levels.

Fig. 7.

Fig. 7

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Analysis of RNA expression levels in HCC sample data from The Cancer Genome Atlas. RNA expression values were measured as fragments per kilobase of transcript per million fragments in HCC samples (n = 361). a-b Associations of CDK4 and CDKN2A expression with survival were analysed (CDK4, n = 54 vs. 75; p < 0.01; hazard ratio, 0.57; 95% CI, 0.40–0.81; CDKN2A, n = 59 vs. 70; p < 0.05; hazard ratio, 0.65; 95% CI, 0.46–0.92; log-rank test). c The RNA expression levels of Rb and cyclin-dependent kinase Inhibitor 2A (CDKN2A) were correlated in HCC samples (n = 361; p < 0.0001; r = −0.2721; Spearman correlation)

Discussion

Dysregulation of the cyclin D-CDK4/6-Rb pathway is one of the most frequently observed alterations in HCC [18, 19]. This signalling pathway is thought to serve as an important target for therapy. Here, we assessed the mechanisms underlying the effect of ribociclib on HCC cells, and specifically addressed the possibility that the antitumour activity of ribociclib may be predicted by the expression of p16 and Rb, two crucial negative regulators of CDK4/6 and Cyclin D activity in determining cell cycle progression (Fig. 8-modified from [16]). In addition, we performed an exploratory analysis to assess the significance of CDK4, CDK6, Rb and p16 expression in human HCC specimens. We found that the antitumour activity of ribociclib was cell line specific and was notably high in cells with a high Rb expression, but lower in cells that exhibited Rb expression levels comparable to those in primary human hepatocytes. However, cells with a low Rb expression consistently exhibited high p16 expression levels and were resistant to the action of ribociclib. These findings are consistent with those from previous studies [20] showing that Rb-expressing cells are sensitive to palbociclib, another recently approved CDK4/6 inhibitor, and with data from other tumour entities including neuroendocrine tumours [28], breast cancer [29] and ovarian cancer [30]. Consistent with the function of CDK4/6 as a major regulator of the cell cycle, we found that loss of cell viability was accompanied by G1-phase cell cycle arrest. In addition, neither apoptosis nor autophagy (which have previously been reported as CDK4/6-independent effects of palbociclib [26]) seem to play a role in the effect of ribociclib. When we assessed the kinetics of Rb expression upon administration of ribociclib, we found that the expression of Rb decreased only in Rb-high/ribociclib-sensitive cells. However, although silencing of Rb caused a loss of cell viability (Fig. 4b), inhibition of Rb did not alter the sensitivity to ribociclib (Fig. 4c), which suggests that Rb, rather than being a mediator of the effect of ribociclib, may be a marker of oncogenic addiction to enhanced CDK4/6 signalling. As Rb is a negative regulator of CDK4/6 activity, its expression may result from a negative feedback mechanism of compensatory regulation of CDK4/6 activation. The observation that p16 and Rb may have opposing functions in terms of their effect on ribociclib sensitivity is interesting and indicates that these two molecules could serve as a combined biomarker for the sensitivity of HCC cells to CDK4/6 inhibitors. Their opposing roles in HCC seem to be corroborated by our mRNA expression analysis in primary patient samples, which showed that p16, like CDK4 but in contrast to Rb, possesses unfavourable prognostic significance. In addition, similar to the correlative variation of Rb and p16 at the protein level, we found that the mRNA expression level of p16 in the patient samples was inversely correlated with that of Rb (Fig. 7). In addition to exhibiting antiproliferative effects as a single agent, we found that ribociclib synergistically potentiated the effect of sorafenib and, as such, may be used either alone or in combination with sorafenib. In summary, we provide preclinical evidence that ribociclib exerts a potent antineoplastic effect on HCC cells and that Rb and p16 may serve as potential efficacy biomarkers. Due to its synergistic interaction with sorafenib and its maintained effect in sorafenib-resistant cells, we suggest that ribociclib may be considered a potential second-line treatment option for HCC alone or in combination with sorafenib.

Fig. 8.

Fig. 8

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The CDK4/6–Rb pathway and its inhibition via ribociclib. Generally accepted mechanism of action of ribociclib (modified from [16])

Electronic supplementary material

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Acknowledgements

We thank Sebastian Reiter for his support with the illustrations. This study was funded by the Georg und Traud Gravenhorst-Stiftung and supported by the Human Tissue and Cell Research Foundation, a non-profit foundation regulated by German civil law, which facilitates research with human tissue.

Abbreviations

HCC

hepatocellular carcinoma

CDK

cyclin-dependent kinase

Rb

retinoblastoma protein

pRb

phospho-retinoblastoma protein

FACS

fluorescence-activated cell sorting

CDKN2A

cyclin-dependent kinase Inhibitor 2A

srHepG2

sorafenib-resistant HepG2

AMPK

AMP-activated protein kinase

PP5

protein phosphatase 5

β-GAL

β-galactosidase

Author’s contributions

FPR and EdT acted as the submission’s guarantors. FPR, AZ, RW, AO, LY and MS performed the research. FPR, GD, AZ, RW, AO, SH, ST, LY, IT, SM and TA collected and analysed the data. FPR and EdT designed the study and wrote the paper. FPR, AG, JM and EdT contributed to the design of the study. All authors approved the final version of the article, including the author list.

Funding

This study was funded by the Georg und Traud Gravenhorst-Stiftung. The funding agency was not involved in the study design and the data collection, analysis and interpretation.

Compliance with ethical standards

Ethical approval

All procedures performed involving human participants were in accordance with the ethical standards of the institutional and/or national research committees, as well as with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This article does not contain any studies with animals performed by any of the authors.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no conflicts of interest.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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