Proprotein Convertase Subtilisin/Kexin Type 9 Promotes Gastric Cancer Metastasis and Suppresses Apoptosis by Facilitating MAPK Signaling Pathway Through HSP70 Up-Regulation

Abstract

Objective: To examine the effect of proprotein convertase subtilisin/kexin type 9 (PCSK9) on gastric cancer (GC) progression and prognosis, and to explore the underlying mechanism.

Methods: PCSK9 expression levels in human GC tissues were determined by quantitative real-time PCR, western blotting, and immunohistochemical assay. PCSK9 serum levels were detected by enzyme-linked immunosorbent assay. The relationships of PCSK9 and GC progression and survival were analyzed using the Chi-square test, Kaplan-Meier analysis, and Cox proportional hazards model. The effect of PCSK9 on cell invasion, migration, and apoptosis were determined in human GC cell lines and mouse xenograft model separately using PCSK9 knockdown and overexpression strategies. The PCSK9 interacting molecules, screened by co-immunoprecipitation combined with LC-MS/MS, were identified by immunofluorescence localization and western blotting. Additionally, the mitogen-activated protein kinase (MAPK) pathway was assessed by western blotting.

Results: PCSK9 mRNA and protein levels were significantly elevated in GC tissues compared with the paired normal tissues at our medical center (P < 0.001). Notably, the up-regulation of PCSK9 expression in GC tissues was related to tumor progression and poor survival. GC patients had higher serum levels of PCSK9 than the age-matched healthy controls (P < 0.001); PCSK9 promoted invasive and migratory ability and inhibited apoptosis in GC cells with no apparent affection in cell proliferation. The silencing of PCSK9 reversed these effects, suppressing tumor metastasis in vitro and in vivo. Furthermore, PCSK9 maintained these functions through up-regulating heat shock protein 70 (HSP70), ultimately facilitating the mitogen-activated protein kinase (MAPK) pathway.

Conclusion: Collectively, our data revealed that high PCSK9 expression levels in GC tissue were correlated with GC progression and poor prognosis and that PCSK9 could promote GC metastasis and suppress apoptosis by facilitating MAPK signaling pathway through HSP70 up-regulation. PCSK9 may represent a novel potential therapeutic target in GC.

Keywords: MAPK pathway; gastric cancer; heat shock protein 70; prognosis; proprotein convertase subtilisin/kexin type 9.

Figure 1

Increase of PCSK9 expression in GC. (A) PCSK9 mRNA expression levels assessed in paired GC and adjacent normal tissues (P < 0.0001; n = 60) (B) PCSK9 protein expression levels measured in GC tissues and related adjacent non-tumorous tissues in representative eight patients by western blotting; β-actin served as the internal control. (C) Comparison of relative PCSK9 protein expression in 40 GC tissues and adjacent non-tumorous tissues by western blotting (P < 0.001). (D) Serum levels of PCSK9 analyzed by ELISA in GC patients (n = 60) and healthy volunteers (HV; n = 30; P < 0.0001). (E) Representative IHC image of PCSK9 protein in GC and adjacent normal tissues. (F) Distribution of PCSK9 IHC score in GC and adjacent normal tissues (P < 0001). (G) IHC score in GC and adjacent normal tissues (n = 155; P < 0.001). (H) Overall survival curve for GC patients with high vs. low expression of PCSK9 IHC score generated with Kaplan-Meier methods (P < 0.01).

Figure 2

PCSK9 promoted migration and invasion and inhibited apoptosis of GC cells in vitro. (A) Relative PCSK9 expression in a panel of GC compared with GES1 cells, as determined by quantitative real-time PCR. (B) Western blotting of PCSK9 expression in eight cell lines; β-actin served as the internal control. (C, D) PCSK9 levels verified by western blotting; β-actin as the internal control. (E) Wound healing assay in SGC-7901 with/without PCSK9 knockdown; scale bar 100 μm. (F) Wound healing assay in MGC-803 cells with/without PCSK9 overexpression; scale bar 100 μm. (G) Transwell migration assay of SGC-7901 with/without PCSK9 knockdown; scale bar 100 μm. (H) Transwell migration assay of MGC-803 with/without PCSK9 overexpression; scale bar 100 μm. (I) Transwell invasion assay of SGC-7901 with/without PCSK9 knockdown; scale bar 100 μm. (J) Transwell invasion assay of MGC-803 with or without PCSK9 overexpression; scale bar 100 μm. (K, L) The cells collected and stained with FITC-Annexin V and propidium iodide; the percentage of apoptotic cells analyzed by flow cytometry assay; representative images presented. Data are expressed as mean ± S.D.; **P < 0.01, ***P < 0.001; WT, wild type; NC, negative control; OE, overexpression.

Figure 3

PCSK9 interacting with HSP70 and modulating MAPK pathway. (A) HSP70 identified by LC-MS/MS as interactor of PCSK9. (B) PCSK9 interacting with HSP70 in GC cell line through co-immunoprecipitation. (C) Confocal laser-scanning detecting the expression of PCSK9 and HSP70 in GC cells. (D) Correlations of PCSK9 and HSP70 protein expression in GC tissues based on immunoscoring. (E) The cells pretreated with TRC051384(10 μM) for 4 h; wound healing assay conducted in PCSK9 shRNA SGC-7901 cells with/without HSP70 agonist and NC cells. (F) The cells pretreated with apoptozole (2 μM) for 24 h; wound healing assay conducted in MGC-803 OE cells with/without apoptozole and MGC-803 control cells. (G) Transwell migration assay of PCSK9 shRNA SGC-7901 cells with/without HSP70 restoration and NC cells. (H) Transwell migration assay of MGC-803 OE cells with/without apoptozole and MGC-803 control cells. (I) Transwell invasion assay of PCSK9 shRNA SGC-7901 cells with/without HSP70 restoration and NC cells. (J) Transwell invasion assay of MGC-803 OE cells with/without apoptozole and MGC-803 control cells. (K) Apoptosis of SGC-7901, measured by flow cytometry. (L) Apoptosis of MGC-803 cells, measured by flow cytometry. (M) PCSK9 up-regulating HSP70 expression and activating MAPK pathway (Right: SGC-7901 cells; Left: MGC-803 cells); β-actin served as the internal control. The statistical significance between different groups was calculated with Student’s t-test; data expressed as mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001; NC, negative control; A, agonist; I, inhibitor.

Figure 4

Silencing of PCSK9 suppressing the GC tumor metastasis in a mouse xenograft model and HSP70 agonist reversing this countervailing effect. (A) Mouse weight measured once a week at the indicated time points after injection with PCSK9 knockdown and control SGC-7901 cells until the 5^th week. (B) Representative images of lungs and PET scan of different groups (white circles represent suspected lesions). (C) Representative images of hematoxylin and eosin (H&E) staining of metastatic lung nodules from different groups. (D) Metastatic nodules in lungs of orthotopic xenograft mice model calculated (n = 10/group); data presented as mean ± S.D.; NS, not significant; **P < 0.01, ***P < 0.001.