Impaired degradation of YAP1 and IL6ST by chaperone-mediated autophagy promotes proliferation and migration of normal and hepatocellular carcinoma cells

Abstract

Impaired degradation of the transcriptional coactivator YAP1 and IL6ST (interleukin 6 cytokine family signal transducer), two proteins deregulated in liver cancer, has been shown to promote tumor growth. Here, we demonstrate that YAP1 and IL6ST are novel substrates of chaperone-mediated autophagy (CMA) in human hepatocellular carcinoma (HCC) and hepatocyte cell lines. Knockdown of the lysosomal CMA receptor LAMP2A increases protein levels of YAP1 and IL6ST, without changes in mRNA expression. Additionally, both proteins show KFERQ-dependent binding to the CMA chaperone HSPA8 and accumulate into isolated lysosomes after stimulation of CMA by prolonged starvation. We further show that LAMP2A downregulation promotes the proliferation and migration in HCC cells and a human hepatocyte cell line, and that it does so in a YAP1- and IL6ST-dependent manner. Finally, LAMP2A expression is downregulated, and YAP1 and IL6ST expression is upregulated, in human HCC biopsies. Taken together, our work reveals a novel mechanism that controls the turnover of two cancer-relevant proteins and suggests a tumor suppressor function of CMA in the liver, advocating for the exploitation of CMA activity for diagnostic and therapeutic purposes.Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; ATG7: autophagy related 7; CMA: chaperone-mediated autophagy; eMI: endosomal microautophagy; HCC: hepatocellular carcinoma; HSPA8: heat shock protein family A (Hsp70) member 8; IL6ST: interleukin 6 cytokine family signal transducer; JAK: Janus kinase; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; MAPK8: mitogen-activated protein kinase 8; P6: pyridine 6; SQSTM1: sequestosome 1; TUBA: tubulin alpha; VDAC1: voltage dependent anion channel 1; VP: verteporfin; YAP1: Yes1 associated transcriptional regulator.

Keywords: Chaperone-mediated autophagy; IL6ST; KFERQ motif; LAMP2A; YAP1; hepatocellular carcinoma; migration; proliferation; protein degradation.

Figure 1.

YAP1 and IL6ST are degraded via the lysosomal pathway. (A,B) Treatment of Hep3B cells for 6 h with the lysosomal inhibitor(s) NH₄Cl (20 mM) and leupeptin (50 μM) (A) or with a combination of the two for the indicated time (B) leads to the accumulation of YAP1 and IL6ST in Hep3B cells. SQSTM1 is shown as a positive control. The bottom panel show the fold change of YAP1 and IL6ST over their expression in untreated cells, set as 1. (C,D) Treatment of Hep3B cells for 6 h with the indicated concentration of the proteasome inhibitor MG132 (C) or for the indicated time with 5 μM MG132 (D) has moderate to no effects on YAP1 and IL6ST levels. The bottom panel show the fold change of YAP1 and IL6ST over their expression in cells treated with DMSO, set as 1. (E,F) Silencing of either ATG5 or ATG7 does not cause the accumulation of YAP1 and IL6ST in Hep3B cells. SQSTM1 is shown as a positive control. The bottom panels show the fold change of YAP1 and IL6ST over their expression in cells transfected with siScr, set as 1. (G) Treatment of Hep3B cells for 24 h with the indicated concentrations of the macroautophagy-inducing drug rapamycin fails to reduce the levels of YAP1 and IL6ST in Hep3B cells. SQSTM1 is shown as a positive control. The bottom panel shows the fold change of YAP1 and IL6ST over their expression in cells treated with DMSO, set as 1. In all panels, the intensity of the bands of interest was normalized to TUBA prior to fold-change calculations. Data are plotted as the mean ± SEM of n = 3 biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

Figure 2.

Modulation of CMA influences YAP1 and IL6ST protein levels. (A) Prolonged starvation reduces protein levels of YAP1 and IL6ST in Hep3B cells. The panel on the right shows the fold change of YAP1 and IL6ST over their expression in cells grown in FM, set as 1. (B) Overnight starvation reduces protein levels of YAP1 and IL6ST in mouse livers. The panel on the right shows the fold change of YAP1 and IL6ST over their expression in fed mice; lane 1 was set as 1. (C) Treatment of Hep3B cells with 10 μM of AKT inhibitor decreases YAP1 and IL6ST levels. The panel on the right shows the fold change of YAP1 and IL6ST over their expression in cells treated with DMSO, set as 1. (D) Treatment of Hep3B for 24 h with the indicated concentrations of the atypical RARA receptor antagonist AR7 decreases YAP1 and IL6ST levels. The panel on the right shows the fold change of YAP1 and IL6ST over their expression in cells treated with DMSO, set as 1. (E) Silencing of LAMP2A causes the accumulation of YAP1 and IL6ST in Hep3B cells. The panel on the right shows the fold change of YAP1 and IL6ST over their expression in cells transfected with siScr, set as 1. (F) Silencing of LAMP2A has no effect on the mRNA levels of YAP1 and IL6ST. Data are plotted as the mean ± SEM of n = 3 biological replicates. ACTB was used as a housekeeping gene. (G) Silencing of LAMP2A prevents starvation-induced YAP1 and IL6ST degradation. Cells were cultured without serum for 24 h. The panel on the right shows the fold change of YAP1 and IL6ST over their expression in cells transfected with siScr, set as 1. The intensity of the bands of interest was normalized to TUBA (panels A-E and G), prior to fold-change calculations. Data are plotted as the mean ± SEM of n = 3 biological replicates. *p < 0.05, **p < 0.01 vs control.

Figure 3.

YAP1 and IL6ST are bona fide CMA substrates. (A,B) Graphical representation of the position of the putative KFERQ motif within the human YAP1 (A) and IL6ST (B) sequences. (C) FLAG-YAP1 WT, but not FLAG YAP1 AALLR, is detectable in HSPA8 immunoprecipitates. (D) More HSPA8 is detectable in HA-IL6ST WT than in IL6ST AAERF immunoprecipitates. (E,F) Mutation of the KFERQ motif increases the expression of YAP1 (E) and (F) IL6ST and renders them insensitive to treatment with lysosomal inhibitors (NL; 20 mM NH₄Cl and 50 μM leupeptin, 6 h). The left bottom panel shows the fold change of YAP1 (E) and IL6ST (F) over the expression of the untreated WT, set as 1. The right bottom panel shows the fold change of YAP1 (E) and IL6ST (F) over the expression of the same construct in the untreated cells, set as 1. (G,H) Starvation induces complex formation between YAP1 (G) or IL6ST (H) and LAMP2A. (I) Starvation promotes the accumulation of YAP1 and IL6ST, and the canonical CMA substrate GAPDH in lysosomes isolated from Hep3B cells. The bottom panel shows the fold change of YAP1 and IL6ST over their expression in cells grown in FM. (J) Silencing of LAMP2A, but not ATG7, reduces starvation-induced lysosomal localization of YAP1 and IL6ST. Cells were cultured without serum for 24 h. The bottom panel shows the fold change of YAP1 and IL6ST over their expression in cells transfected with siScr. The intensity of the bands of interest was normalized to TUBA (panels E and F) or LAMP1 (panels I and J) prior to fold-change calculations. Data are plotted as the mean ± SEM of n = 3 biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 vs control.

Figure 4.

Inhibition of CMA promotes cell proliferation and migration. (A,B) Inhibition of CMA increases proliferation of HuS cells, measured by live cell counting (A) and crystal violet staining (B). (C,D) Inhibition of CMA increases proliferation of Hep3B cells, measured by cell counting (C) and crystal violet staining (D). (E,F) Inhibition of CMA increases migration of HuS cells, measured by the wound healing (E) and transwell (F) assays. (G,H) Inhibition of CMA increases migration of Hep3B cells, measured by the wound healing (G) and transwell (H) assays. Data are plotted as the ± SEM of n = 3 biological replicates. *p < 0.05, **p < 0.01 vs siScr. (I) LAMP2A, YAP1 and IL6ST protein expression in human HCC samples (T) compared to matched adjacent normal tissues (N). (J) Box and whiskers plot of densitometry analysis of LAMP2A, YAP1 and IL6ST protein expression normalized to ACTB in n = 12 human HCC samples and matched adjacent normal tissues. Data are compared using the paired samples Wilcoxon test for not normally distributed data.

Figure 5.

YAP1 and IL6ST contribute to the growth advantage and increased motility induced by CMA downregulation. (A,C) Silencing of either YAP1 or IL6ST restrains the increased proliferation of LAMP2A knockdown HuS (A) and Hep3B (C) cells. (B,D) Silencing of either YAP1 or IL6ST restrains the increased migration of LAMP2A knockdown HuS (B) and Hep3B (D) cells, measured by the wound healing assay. Data are plotted as the ± SEM of n = 3 biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001 vs siScr.