Knockdown of Golgi phosphoprotein 73 blocks the trafficking of matrix metalloproteinase‐2 in hepatocellular carcinoma cells and inhibits cell invasion

Abstract Golgi phosphoprotein 73 (GP73) has been regarded as a novel serum biomarker for the diagnosis of hepatocellular carcinoma (HCC) in recent years. It has been reported that the upregulation of GP73 may promote the carcinogenesis and metastasis of HCC; however, the mechanisms remain poorly understood. In this study, GP73 correlates positively with matrix metalloproteinase‐2 (MMP‐2) in HCC‐related cells and tissues. Further studies indicate that the knockdown of GP73 blocks MMP‐2 trafficking and secretion, resulting in cell invasion inhibition. Additionally, the knockdown of GP73 induces the accumulation of intracellular MMP‐2, which inhibits the phosphorylation of Src at Y416 and triggers the inhibition of SAPK/JNK and p53‐p21 signalling pathways through a negative feedback loop. Finally, the transactivation of MMP2 was inhibited by the reduction in E2F1. This study reveals that GP73 plays functional roles in the trafficking and equilibrium of epithelial‐mesenchymal transition (EMT)‐related secretory proteins and that GP73 serves as a new potential target for combating the metastasis of HCC.


| INTRODUCTION
Hepatocellular carcinoma (HCC) is the third leading cause of cancerrelated death worldwide, with limited treatment options. [1][2][3] HCV infections increase the occurrence of HCC, and statistically, more than 80% of HCC patients are HBV or HCV infected. 2,3 Recurrence and metastasis are the main causes of HCC-related deaths. 4 HCC cells metastasize rapidly through the vasculature, and intrahepatic metastasis is the common form of metastasis. 5 Robust estimates suggest that approximately 70% of HCC patients will relapse within 5 years following surgery. 6 Therefore, it is critical to diagnose and control HCC metastasis as soon as possible to extend the survival time of HCC patients.
GP73 is a highly phosphorylated protein located in the Golgi apparatus that can also be cleaved and secreted into serum. 7,8 GP73 is highly expressed in several kinds of cancers, including HCC, nonsmall cell lung cancer, breast carcinoma, pancreatic carcinoma, melanoma, and prostate carcinoma. [9][10][11][12][13] As the expression of GP73 was positively associated with the process of cancers, this protein has been regarded as a novel serum biomarker in the diagnosis of cancers, especially in HCC diagnosis. 9,14,15 Recently, it was also reported that serum GP73 facilitates the progression of HBV-related acute and chronic hepatitis, acute-onchronic liver failure, fibrosis, and other immunologically mediated liver diseases. [16][17][18][19][20] Furthermore, some studies even indicate that GP73 is a suitable diagnostic biomarker not only for HCC but also for liver cirrhosis. 21,22 Although studies indicate that GP73 is a remarkable biomarker in clinical diagnostics, its function remains unclear. In recent years, some studies have indicated that highly expressed GP73 promotes the migration and invasion of HCC, 8,23 but the molecular processes are far more complex.
MMP-2 is a critical member of the matrix metalloproteinase family, which is activated on the plasma membrane and secreted into the extracellular space, resulting in fibrillin degradation and extracellular matrix damage. [24][25][26] MMP-2 is often highly expressed in cancer cells, facilitating cell invasion. The transcription, activation, secretion, and function of MMP-2 have been studied in recent years, but the process of intracellular MMP-2 trafficking is still poorly understood. 24 In this study, the knockdown of GP73 blocked intracellular MMP-2 trafficking, resulting in cell invasion inhibition. Moreover, the knockdown of GP73 triggered SAPK/JNK and p53-p21 signalling pathways inhibition, which modulated the equilibrium of intracellular MMP-2 by regulating the transactivation of MMP2. These studies provided a potential target for combating metastatic HCC.

| Immunofluorescence and confocal microscopy
Each well of a 24-well plate was seeded with 2 × 10 4 cells and incubated for 24 hours. The remaining procedures were performed as previously reported. 23

| Transwell cell invasion assay
The cell invasion assay was performed using a 6.5 mm Transwell plate with 8.0 μm pore polycarbonate membrane-coated inserts (Corning, NY, USA). The upper chambers were coated with 15% Matrigel from Corning. The cell invasion assay was performed as described. 28 2.9 | Anchorage-independent cell colony formation assay Anchorage-independent proliferation was examined in MHCC-97H cell lines following GP73 silencing. For this assay, the cells were seeded onto 6-well plates (1 × 10 4 cells/mL) and transfected with siGP73s. The cells were digested immediately after transfection and processed as previously described. 29 The mixture was then incubated at 37°C and 5% CO 2 for 6 days, and the colonies were counted.

| Fluorescence resonance energy transfer
For the fluorescence resonance energy transfer (FRET) assay, the primers sequences for GP73 and MMP-2 expression are shown in Table S1B. The forward and reverse primers were annealed and cloned into pcDNA3.1-CFP (GP73) and pcDNA3.1-YFP (MMP-2) vectors through HindIII and SacI sites. 293T cells were seeded onto 24-well plates and transfected with 4 μg of plasmid in proportions of 1:1 using Lipofectamine 3000. Forty-eight hours later, protein-protein interactions were examined using a Nikon A1 confocal microscope (Nikon Corporation, Tokyo, Japan) with FRET.

| Immunoprecipitation
The cells were lysed in 500 μL of RIPA lysis buffer after reaching approximately 90% confluence in 15 cm dishes. The samples were centrifuged to remove insoluble debris, and the supernatant was split into 2 equal aliquots. Target-specific antibodies and anti-rabbit IgG antibodies were added. Immunoprecipitation was performed using Pierce ™ Protein A/G Magnetic Beads (Thermo Fisher) according to the manufacturer's instructions.

| Isolation of vesicles and exosomes
For vesicle isolation, cells cultured in 15 cm dishes were digested and centrifuged at 1000× g for 5 minutes. The pellet was resuspended in 1 mL 1× PBS and ground using a homogenizer. The homogenate was centrifuged at 2000× g for 5 minutes. The supernatant was collected and the vesicles were isolated by PEG6000 and ultracentrifugation as previously described. 30 For exosome isolation, the cells were cultured using serum-free DMEM for 24 hours in 5% CO 2 at 37°C. Cell culture media were collected, and the exosomes were isolated using the Exosome Isolation Kit (Thermo Fisher) following the manufacturer's instructions.
Forty-eight hours after transfection, the movement track of fusion proteins was examined using Nikon A1R confocal microscope (Nikon Corporation). Images were captured every 5 seconds for 10 minutes.

| Mapping of the binding site of GP73/MMP-2 in vitro
Truncated mutants were constructed based on the template of pCMV3-GOLM1-flag. PCR was performed with the primers shown in Table S1C. Truncated mutants and pCMV-MMP2 were transfected into 293T cells. Immunoprecipitation assays were performed as previously described. 23

| Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) analysis was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) following the manufacturer's instructions. DNA-protein complexes were precipitated using a specific antibody against E2F1.
Immunoprecipitated DNA fragments and input DNA were used as templates for chromatin immunoprecipitation and PCR (ChIP-PCR) using PrimeSTAR GXL (TaKaRa). The primers used in the ChIP-PCR analysis are listed in Table S1D.   Figure 1F). Strikingly, the correlation of GP73 and MMP-2 was closer in serum than that in HCC tissues (R 2 = 0.4698) ( Figure 1G). The data above imply that the expression of GP73 might be associated with the generation or secretion of MMP-2.

| Knockdown of GP73 blocks trafficking of intracellular MMP-2 and inhibits cell invasion
To investigate the association of GP73 and MMP-2, we silenced endogenous GP73 in HepG2 and MHCC-97H cells by using two pairs of GP73-specific siRNAs (siGP73s). Immunoblotting and immunofluorescence assays showed that the knockdown of GP73 upregulated intracellular MMP-2 (Figure 2A 32 we measured the expression of cytosolic and nuclear p-JNK1/2 (T183/Y185) after HepG2 cells were transfected with siGP73 for 72 hours. Surprisingly, the knockdown of GP73 not only inhibited the phosphorylation of p-JNK1/2 (T183/ Y185) but also blocked its nuclear translocation ( Figure 5A, B).
Importin-3 and importin-7 participated in the nuclear translocation of p-JNK1/2 (T183/Y185), 33 and it was found that expression of importin-7 was reduced in HepG2 siGP73 groups compared with siNC group ( Figure 5C). Coimmunoprecipitation and immunoblotting analysis further verified that p-JNK1/2 (T183/Y185) interacted with importin-7 ( Figure 5D). 34 Because the nuclear translocation of p-JNK1/2 (T183/Y185) was inhibited, p53-p21 signalling pathways were blocked, and the phosphorylation of p-Rb (S780) was inactivated, which led to a reduction in the expression of the transcription factor E2F1 ( Figure 5E). 35,36 3.4 | Reduction in E2F1 inhibits transactivation of The fold enrichment decreased in the siGP73-72 hours group compared with that in the siGP73-24 group, which proved that the negative feedback loop was activated and inhibited the transcription of MMP2 ( Figure 6C, D). Additionally, luciferase reporter analysis further demonstrated that transcriptional activity was inhibited in the siGP73-72 hours group, which is consistent with the results derived from the ChIP analysis ( Figure 6E).
The data above proved that accumulated intracellular MMP-2 activated a negative feedback loop and inhibited the SPAK/JNK and p53-p21 signalling pathways after GP73 silencing, resulting in the inhibition of MMP2 transactivation ( Figure 6F). In this way, the equilibrium of intracellular MMP-2 was maintained.

| DISCUSSION
In recent years, 90% of HCC-related mortality incidences correlate with metastasis. The mechanism of HCC metastasis remains complex. GP73, as a serum biomarker, has been comprehensively used in the diagnosis of HCC in recent years and has been associated with the carcinogenesis and metastasis of HCC. The knockdown of GP73 inhibited the expression of EMT-related factors, such as MMP-7, CD44 and EGFR. 8,23 However, the regulating and coordinating mechanisms are still poorly understood.
Because GP73 is located in the cis-Golgi apparatus, mechanistically, it is supposed that highly expressed GP73 might promote the metastasis of HCC through regulating the trafficking of EMT-related membrane and secretory proteins. Therefore, in our preliminary work, the content of GP73 and other EMT-related proteins was measured in exosomes derived from 5 normal or liver cancer cell lines to discover a dominant EMT-related secretory protein closely p53-p21 signalling pathways. However, it is difficult to find the reason for the reduction in importin-7. We deem that the knockdown of GP73 might block the trafficking of other membrane receptors, which inhibits the transcription of IPO7. Finally, the transactivation of MMP2 is suppressed because of the inhibition of E2F1, which maintains the equilibrium of intracellular MMP-2.
The evidence above reveals that GP73 plays a role in the trafficking and equilibrium of EMT-related secretory proteins. In addition, we propose that MMP-2 is not the only trafficking substrate of GP73 because the knockdown of GP73 strongly inhibits cell proliferation and invasion of liver cancer cell lines, which implies that not only the trafficking of MMP-2 is inhibited. We have identified more trafficking substrates of GP73, and these proteins might be reported in forthcoming studies.
MMPs, especially MMP-2 and MMP-9, are highly expressed in pathological tissues derived from patients with metastatic HCC after SIRT treatment. 40 In recent years, patients with metastatic HCC have been treated with personalized management and tailored therapy. [41][42][43] GP73 might be a potential target in tailored therapy targeting metastatic HCC.
This study also indicated that GP73 correlates positively with MMP-2 in serum derived from HCC patients. GP73 is correlated with the process of HCC, and MMP-2 is correlated with cell invasion. Because these proteins are closely related to each other, it is supposed that they can serve as serum indicators of metastatic HCC. Unfortunately, insufficient serum samples from HCC patients were collected in this study, and it is illogical to prove that these proteins can serve as proper biomarkers in the diagnosis of metastatic HCC. However, we propose that serum GP73 and MMP-2 can be excellent serum indicators for the diagnosis of metastatic HCC.
In summary, this study indicates that GP73 plays critical roles in MMP-2 trafficking. The knockdown of GP73 blocks the secretion of MMP-2 and suppresses the transactivation of MMP2 by inhibiting