Sec62 promotes gastric cancer metastasis through mediating UPR-induced autophagy activation

Abstract

Background and aims: Sec62 is a membrane protein of the endoplasmic reticulum that facilitates protein transport. Its role in cancer is increasingly recognised, but remains largely unknown. We investigated the functional role of Sec62 in gastric cancer (GC) and its underlying mechanism.

Methods: Bioinformatics, tissue microarray, immunohistochemistry (IHC), western blotting (WB), quantitative polymerase chain reaction (qPCR), and immunofluorescence were used to examine the expression of target genes. Transwell, scratch healing assays, and xenograft models were used to evaluate cell migration and invasion. Transmission electron microscopy and mRFP-GFP-LC3 double-labeled adenoviruses were used to monitor autophagy. Co-immunoprecipitation (CO-IP) was performed to evaluate the binding activity between the proteins.

Results: Sec62 expression was upregulated in GC, and Sec62 upregulation was an independent predictor of poor prognosis. Sec62 overexpression promoted GC cell migration and invasion both in vitro and in vivo. Sec62 promoted migration and invasion by affecting TIMP-1 and MMP2/9 balance. Moreover, Sec62 could activate autophagy by upregulating PERK/ATF4 expression and binding to LC3II with concomitant FIP200/Beclin-1/Atg5 activation. Furthermore, autophagy blockage impaired the promotive effects of Sec62 on GC cell migration and invasion, whereas autophagy activation rescued the inhibitory effect of Sec62 knockdown on GC metastasis. Notably, Sec62 inhibition combined with autophagy blockage exerted a synergetic anti-metastatic effect in vitro and in vivo.

Conclusion: Sec62 promotes GC metastasis by activating autophagy and subsequently regulating TIMP-1 and MMP2/9 balance. The activation of autophagy by Sec62 may involve the unfolded protein response (UPR)-related PERK/ATF4 pathway and binding of LC3II during UPR recovery involving FIP200/Beclin-1/Atg5 upregulation. Specifically, the dual inhibition of Sec62 and autophagy may provide a promising therapeutic strategy for GC metastasis.

Keywords: EMT; ER stress; ER-phagy; Protein translocation machinery; Unfolded protein response (UPR).

Fig. 1

Upregulation of Sec62 in GC predicts poor survival. Volcano plots of mass spectrometry data showed the top 10 upregulated molecules identified in GC tissues versus controls (A). Sec62 mRNA expression was significantly higher in GC than in other human malignancies (B) and adjacent normal stomach tissues (C, D) in the GEPIA database. Correlation of Sec62 mRNA expression in GC patients with OS in KM-plotter database (E). Quantitative PCR (F) and WB (G) assays suggested that Sec62 expression in GC cell lines was much higher than that in GES. In GC tissues and adjacent normal tissues, qRT-PCR (H) and WB (I) assays also confirmed higher Sec62 levels in GC tissues than in normal controls. IHC assays of Sec62 protein expression in GC and adjacent normal tissue (J). Chi-squared test for Sec62 expression in GC and adjacent normal tissues (K). Kaplan–Meier analysis using tissue microarray data showed a correlation between Sec62 protein levels and the OS of GC patients (L). **p < 0.01, ***p < 0.0001

Fig. 2

Upregulation of Sec62 promotes GC cells migration and invasion in vitro and in vivo. After lentivirus transfection, WB assays verified the protein expression of Sec62 in three GC cell lines, namely, AGS (A), MKN45 (B) and GES (C). Transwell assays showed that Sec62 significantly promoted cell migration and invasion in AGS (D), MKN45 (E), and GES (F) cell lines. Scratch healing assays confirmed that upregulation of Sec62 enhanced GC cell migration in GC cell lines (G–I). Representative images of lung tissues from nude mice (J). Hematoxylin and eosin staining of the lungs and liver (K). The incidence of lung metastasis (L) and the number of metastatic nodules in the lungs (M) between the two groups**p < 0.01, ***p < 0.0001

Fig. 3

Sec62 affects TIMP-1 and MMP 2/9 expression instead of EMT. Western blotting showed that Sec62 expression was positively associated with MMP2 and MMP9 in cell lysates, while no correlation was found between Sec62 and EMT markers such as E-cadherin or vimentin (A, B). Immunofluorescence staining confirmed no correlations between Sec62 and E-cadherin or vimentin in AGS (C), MKN45 (D), and GES (E) cell lines. ELISA showed that Sec62 expression was positively correlated with the activity of MMP2 and MMP9, and negatively associated with TIMP-1 activity in GC cell culture supernatant (F–H). Gelatin zymography demonstrated Sec62 upregulated the activity of MMP2/9 in the supernatant of cell medium (I). *p < 0.05, **p < 0.01, ***p < 0.0001

Fig. 4

Sec62 regulates PERK/ATF4 expression and activates autophagy accompanied by FIP200/Beclin-1/Atg5 upregulation. The WB assay demonstrated that Sec62 could affect PERK and ATF4 expression and showed no effect on IRE1, JNK, and ATF6 (A, B). Sec62 correlated with autophagy activation indicated by LC3 II, and P62 was identified via WB assays (C, D). Transmission electron microscopy revealed more autophagolysosomes (red arrows) or autophagosomes (blue arrows) in GC cells with higher Sec62 expression (E). Immunofluorescence assays after mRFP–GFP–LC3 double-labeled adenovirus transfection indicated increased LC3II expression (red or yellow dots) in GC cells with higher Sec62 levels (F, G). The CO-IP assay showed that exogenous FLAG-tagged Sec62 (the lentivirus bears a FLAG tag) is associated with endogenous LC3II (H). Immunofluorescence co-localisation assays also supported the relationship between Sec62 and LC3II. The Manders Overlap Coefficient (MOC) was 0.937 (I). Western blotting revealed a positive correlation between Sec62 and FIP200, Beclin-1, and Atg5 expression (J–K). *p < 0.05, **p < 0.01, ***p < 0.0001

Fig. 5

Autophagy blockage induced by HCQ was confirmed and correlated with repressed MMP2 and MMP9 expression. After HCQ (25 uM) treatment for 24 h, WB blotting showed that autophagy was blocked, as indicated by LC3II and P62. In addition, both MMP2 and MMP9 expression was also downregulated after HCQ treatment (A–C). Transmission electron microscopy showed more autophagosomes (blue arrows) and lysosomes (yellow arrows) in the HCQ-treatment groups (D–F). Immunofluorescence assays demonstrated upregulated LC3II expression (yellow dots) in GC cells after MRFP-GFP-LC3 adenovirus transfection (G–I). HCQ hydroxychloroquine, blue arrows: autophagosomes; red arrows, autophagolysosomes; yellow arrows, lysosomes; *p < 0.05, **p < 0.01, ***p < 0.0001

Fig. 6

Autophagy blockage inhibited GC metastasis in vitro and in vivo. Sec62 inhibition combined with autophagy blockage exerts synergetic anti-metastatic effects on GC cells. Transwell assays demonstrated the inhibitory effects of autophagy blockage induced by HCQ on GC cell migration and invasion (A–C and G–I). Scratch healing assays confirmed that GC cell migration was inhibited after HCQ treatment (D–F and J–L). In vivo rescue experiments using HCQ (50 mg/kg) intraperitoneal injection of nude mice verified that autophagy inhibition could rescue the promotive effect of Sec62 on GC metastasis. Hematoxylin and eosin staining of the lungs (M). The incidence of lung metastasis (N) and the number of metastatic nodules in the lungs (O) in diverse groups. HCQ hydroxychloroquine. *p < 0.05, **p < 0.01, ***p < 0.0001

Fig. 7

Schematic diagram for the pro-metastatic role of Sec62 in GC. Schematic diagram of the role of Sec62 in GC metastasis. Sec62 activation of autophagy may involve two pathways, namely, the UPR-related PERK/ATF4 pathway and binding of LC3II during UPR recovery involving FIP200/Beclin-1/Atg5 upregulation. Autophagy subsequently promotes GC metastasis by regulating the TIMP-1 and MMP2/9 balance