The level of caveolin-1 expression determines response to TGF-β as a tumour suppressor in hepatocellular carcinoma cells

Abstract

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification.

Figure 1

CAV1 expression interferes with TGF-β-induced apoptosis in HCC cell lines. HLE parental, HLE shControl, HLE shCAV1, Huh7 parental, Huh7 +pControl and Huh7 +pCAV1 were treated with TGF-β (5 ng/ml) at the times shown after previous FBS starvation (2% FBS; 4 h). (a) Immunoblot of total protein extracts; α-TUBULIN as loading control. A representative experiment is shown. (b and c) Cell viability measured using Trypan blue staining and expressed as percentage of non-viable cells (N=4 for b and N=3 for c). (d and e) Caspase-3 activity, expressed as fold induction versus an untreated control (N=6 for d and N=5 for e). Results are expressed as mean±S.E.M. Statistical comparison uses two-way ANOVA with Sidak post-hoc test as shown in the figure: *P<0.05, **P<0.01, ***P<0.001

Figure 2

CAV1 expression levels in HCC cell lines determine the clonogenic ability in presence of TGF-β and alter their migratory capacity. (a and b) HLE parental, HLE shControl, HLE shCAV1, Huh7 parental, Huh7 +pControl and Huh7 +pCAV1 were treated with TGF-β (5 ng/ml) for 1 week in complete medium (10% FBS). Crystal violet stained colonies indicate clonogenic growth; a representative experiment is shown (left), and quantification is presented from three independent experiments (right). (c and d) Cell migration in real-time was analysed by the xCELLigence RTCA. Cells were treated with TGF-β (2 ng/ml) for 72 h. Migration rate was determined by analysing the slope of the lineal interval, between 10 and 35 h in HLE cells and between 15 and 35 h in Huh7 cells (N=6 from two independent experiments). Results are expressed as mean±S.E.M. Statistical comparison uses two-way ANOVA with Sidak post-hoc test as shown in the figure: *P<0.05, **P<0.01, ***P<0.001

Figure 3

Expression of BCL-2 family genes is altered by CAV1 in HCC. HLE shControl, HLE shCAV1, Huh7 +pControl and Huh7 +pCAV1 were treated with TGF-β (2 ng/ml) at the times shown after previous FBS starvation (2% FBS; 4 h). (a and b) Relative expression levels of BMF and BIM; L32 was used as house-keeping gene (N=2). Results are expressed as mean±S.E.M. (c) Upper panel: immunoblot of total protein extracts; β-ACTIN is presented as loading control. A representative experiment is shown (N=3). Lower panel: densitometric analysis of BIM relative levels; results are mean±S.E.M. of three independent experiments and are expressed as absolute values. Statistical comparison was done by two-way ANOVA with Sidak post-hoc test as shown in the figure: *P<0.05, **P<0.01, ***P<0.001

Figure 4

NOX4 upregulation after TGF-β treatment in HCC cell lines is decreased by CAV1. HLE shControl, HLE shCAV1, Huh7 +pControl and Huh7 +pCAV1 were treated with TGF-β (2 ng/ml) at the times shown after previous FBS starvation (2% FBS; 4 h). (a and b) Relative expression levels of NOX4; L32 was used as house-keeping gene (N=3). Results are expressed as mean±S.E.M. (c and d) Immunoblot of total protein extracts; β-ACTIN was used as loading control. A representative experiment is shown, whose quantification (densitometry of the specific band relative to the β-ACTIN levels) is incorporated below the band. (e and f) Amplex Ultra Red Fluorescence as a measure of ROS production, expressed as relative percentage versus untreated HLE shControl cells or Huh7 pControl cells (N=6 from two independent experiments). Statistical comparison was done using two-way ANOVA with Sidak post-hoc test as shown in the figure: *P<0.05, **P<0.01, ***P<0.001

Figure 5

CAV1 facilitates EGFR/AKT survival axis in HCC cells. HLE shControl, HLE shCAV1, Huh7 +pControl and Huh7 +pCAV1 were treated with TGF-β (2 ng/ml in a or 5 ng/ml in b) or HB-EGF (20 ng/ml) at the times shown after previous FBS starvation (2% FBS; 4 h). (a and b) Immunoblot of total protein extracts; α-TUBULIN was used as loading control. A representative experiment is shown

Figure 6

Analysis of CAV1 and TGFB1 expression in HCC tumoural and non-tumoural tissues. qRT-PCR analysis of mRNA levels of CAV1 in non-tumoural and tumoural tissue. (a) Detail of the relative expression of each one of the HCC tumour tissues analysed versus its respective surrounding tissue (n=65). Black line represents cut-off (relative expression=1). (b) Each dot represents relative expression of each HCC tumour tissue versus its respective surrounding tissue. Statistical comparison was done using Wilcoxon matched-pairs signed rank test: *P<0.05. (c) Spearman correlation analysis among TGFB1 and CAV1 expression analysed by qRT-PCR in the cohort of 65 samples from HCC patients

Figure 7

CAV1 orchestrates the TGF-β response in HCC. Whereas in healthy liver, CAV1 levels are very low, its expression increases through the hepatocarcinogenic process, being very high in poorly differentiated liver tumour cells (i.e., HLE). In those late stage liver tumour cells, high expression of CAV1 inhibits TGF-β-induced apoptosis through its interaction with the EGFR pathway, which impairs NOX4 upregulation by TGF-β and BMF-dependent apoptosis