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. 2017 May 4;13(5):900-913.
doi: 10.1080/15548627.2017.1291479.

SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

Hong Liu  1 , Yan Ma  1 , Hong-Wei He  1 , Wu-Li Zhao  1 , Rong-Guang Shao  1
Affiliations

SPHK1 (sphingosine kinase 1) induces epithelial-mesenchymal transition by promoting the autophagy-linked lysosomal degradation of CDH1/E-cadherin in hepatoma cells

Hong Liu et al. Autophagy. .

Abstract

SPHK1 (sphingosine kinase 1), a regulator of sphingolipid metabolites, plays a causal role in the development of hepatocellular carcinoma (HCC) through augmenting HCC invasion and metastasis. However, the mechanism by which SPHK1 signaling promotes invasion and metastasis in HCC remains to be clarified. Here, we reported that SPHK1 induced the epithelial-mesenchymal transition (EMT) by accelerating CDH1/E-cadherin lysosomal degradation and facilitating the invasion and metastasis of HepG2 cells. Initially, we found that SPHK1 promoted cell migration and invasion and induced the EMT process through decreasing the expression of CDH1, which is an epithelial marker. Furthermore, SPHK1 accelerated the lysosomal degradation of CDH1 to induce EMT, which depended on TRAF2 (TNF receptor associated factor 2)-mediated macroautophagy/autophagy activation. In addition, the inhibition of autophagy recovered CDH1 expression and reduced cell migration and invasion through delaying the degradation of CDH1 in SPHK1-overexpressing cells. Moreover, the overexpression of SPHK1 produced intracellular sphingosine-1-phosphate (S1P). In response to S1P stimulation, TRAF2 bound to BECN1/Beclin 1 and catalyzed the lysine 63-linked ubiquitination of BECN1 for triggering autophagy. The deletion of the RING domain of TRAF2 inhibited autophagy and the interaction of BECN1 and TRAF2. Our findings define a novel mechanism responsible for the regulation of the EMT via SPHK1-TRAF2-BECN1-CDH1 signal cascades in HCC cells. Our work indicates that the blockage of SPHK1 activity to attenuate autophagy may be a promising strategy for the prevention and treatment of HCC.

Keywords: BECN1/Beclin 1; HepG2 cells; TRAF2; autophagy; invasion and metastasis.

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Figures

Figure 1.
Figure 1.
SPHK1 stimulates the EMT in both HCC tissues and cells. (A, B) The expression of SPHK1 and CDH1 was correlated with malignancy grade in HCC tissues. The expression of SPHK1 was analyzed by staining (IHC) as indicated in Materials and Methods. (C, D) SPHK1 promotes cell migration and invasion. HepG2 cells stably expressing vector or MYC-SPHK1 were plated in the upper chamber of the filters for 24 h. Then, cells that had migrated to the underside of the transwell insert were counted. (E) SPHK1 overexpression decreased the levels of epithelial markers and increased the level of mesenchymal markers. HepG2 cells stably expressing vector or MYC-SPHK1 were harvested and the level of epithelial or mesenchymal markers was detected by western blot analysis. ACTB served as the loading control. The value for the vector was set to 1.0 and the other values were normalized. (F) SPHK1 overexpression decreased the expression of CDH1 and increased the expression of VIM. HepG2 cells stably expressing vector or MYC-SPHK1 were fixed in 4% paraformaldehyde and stained for CDH1 or VIM, and imaged by confocal microscopy, scale bar: 20 μm. Data are presented as the mean ± SE (n = 4).
Figure 2.
Figure 2.
SPHK1 promotes the lysosomal degradation of CDH1. (A) SPHK1 decreased the expression of CDH1. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector and protein lysates were analyzed by immunoblotting with the indicated antibodies. (B) SPHK1 did not affect the level of CDH1 mRNA in HepG2 cells. Cells were transfected with the indicated concentrations of MYC-SPHK1 or vector, and total RNA was isolated. CDH1 mRNA was analyzed by fluorescent quantitative RT-PCR, as indicated in Materials and Methods. (C) SPHK1 inhibits the degradation of CDH1 in HepG2 cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with pCMV6-CDH1 for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were detected by western blotting using an anti-CDH1 antibody. (D) SPHK1 did not affect the proteasomal degradation of CDH1. HepG2 cells were transfected with pCMV6-CDH1 for 24 h. Cells were treated with MG132 (10 μmol/L) for 2 h, and then also treated with CHX for the indicated times. Immunoblotting was performed with the indicated antibody. (E) SPHK1 accelerated the lysosomal degradation of CDH1. Cells were transfected with pCMV6-CDH1 for 24 h. HepG2 cells were treated with CQ (100 μmol/L) for 12 h, and then CHX was added for the indicated times. Immunoblotting was performed with the indicated antibody. Data are presented as the mean ± SE (n = 4). NS, nonsignificant; CHX, cycloheximide; CQ, chloroquine.
Figure 3.
Figure 3.
SPHK1 stimulates autophagy in HepG2 cells. (A, B) SPHK1 increased the number of autophagosomes (APs) in HepG2 cells. Electron microscopy revealed typical autolysosomes as observed in SPHK1-overexpressing cells (indicated by the red arrowhead). Typical mitochondrion is indicated by the green arrowhead. Magnification x 10,000–50,000. The number of autophagosomes was quantified as described in Materials and Methods. (C) SPHK1 overexpression upregulated the expression of autophagy-related proteins. HepG2 cells stably expressing vector or MYC-SPHK1 were harvested and autophagy-related proteins were detected by western blot analysis. (D) SPHK1 overexpression aggregated MAP1LC3 foci. HepG2 cells stably expressing vector or MYC-SPHK1 were fixed in 4% paraformaldehyde, stained with fluorochromes, and imaged by confocal microscopy, scale bar: 20 μm. MAP1LC3 foci per cell were quantified as described in Materials and Methods. (E) Treatment with CQ augmented the expression of MAP1LC3-II. HepG2 cells expressing either vector or MYC-SPHK1 were treated with or without CQ (100 μmol/L) for 12 h, and the expression of MAP1LC3 was measured by western blotting. Data are presented as the mean ± SE of 4 independent assays. N, nucleus; CQ, chloroquine.
Figure 4.
Figure 4.
SPHK1 induces the EMT by stimulating autophagy. (A) The inhibition of autophagy recovered the expression of epithelial markers and mesenchymal markers in SPHK1-overexpressing HCC cells. HepG2 cells stably expressing vector or MYC-SPHK1 were treated with autophagic inhibitors MHY1485 (2 μmol/L, 6 h), SBI-0206965 (10 μmol/L, 2 h), 3-MA (10 mmol/L, 6 h) and CQ (100 μmol/L, 12 h) and then harvested. EMT-related protein expression was detected by western blot analysis. (B) Suppression of autophagy reduced cell migration in SPHK1-overexpressing HCC cells. HepG2 cells stably expressing vector or MYC-SPHK1 were plated in the upper chamber of transwell filters for 24 h and then treated with 3-MA (10 mmol/L, 6 h) or CQ (100 μmol/L, 12 h). Then cells migrating to the underside of the transwell insert were measured. (C) Suppression of autophagy reduced the invasion of SPHK1-overexpressing cells. The transwell invasion assay was performed as described in (B), except that the chambers were coated with basement membrane Matrigel. (D, E) Statistical analysis of cells per field in (B) and (C). The cells per field were quantified as described in Materials and Methods. MHY, MHY1485; SBI, SBI-0206965; CQ, chloroquine.
Figure 5.
Figure 5.
SPHK1 activates autophagy through TRAF2. (A, B) Silencing TRAF2 decreased the dots of MAP1LC3 in SPHK1-overexpressing cells. HepG2 cells were transfected with control-siRNA or si-TRAF2 and then fixed in 4% paraformaldehyde, stained with fluorochromes, and imaged by confocal microscopy; scale bar: 20 μm. (C) TRAF2 is necessary for the activation of autophagy when SPHK1 is overexpressed. The HepG2 cells were transfected with control-siRNA or si-TRAF2. Cell lysates were collected, and the expression of autophagy-related proteins was analyzed by western blotting. Data are presented as the mean ± SE of 4 independent assays. Con-siRNA, control-siRNA.
Figure 6.
Figure 6.
SPHK1 induces the lysine 63-linked ubiquitination of BECN1 through TRAF2. (A-C) SPHK1 induced the lysine 63-linked ubiquitination of BECN1 and did not affect the lysine 48-linked ubiquitination of BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with a plasmid encoding HA-BECN1. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-ubiquitin (A), anti-K63-specific ubiquitin (B) or anti-K48-specific ubiquitin (C). (D) TRAF2 was imperative for SPHK1 to induce the lysine 63-linked ubiquitination of BECN1. HepG2 cells were cotransfected with a plasmid encoding HA-BECN1 and control-siRNA (or si-TRAF2); then, the cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then examined with anti-K63-specific ubiquitin antibody. WT, wild type; Ub, ubiquitin; Con-siRNA, control-siRNA.
Figure 7.
Figure 7.
SPHK1 stimulates the lysine 63-linked ubiquitination of BECN1 to trigger autophagy by binding to TRAF2. (A) BECN1 interacted with TRAF2. Whole cell lysates were extracted from HepG2 cells and then immunoprecipitated with anti-BECN1 antibody or an equal amount of mouse IgG and blotted with anti-TRAF2 antibody. (B) SPHK1 bound to TRAF2 and BECN1. Whole cell lysates were extracted from HepG2 cells stably expressing MYC-SPHK1 and then immunoprecipitated with anti-MYC (SPHK1) antibody or an equal amount of mouse IgG and blotted with anti-BECN1 or anti-TRAF2 antibody. (C) Silencing TRAF2 inhibited the binding of SPHK1 and BECN1. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with a plasmid encoding HA-BECN1 and control-siRNA (or si-TRAF2). Whole cell extracts were immunoprecipitated with anti-MYC (SPHK1) antibody and blotted with an anti-HA (BECN1) antibody. (D) Deletion of the RING domain of TRAF2 reduced the lysine 63-linked ubiquitination of BECN1 and blocked the interaction of BECN1 with TRAF2 in SPHK1-overexpressing cells. HepG2 cells were cotransfected with a plasmid encoding HA-BECN1 and the indicated constructs of Flag-TRAF2. The cell lysates were extracted and immunoprecipitated using an anti-HA antibody. The precipitates were then analyzed with anti-K63-specific ubiquitin or anti-Flag antibody. (E) The deletion of the RING domain of TRAF2 did not induce autophagy. HepG2 cells were transfected with the indicated constructs of Flag-TRAF2 and the cell lysates were extracted. Autophagy-related proteins were detected by western blot analysis. Data are presented as the mean ± SE of 4 independent assays. Con-siRNA, control-siRNA; FL, full-length.
Figure 8.
Figure 8.
SPHK1 induces the EMT by stimulating BECN1 in HepG2 cells. (A, B) Silencing BECN1 blocked cell migration and invasion in SPHK1-overexpressing cells. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si-BECN1 and plated in the upper chamber of the filters for 24 h. Then cells migrating to the underside of the transwell insert were counted. (C) SPHK1 overexpression regulated the expression of EMT-related markers through stimulating BECN1. HepG2 cells stably expressing vector or MYC-SPHK1 were transfected with control-siRNA or si-BECN1 for 24 h and then harvested for cell lysate extraction. The level of epithelial or mesenchymal markers was detected by western blot analysis. (D) Silencing BECN1 blocked the degradation of CDH1 in SPHK1-overexpressing cells. HepG2 cells stably expressing MYC-SPHK1 were cotransfected with pCMV6-CDH1 and si-BECN1 (or control-siRNA) for 24 h and then treated with CHX (20 μmol/L) for the indicated times. The cell lysates were analyzed by western blotting using an anti-CDH1 antibody. (E) Schematic diagram of the mechanism of SPHK1-mediated autophagy and lysosomal CDH1 degradation that stimulates the EMT in HepG2 cells. Con-siRNA, control-siRNA.
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