VRK2 promotes HCC metastasis through stabilizing ATG5 to activate autophagy-mediated degradation of ZO-1

VRK2 promotes HCC metastasis through stabilizing ATG5 to activate autophagy-mediated degradation of ZO-1 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article VRK2 promotes HCC metastasis through stabilizing ATG5 to activate autophagy-mediated degradation of ZO-1 Mao Ye, Jingying Zhang, Binglong Bai, Maolin Zhang, Tiantian Wang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6357129/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Vaccinia-related kinase 2 (VRK2) is involved in the process of multiple cancers, but its role in the metastasis of hepatocellular carcinoma (HCC) is largely unknown. Here, we have found that VRK2 was markedly overexpressed in HCC, high expression of VRK2 was positively correlated with poor prognosis. Functionally, silencing VRK2 caused a decrease of HCC cell’s invasion and metastasis in vitro and vivo, whereas VRK2 overexpression increased the invasion and metastasis abilities of HCC cells. What’s more, we revealed that the pro-metastatic function of VRK2 was mediated by tight junction protein ZO-1 (Zonula occluden-1), ZO-1 overexpression attenuated the increase of HCC cell’s invasion and metastasis abilities induced by VRK2 overexpression. Notably, we surprised to find that VRK2 promoted the degradation of ZO-1 protein through autophagic degradation pathway instead of known ubiquitin proteasome pathway. Mechanically, phosphorylation mass spectrometry identified ATG5, a E3 conjugating enzyme which catalyze the lipidation of LC3, as a new substrate of VRK2. VRK2 phosphorylates ATG5 at serine 106 and protects it from ubiquitin-dependent proteasomal degradation by enhancing the interaction of ATG5 with USP13, promote autophagy-mediated degradation of ZO-1. The regulatory axis involving VRK2, ATG5, autophagy and ZO-1 also existed in HCC tissue further proved that VRK2 might be a promising therapeutic target for HCC. In summary, our research shows that the crosslink between VRK2 and autophagy plays an important role in HCC metastasis, thus providing a new theoretical basis for treatment of HCC by targeting VRK2. VRK2 Tight junctions Autophagy ZO-1 HCC Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Disruption of tight junctions cause the looseness of cell-to-cell contact and provide a space for the motility of cancer cells, which are essential events in the invasion and metastasis process [1]. Studies in many experimental models have confirmed that tight junction proteins play an important role in cancer metastasis [2]. Zonula occluden-1(ZO-1), the first identified tight junction protein with a molecular weight of 195 kDa, acts as a peripheral scaffolding protein in tight junction complex, and alters the assembly, maintenance, and barrier function of the tight junction complex [3]. Depletion of ZO-1 leads to tight junction disruption [4]. ZO-1 is down-regulated in various metastatic cancers [5, 6], previous studies have shown that low expression of ZO-1 is associated with metastasis in HCC [7, 8], but the regulatory mechanism is largely unknown. Thereby, its paramount important to understand the molecular mechanism leading to the abnormal low expression of ZO-1 in HCC, as it may be the development of new therapies for metastatic HCC. Autophagy is a highly conserved process which cells use to deliver damaged or macromolecular protein to the lysosome for degradation, dysregulation of autophagy is implicated in various cancers include HCC [9]. The process of autophagy is controlled by a highly regulated set of signaling events, studies of autophagy mainly focus on the regulative kinases and their phosphorylation initially [10]. VRK2, as a serine-threonine kinase, has been regarded as a central regulator of autophagy [11]. It is worth noting that the key of autophagy pathway is the lipidation of LC3 proteins and their anchoring to autophagosome membranes [12]. This lipidation system include E1 activating enzyme ATG7, E2 conjugating enzyme ATG3 and E3 ligases ATG12-ATG5/ATG16L1complex [13]. However, it is not clear whether and how the process of the LC3 lipidation might be modified by VRK2. VRK2 is involved in various cellular processes, including immune response, cell cycle, apoptosis and chromatin instability [14]. In recent decade, the role of VRK2 in promoting cancer progression has attracted wide attention, a lot of studies have confirmed that VRK2 is highly expressed in tumors and promotes proliferation, invasion and metastasis of cancer cells [15, 16]. Previous studies have identified that VRK2 can interact with Akt1 and Akt2 in mammalian cells, VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction, downregulation of VRK2 eliminated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB, ultimately inhibition of autophagy induction [11]. Besides, Chen has revealed that, VRK2 promotes the phosphorylation of Bcl-2 by activating JNK1/MAPK8, enhancing the dissociation of Bcl-2 from Beclin-1 and promoting the formation of the Beclin-1-Atg14-Vps34 complex, which facilitates autophagy, and imbalance between apoptosis and autophagy leading to sorafenib chemoresistance of HCC [17]. These studies bear out that VRK2 a specific and important oncoprotein in HCC. There is still a lack of more experimental data to clarify its impact and regulatory mechanism on the metastasis of HCC. Another interesting question is whether there are other pathways involved in the regulation of autophagy by VRK2 and what’s the mechanism. According to this, we speculate whether VRK2 through participates in regulating the autophagy by other pathways, destroying the tight junction of HCC cells, thus promotes the metastasis of HCC. In this study, we sought to clarify the impact of VRK2 on HCC and its molecular mechanism. We found that over expression of VRK2 was associated with metastasis and poor prognosis of HCC. Moreover, our functional studies provided the first evidence that VRK2 promotes HCC invasion and metastasis by promote the degradation of ZO-1. VRK2 regulated the degradation of ZO-1 through the autophagy instead of known classical proteasome pathway. Further investigations indicated that VRK2 could directly phosphorylates ATG5 at serine 106 and stabilize ATG5 through enhancing the interaction with USP13 to activate autophagy mediated degradation of ZO-1. Materials and Methods Clinical specimens Between January 2017 and January 2020, human HCC specimens and paired adjacent normal tissues were collected from 102 patients at the Second Affiliated Hospital School of Medicine Zhejiang University. Informed consent was obtained from all patients with approval by the Medical Research Ethics Committee of Second Affiliated Hospital School of Medicine Zhejiang University. Cell culture, plasmids, and reagents The human HCC cell lines Li-7, HuH-7, Hep 3B, Hep G2, SK-HEP-1, MHCC97H and normal liver cell line L-O2 were purchased from the Shanghai Institute of Cell Biology, China. All cells were cultured in the recommended DMEM(Gibco) supplemented with 10% fetal bovine serum and were exposed to 100 U/mL penicillin and streptomycin at 37°C in 5% CO2. VRK2 short hairpin RNA (shVRK2) plasmid, ligated in pGV248 vector, VRK2 overexpressed plasmid, ZO-1 overexpressed plasmid, ATG5 overexpressed plasmid, were purchased from Shanghai GeneChem Technologies (Shanghai, China). Westernblot and Co-immunoprecipitation For westernblot analysis, equal amounts of protein were isolated and fractionated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins were transferred to PVDF membranes, blocked with 5% nonfat milk. The membranes were then incubated with primary antibodies according to the recommended concentration at 4 °C overnight, followed by HRP-conjugated secondary antibodies, washed three times with 1x TBST, then, The results were obtained via chemiluminescence using Quantity-One software(Bio-Rad, Hercules, CA, USA). For co-immunoprecipitation, cells were harvested in western and IP lysis buffer, rotated in rotator at 4℃ for 2h, centrifuged at10,000rpmfor10min to get rid of cellular debris, then, the supernatant was incubated with primary antibody at 4 ºC for 2h and mixed with protein A/GPLUS-Agarose overnight, the immuno-complexes and collected and washed five times by western and IP lysis buffer after centrifugation, the pellet was mixed with SDS-PAGE sample buffer and boiling for 10 min following by western blot and autoradiography. Isobaric tags for relative and absolute quantitation (iTRAQ) Cells were collected and extracted the proteins using extraction buffer (50 mM phosphate-buffered saline, 100 mM NaCl, 1 mM PMSF, and 1 mM EDTA), Groups N1, N2 and N3 (N = controls) and groups Over1, Over2 andOver3 (Over VRK2) were formed. After precipitated with acetone, the protein samples were dissolved in TEAB buffer, and then reduced, alkylated, trypsin-digested, and labeled according to the manufacturer s instructions. Samples from N1, N2 and N3 were labeled with iTRAQ tags 113,114 and 115, samples from Over1, Over2 and Over3were labeled with iTRAQ tags 116, 117 and 118 respectively. After the combination of six labeled samples, the iTRAQ-labeled peptides were fractionated by high-performance liquid chromatography (HPLC), and analyzed by LC-MS/MS. The MS raw data were analyzed with Proteome Discoverer Software version 2.1 (Thermo Fisher Scientific). Quantitative RT-PCR Total RNA of liver cancer cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR for mRNA detection was performed using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa,RR047A) and TB GREEN Premix Ex Taq (TaKaRa,RR420A). Relative quantification of the mRNA levels was performed using the comparative Ct method, and GAPDH was used as the reference gene for mRNA expression, each experiment was run in triplicate and a mean value was used for the comparison of each group. Primer sequences are listed in Supporting Data section. In vitro migration and invasion assay For the migration assay, the HCC cells were washed with PBS and resuspended in serum-free medium, then, counting 1×105 HCC cells add into the upper chambers (pore size of 8 μm, BD Biosciences); for the invasion assay, the cells were seeded in a Matrigel-coated chamber (BD Biosciences, Bedford, MA, USA). The, incubated in the 24 plates, which filled with DMEM(Gibco) supplemented with 10% fetal bovine serum. 24h later, the cells passed through the polycarbonate membrane and adhered to the lower chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, the images were extracted with a light microscope. Ubiquitination assay HCC cells were exposed to MG132 (15 mmol/L) for 12 h, and then the cell lysate was immunoprecipitated with an anti-ATG5 antibody. The ubiquitination of ATG5 was detected by an anti-ubiquitin antibody. Immunohistochemistry (IHC) and Hematoxylin & eosin (H&E) HCC and adjacent normal tissues were fixed with formalin, then, dehydration with xylene and graded alcohol and embedded in paraffin. A slide was subjected to antigen retrieval in 0.01 M citrate buffer, blocked in goat serum for 30 min and incubated with antibody overnight at 4 °C in optimal dilution, then, slides were further incubated with biotin-conjugated secondary antibody for 30 minutes in 37℃and developed by 3,3-diaminobenzidine(DAB) following by hematoxylin staining. The stained IHC slides were scanned and the scores are as follows: 0, 1, 2, and 3 points indicated no, minimal staining, moderate staining and strong staining, respectively. For HE, the slides were stained with hematoxylin & eosin (H&E) after deparaffinized to detect morphologic changes. Transmission electron microscopy (TEM) Cells were collected and washed twice with phosphate buffer(pH 7.4), fixed with 2.5% glutaraldehyde for 2h at 37℃and stored at 4℃until embedding. Then, the cells were fixed with 1% osmic acid for 2h at room temperature, washed with PBS three times for fifteen minutes，dehydrated with discontinuous density gradient ethyl alcohol(50％,70％,80％,90％,95％,100％) and acetone, samples were then embedded in embedding medium for 12h in 37℃. Ultrathinsections (60–80 nm) were cut using an ultramicrotome (Leica UC7), stained with both uranyl acetate and lead citrate, finally, images were examined with transmission electron microscope (HT7700). Live-cell fluorescence for autophagic flux The mRFP-GFP-LC3 lentivirus particles(NM_022818) were purchased from Shanghai Gene chem. Co., LTD (Shanghai, China). Liver cancer cells were infected with mRFP-GFP-LC3 lentivirus particles for 12h and cultured for another 24 h before observation. Images were obtained using a confocal laser scanning microscope(Zeiss LSM 900). Immunofluorescence(IF) Cells were seeded on a confocal dish for 36h, fixed with 4% paraformaldehyde for 30 min and washed three times with PBS, using 0.5%TritonX-100 to penetrate the nucleus before blocking with goat serum. Then, cells were incubated with primary antibody according to the recommended concentration at 4 °C overnight, washed with PBS and followed by incubated again with FITC-conjugated anti-goat IgG and TRITC-conjugated anti-rabbit IgG and DAPI in dark environment, then, washed three times and the fluorescent change of the cells were observed using a confocal laser scanning microscope(Zeiss LSM 900). Pulmonary metastasis assay For tail vein injection assay, eight-week-old athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd and fed in specific pathogen-free conditions, All the animal experiments was approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital School of Medicine Zhejiang University (No. [2024] 393). 1*10 6 cells with GFP fluorescence resuspended in100 ml of phosphate-buffered saline On the super-clean table, the mice were fixed in the mouse fixator, the root of the tail was pressed, the tail was wiped with an alcohol cotton ball to disinfect and expand the blood vessels, the cell suspension was absorbed with a 1 mL syringe, and the needle was injected into the middle third of the tail, and the speed was controlled to prevent the embolization of the mice. 0.1 mL per mice. A month later, Vivo imaging system was used to observe the lung metastasis of tumor, and the lungs were harvested after the nude mice were anesthetized, and the specimen was fixed with formalin for the further experiments. Statistical analysis Continuous variables are presented as mean ± standard error and categorical variables are presented as frequency. Two-tailed unpaired Student's t-test was used for the comparison of continuous variable and and one-way ANOVA was used for analyzing the comparison among multiple groups. Chi-square test was used for the comparison of categorical variable, survival plots were generated using the Kaplan–Meier method. All date were analyzed by GraphPad Prism 9.5.0 (GraphPad Software, USA) from at least three independent experiments. P < 0.05 was considered significant. Results VRK2 is highly expressed in HCC and is closely related to poor prognosis To explore the relationship between VRK2 and HCC, we initially examined the expression of VRK2 in HCC. Data from Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) showed that，VRK2 expression was upregulated in HCC tissues (T) compared with non-tumour tissues (N) (Fig. 1A). Notably, VRK2 expression was negatively associated with both Overall Survival (OS) and Disease Free Survival (DFS) (Fig. 1B and C). In addition, we examine the VRK2 expression in 102 pairs tissue specimens as well as the corresponding adjacent non-tumour tissues, IHC results showed that the protein expression of VRK2 was upregulated in 81.37% (83 of 102) HCC tissues (Fig. 1D and E). Furthermore, western blotting results exhibited that VRK2 protein expression in 10 fresh HCC tissues was consistent with the IHC results (Fig. 1 F). These results indicated that the expression of VRK2 was upregulated in HCC. Next, to determine whether VRK2 might been effective target for predicting HCC patient’s survival in 102 patients. A Kaplan–Meier analysis showed that the OS and DFS of patients with high protein expression of VRK2 was significantly poorer than low expression group (Fig. 1 G and H). Taken together, these data demonstrated that VRK2 was overexpressed and associated with poor prognosis in HCC. VRK2 could regulate the invasion and metastasis of HCC cells in vitro and in vivo To explore whether VRK2 could regulate the invasion and metastasis of HCC. We first explore the levels of VRK2 in a variety of HCC cells by Western blotting. The results suggested that the expression of VRK2 in HCC cells was higher than that in normal liver cell (Supplementary Fig. S1A). Next, we investigate whether VRK2 regulated the migration and invasion abilities of HCC cells in vitro . We transfected two VRK2-specific short hairpin RNA (shVRK2-1, shVRK2-2) into Hep 3B and HuH-7 cells to construct cells stably knocking down VRK2 (Fig. 2A). The real-time cellular analysis, transwell migration and invasion assays revealed that down-regulation of VRK2 caused a decrease of Hep 3B and HuH-7 cells’ migration and invasion abilities (Fig. 2B-D). And then, we further examined the effects of VRK2 on HCC lung metastasis in vivo . In the tail vein injection model of Hep 3B cell, VRK2 inhibition can significantly reduce the probability of lung metastasis, the number of metastatic nodules and the area of metastatic foci (Fig.2E-H). In contrast, ectopic expression plasmid (Flag-VRK2)-mediated up-regulation of VRK2 markedly increased probability of lung metastasis, the number of metastatic nodules and the area of metastatic foci in Hep G2 cancer cell (Supplementary Fig. S1B-I). In summary, these data got from in vitro and in vivo experiments demonstrated that VRK2 promoted the metastasis of HCC. ZO-1 is a key factor for VRK2 promoting invasion in HCC Then, we want to explore the molecular event of VRK2 regulating HCC metastasis. We applied iTRAQ (isobaric tags for relative and absolute quantification）to detect proteomic changes in VRK2 overexpressed Hep G2 cell. Biological pathway analyses of the differentially expressed proteins revealed that top dysregulated protein set in VRK2 overexpressed cells was related to aspect of tight junctions (Fig. 3A). Among the tight junction proteins, we pay attention to ZO-1, because ZO-1 act as a peripheral scaffolding protein in tight junction complex, the change of ZO-1 was the second (Fig. 3B), and researches have also showed that ZO-1 played a crucial role in the metastatic process of HCC [18-20], Therefore, we speculated that the pro-metastasis function of VRK2 might be related to ZO-1. To test this hypothesis, we firstly investigated the correlation between the expression of VRK2 and ZO-1 in HCC cells. Western blotting analysis showed that VRK2 was highly expressed while ZO-1 was lowly expressed in HCC cells, and there is a negative correlation between them (Fig. 3C). Besides, the down-regulation of VRK2 significantly increased the protein expression of ZO-1 in Hep 3B and HuH-7 cells (Fig. 3D), whereas up-regulation of VRK2 decreased the protein expression of ZO-1 in Hep G2 cell (Supplementary Fig. S2A). But, qRT-PCR analysis showed that VRK2 had no influence on mRNA expression of ZO-1(Fig. 3E and Supplementary Fig. S2B). Furthermore, down-regulation of ZO-1 inhibited the increase of ZO-1 protein expression, and then rescuing the decrease of migration and invasion abilities in VRK2-silencing Hep 3B cell (Fig. 3F-J). In addition, re-expression of ZO-1 rescued the decrease of ZO-1 protein expression by VRK2, and then attenuating the increase of HCC cells’ migration and invasion abilities induced by VKR2 overexpression (Supplementary Fig. S2C-G). Collectively, our results confirmed that ZO-1 was the key factor for the pro-metastasis function in HCC of VRK2. VRK2 promotes autophagy-mediated degradation of ZO-1 in HCC cells The previous study has reported that ZO-1 is degraded by autolysosome, we next explore whether VRK2 accelerate the degradation of ZO-1 through autophagy. Firstly, we first observed the expression of ZO-1 protein in HCC cell, which was added with autophagy inhibitor 3-methyladenine(3-MA) or chloroquine (CQ). Our results show that treatment with the lysosome inhibitor for the indicated time caused significant accumulation of the endogenous ZO-1 protein in Hep 3B cell (Fig. 4A). We also observed that Hep G2 cell was treated with Rapamycin or hungry to induce autophagy, both of the autophagy inducers increased LC3-II expression and decreased the protein expression of ZO-1 in a time-dependent manner (Fig. 4B and C). To further prove whether ZO-1 is degraded by autolysosome. Co-IP and immunofluorescence result suggested that ZO-1 and LC-3B can combine with each other (Fig. 4 D and E). Together, these data demonstrated that ZO-1 is degraded by autophagy-lysosomal pathway. Next, to determine whether VRK2 regulate the degradation of ZO-1 through autophagy-lysosomal pathway, we transfected the shVRK2 and Flag-VRK2 plasmids into Hep G2 cell and detected the effects of variable VRK2 on ZO-1 expression, the degradation dynamics assay showed that the half-life of the ectopically expressed ZO-1 was significantly decreased in the VRK2-overexpressing Hep G2 cell compared with that in the control cells, whereas the half-life of the ectopically expressed ZO-1 was significantly increased in the shVRK2 HCC cells (Fig. 4F). These results suggest that VRK2 was involved in the degradation of ZO-1. To further confirm VRK2 regulate the degradation of ZO-1 through autophagy-lysosomal pathway, we transfected Flag-VRK2 plasmids into Hep G2 cell to detect the effect of VRK2 on the degradation of ZO-1, either with or without the autophagy inhibitor 3-MA or Bafilomycin A1. Our results also showed that the reduction or increase of VRK2 had no effect on the degradation rate of ZO-1 after treating Hep G2 cell with autophagy inhibitors 3-MA or Bafilomycin A1 (Fig. 4G and H). VRK2–mediated ATG5 Ser106 phosphorylation is necessary for the stabilization of ATG5 and activate autophagy We further explored whether VRK2 activates autophagy and the regulatory axis involving VRK2, autophagy and ZO-1 in HCC. Studies have confirmed that autophagy deliver proteins and organelles to the lysosome for degradation, it is the key for autophagy-lysosomal degradation pathway, and enhanced autophagy can promote autophagy-lysosomal degradation. Thereby, we investigated the effects of VRK2 on the regulation of autophagy in HCC cells. Knockdown the expression of VRK2 decreased the ratio of LC3-II/LC3-I and increased the expression of SQSTM1 in Hep 3B cell (Fig. 5A). Furthermore, live-cell fluorescence for autophagic flux assays also showed that down-regulation of VRK2 expression caused a blockage of autophagy flux (Fig. 5B). Electron microscopy revealed that the number of autophagosomes was decreased in VRK2-silencing cells (Fig. 5C). Moreover, overexpression of VRK2 decreased the expression of SQSTM1, increased the number of autophagosomes and the level of LC-3BII, and promoted autophagy flux (Supplementary Fig. S3A - C). These results showed that VRK2 increased autophagy activity. As VRK2 is a serine-threonine protein kinase that reportedly stabilization of proteins through its catalytic activity. We conducted further investigations to determine if the phosphorylation of autophagy-related genes was influenced by VRK2. The result of phosphorylation mass spectrometry showed that, ATG5, one of the autophagy-related genes, could be phosphorylated at a novel site of ser106(Fig. 5D). To determine whether the catalytic activity of VRK2 is necessary for ATG5 stability, a VRK2 kinase-dead mutant plasmid was constructed by generating the point mutation of Lys61 (K61) to K61A, which eliminated the enzymatic activity and effectively prevented autophosphorylation of the mutant. Interestingly, we found that WT VRK2, but not VRK2 (K61A), had the ability to increase the expression of ATG5 (Fig. 5E). Subsequently, to determine whether VRK2 directly phosphorylates ATG5, we performed an in vivo kinase assay and found that exogenous expression of WT VRK2 but not VRK2 (K61A) increased the phosphorylation level of ATG5 at the S106 site (Fig. 5F). In addition, by employing an in vitro kinase reaction, we also observed a similar result (Fig. 5G). A previous report demonstrated that USP13, an deubiquitinating enzymes (DUBs), has the capability to deubiquitinate and decrease the degradation of ATG5 by integrate with M1 region (amino acids 1–184) of ATG5, and PAK1-mediated phosphorylation at residue T101 is critical for the binding and deubiquitination of ATG5 with USP13 [21]. Ser106 site of ATG5 also located in M1 region, so we hypothesize that, VRK2-mediated ATG5 ser106 phosphorylation may have played an important role in the regulation of the ATG5-USP13 protein interaction, ultimately protects ATG5 from ubiquitination-dependent degradation and promote autophagy. We use Co-IP to confirm our hypothesis, and the result revealed that the interaction of ATG5 and USP13 was dramatically increased in VRK2 overexpressing cells but was greatly reduced inVRK2 downregulated cells (Fig. 5H and I). Finally, we find that, WT-VRK2 but not VRK2 K61A could decreased the ubiquitination of ATG5(Fig. 5J). Together, our results confirmed for the first time that VRK2 affected autophagy degradation of ZO-1 by phosphorylating ATG5 at serine 106. The regulatory axis involving VRK2, ATG5, autophagy and ZO-1 was confirmed to exist in HCC tissue To verify whether the above regulatory axis also exists in HCC tissues, we detected the expression of VRK2, ATG5, LC3-Ⅱ/LC3-Ⅰ ratio and ZO-1, and analyzed their correlations in 53 pairs of fresh HCC tissues. We found that VRK2 and ATG5 were highly expressed, and LC3-Ⅱ/LC3-Ⅰ ratio was high while ZO-1 was lowly expressed in tumor tissue specimens analyzed by western blotting (Fig. 6A-E). Further analysis revealed that the expression level of VRK2 was positively correlated with ATG5 but was negatively correlated with ZO-1(Fig. 6F-G). In addition, the expression level of ZO-1 was also negatively correlated with LC3-Ⅱ/LC3-Ⅰ ratio (Fig. 6H). Furthermore, continuous section IHC staining revealed that VRK2, ATG5 and LC3 expression were high, but ZO-1 expression level was low in tumor tissue as compared to the adjacent non-tumor tissue (Fig. 6I). Our results indicated that the regulatory axis which VRK2 activated autophagic degradation of ZO-1 by up-regulating ATG5 expression also existed in HCC tissues. Discussion HCC is one of the most common malignant tumors. According to Global Cancer Statistics 2020, there are approximately 1 million new cases of HCC worldwide each year, and causing about 830,000 people die of HCC [22]. Liver cancer ranks fifth in terms of global incidence and second in terms of mortality for men [23]. Due to early detection and systemic therapy of surgery combined with adjuvant chemotherapy, targeted treatment or immunotherapy, the mortality rate of HCC has declined in the last three decades [24, 25]. However, the 5-year survival rate of patients with advanced HCC is still low, which is mainly due to tumor metastasis [26]. Therefore, it is important to understand the molecular mechanisms underlying HCC’s metastasis and identify an effective target for the prevention and treatment. Recent studies have focus on the relationship of HCC and autophagy. For example, a lot of studies have confirmed that autophagy promotes cancer cells’ proliferation, tumor progression, chemotherapy resistance and inhibits apoptosis [27-29], and the autophagy process were regulated by the process of phosphorylation [30, 31]. Here, we also find that, VRK2, as a serine-threonine kinase, promotes HCC metastasis through autophagy-mediated degradation of ZO-1. Vaccinia-related kinase 2 (VRK2) is one of the serine-threonine kinase members, previous studies have demonstrated that VRK2 can stabilize substrates through phosphorylating proteins and antagonizing proteasome degradation pathway [17, 32], VRK2 could disturb the apoptosis-autophagy balance leading to the resistance of sorafenib in HCC, but the role of VRK2 in autophagy-lysosomal degradation pathway is unclear. In the present study, we have found that high expression of VRK2 accompanied with increased autophagic activity was correlated with HCC metastasis and poor survival. Our results revealed the important role of VRK2 in regulating autophagy and tumor metastasis in vitro and in vivo. Mechanistically, VRK2 can directly phosphorylate ATG5 and stabilized its expression through antagonizing ubiquitination by enhancing the interaction with USP13, then further promote autophagic flux. The fluent autophagic flux relies on the lipidation of ATG8-family (LC3- and GABARAP-family in higher eukaryotes, here we called ATG8-family collectively), which serve as bridges of ATG8- family transformed from the cytoplasm to autophagy membranes, the crucial step of ATG8 lipidation cascade is the E3 conjugating enzyme ATG12-ATG5/ATG16L1complex [33], ATG3 can form a covalent intermediate with ATG8, which received from E1 activating enzyme ATG7, the ATG3-ATG8 covalent intermediate was linked by the thioester bond between the catalytic Cys of ATG3 and the C-terminus of ATG8, ultimately, under the mediate of ATG3, Atg8 finally transferred to PE to form a ATG8 lipidation (also known as LC3-II) catalyzed by E3 enzyme ATG12-ATG5/ATG16L1complex [34]. In the present study, increased expression of ATG5 stabilized by VRK2 can accelerate the transferred of LC3-Ⅰ to LC3-II, further promote autophagic flux indicated by Western blot, live-cell fluorescence and electron microscope. On the contrary, down-regulation of VRK2 inhibit the autophagy through decrease the transformation of LC3-Ⅰ to LC3-II mediated by ATG5. Autophagy is process of type II programmed cell death, which plays a pivotal impact in the initiation and development of cancers, the dual role of autophagy in whether promoting tumor cell survival or inhibiting tumor cell initiation remains controversial [27, 35]. Hitherto a lot of studies support the view that, autophagy plays a dynamic tumor-suppressive and tumor-promoting role in different stages and contexts of cancer development [36]. Before the tumorigenesis forms, autophagy act as quality-control role to maintaining the normal cell physiology metabolism through recycling damaged or dysfunctional cellular components, sustaining the genomic stability and preventing the tumor initiation. Once the tumor initiated or progress to late stage, autophagy contributes to be essential in the survival and growth of the established tumor cell under the nutritional deficiency and hypoxia environment. In addition, autophagic activities were found to be upregulated during the metastasis of various tumors. Zhao [37] has demonstrated that increased autophagic flux was associated with lymph node and distant metastasis in triple-negative breast cancer (TNBC). As for melanoma [38], the high expression of LC3 and beclin-1, which indicated the enhanced autophagy activities, was significantly associated with lymph and distant metastasis and poor clinical prognosis, the mechanism is related to vasculogenic mimicry (VM). In the present study, our results suggest that, overexpressed VRK2 was associated with HCC metastasis both in vitro and vivo, mechanistically, VRK2 promotes autophagy-mediated degradation of ZO-1, a protein related to tight junctions of cell-to-cell contact, decreased expression of ZO-1 contribute HCC cells detach from the solid tumor, ultimately result in the metastasis of HCC. Zonula occluden-1(ZO-1), is the first identified protein relative to tight junction between cells, it contains various domains (a guanylate kinase domain, a proline-rich region, three PDZ domains and an Src homology 3 domain) that allow its connection with other proteins as well as specialized sites in the plasma membrane, acts as crucial role in tight junction complex to prevent cancer cell migration and invasion. Previous studies have been verified ZO-1 act as metastasis suppressor in various tumor. Quan [39] has demonstrated that ZO-1 is overexpressed in human colorectal cancer examined by colorectal cancer cell lines, colorectal cancer tissues, xenograft tumor model and genetically engineered mouse model, increased levels of ZO-1 inhibit colorectal tumor metastasis both in vitro and in vivo through targeting their downstream protein PTEN and AKT/MDM2. Kim [6] has also revealed that, in gastric cancer, down regulation of ZO-1 promotes the progress of gastric cancer cells. Our study also demonstrated ZO-1 was decreased in HCC regulated by VRK2, VRK2 degrades ZO-1 expression and promote HCC metastasis. Notably, as a membrane protein of approximately 195 kDa, our results surprise to revealed that VRK2 degrades such macromolecular protein of ZO-1 through autophagy-lysosome degradation pathway instead of known ubiquitin proteasome pathway, as autophagy refers to degrade protein mainly include macromolecules, organelles, misfolded proteins and pathogenic bacteria [40]. In summary, our results reveal a novel autophagy regulation mechanism for HCC metastasis, involving the antagonism between VRK2 and ubiquitin in regulating the expression of ATG5, alter the autophagy mediated degradation of ZO-1. VRK2 expression was positive correlation with poor prognosis, and promote HCC metastasis in vitro and vivo . ZO-1 was the key regulator which mediated VRK2 influence the biological behavior of HCC. VRK2 promote autophagy mediated degradation of ZO-1 through antagonizing AGT5 ubiquitination by phosphorylating ATG5 at serine 106 to stabilized its expression. Our study suggested that VRK2 may become a novel target for HCC therapy. Declarations Conflict of interest: The authors disclose no conflicts. Acknowledgements ： This study was supported by the National Natural Science Foundation of China (82203075), the Medical and Health Science and Technology Project of Zhejiang Province (No.2023RC169). Ethics approval and consent to participate: This study was approved by the Ethics Committee of Second Affiliated Hospital School of Medicine, Zhejiang University. References Jiang X, Peng M, Liu Q, et al. Circular RNA hsa_circ_0000467 promotes colorectal cancer progression by promoting eIF4A3-mediated c-Myc translation[J]. Mol Cancer,2024,23(1):151. Santos L, Tomatis F, Ferreira H, et al. ENPP1 induces blood-brain barrier dysfunction and promotes brain metastasis formation in human epidermal growth factor receptor 2-positive breast cancer[J]. Neuro Oncol,2025,27(1):167-183. El B Y, Chidiac R, Delisle C, et al. ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis[J]. Nat Commun,2024,15(1):4405. Chen Y, Fang H, Chen H, et al. Bifidobacterium inhibits the progression of colorectal tumorigenesis in mice through fatty acid isomerization and gut microbiota modulation[J]. Gut Microbes,2025,17(1):2464945. Campbell A C, Baik J E, Sarker A, et al. Breast Cancer-Related Lymphedema Results in Impaired Epidermal Differentiation and Tight Junction Dysfunction[J]. J Invest Dermatol,2025,145(1):85-97. Choi K M, Kim B, Lee S M, et al. Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts[J]. Cell Commun Signal,2024,22(1):8. Liu Y, Mo W, Sun W, et al. NFAT2 Induces Tumor Cell Proliferation and Metastasis by Acting as a Transcriptional Co-activator of the TGF-beta1/SMAD Signaling Pathway and Inducing the Epithelial-Mesenchymal Transition in Liver Cancer[J]. Dig Dis Sci,2025. Mao D, Wang H, Guo H, et al. Tanshinone IIA normalized hepatocellular carcinoma vessels and enhanced PD-1 inhibitor efficacy by inhibiting ELTD1[J]. Phytomedicine,2024,123:155191. Yu X, Feng M, Guo J, et al. MLKL promotes hepatocarcinogenesis through inhibition of AMPK-mediated autophagy[J]. Cell Death Differ,2024,31(8):1085-1098. Zhang R, Wang J, Du Y, et al. CDK5 destabilizes PD-L1 via chaperon-mediated autophagy to control cancer immune surveillance in hepatocellular carcinoma[J]. J Immunother Cancer,2023,11(11). Hirata N, Suizu F, Matsuda-Lennikov M, et al. Functional characterization of lysosomal interaction of Akt with VRK2[J]. Oncogene,2018,37(40):5367-5386. Wei F, Wang Y, Yao J, et al. ZDHHC7-mediated S-palmitoylation of ATG16L1 facilitates LC3 lipidation and autophagosome formation[J]. Autophagy,2024,20(12):2719-2737. Killackey S A, Bi Y, Soares F, et al. Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1[J]. Mol Cell,2022,82(15):2815-2831. Shields J A, Meier S R, Bandi M, et al. VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma[J]. Cancer Res,2022,82(21):4044-4057. Chen J, Qiao K, Zhang C, et al. VRK2 activates TNFalpha/NF-kappaB signaling by phosphorylating IKKbeta in pancreatic cancer[J]. Int J Biol Sci,2022,18(3):1288-1302. Zhang J, Lin X T, Yu H Q, et al. Elevated FBXL6 expression in hepatocytes activates VRK2-transketolase-ROS-mTOR-mediated immune evasion and liver cancer metastasis in mice[J]. Exp Mol Med,2023,55(10):2162-2176. Chen S, Du Y, Xu B, et al. Vaccinia-related kinase 2 blunts sorafenib's efficacy against hepatocellular carcinoma by disturbing the apoptosis-autophagy balance[J]. Oncogene,2021,40(19):3378-3393. Kaihara T, Kusaka T, Nishi M, et al. Dedifferentiation and decreased expression of adhesion molecules, E-cadherin and ZO-1, in colorectal cancer are closely related to liver metastasis[J]. J Exp Clin Cancer Res,2003,22(1):117-123. Tornavaca O, Chia M, Dufton N, et al. ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation[J]. J Cell Biol,2015,208(6):821-838. Martin T A. The role of tight junctions in cancer metastasis[J]. Semin Cell Dev Biol,2014,36:224-231. Gao Z, Li C, Sun H, et al. N(6)-methyladenosine-modified USP13 induces pro-survival autophagy and imatinib resistance via regulating the stabilization of autophagy-related protein 5 in gastrointestinal stromal tumors[J]. Cell Death Differ,2023,30(2):544-559. Sung H, Ferlay J, Siegel R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin,2021,71(3):209-249. Cao W, Chen H D, Yu Y W, et al. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020[J]. Chin Med J (Engl),2021,134(7):783-791. Qiu S, Cai J, Yang Z, et al. Trends in Hepatocellular Carcinoma Mortality Rates in the US and Projections Through 2040[J]. JAMA Netw Open,2024,7(11):e2445525. Cavalcante D F, Garcia E M, Farias N, et al. Mortality due to hepatocellular carcinoma associated with hepatitis B and C viruses in the state of Sao Paulo, Brazil[J]. Rev Bras Epidemiol,2022,25:e220004. Chen C, Wang Z, Ding Y, et al. Tumor microenvironment-mediated immune evasion in hepatocellular carcinoma[J]. Front Immunol,2023,14:1133308. Niu X, You Q, Hou K, et al. Autophagy in cancer development, immune evasion, and drug resistance[J]. Drug Resist Updat,2025,78:101170. Wen W, Ertas Y N, Erdem A, et al. Dysregulation of autophagy in gastric carcinoma: Pathways to tumor progression and resistance to therapy[J]. Cancer Lett,2024,591:216857. Wang F, Liao Q, Qin Z, et al. Autophagy: a critical mechanism of N(6)-methyladenosine modification involved in tumor progression and therapy resistance[J]. Cell Death Dis,2024,15(10):783. White J, Choi Y B, Zhang J, et al. Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates[J]. Autophagy,2025,21(1):160-177. Zhang J, Tian Y, Xu X, et al. PLD1 promotes spindle assembly and migration through regulating autophagy in mouse oocyte meiosis[J]. Autophagy,2024,20(7):1616-1638. Zhu H, Li Q, Zhao Y, et al. Vaccinia-related kinase 2 drives pancreatic cancer progression by protecting Plk1 from Chfr-mediated degradation[J]. Oncogene,2021,40(28):4663-4674. Iriondo M N, Etxaniz A, Varela Y R, et al. Effect of ATG12-ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion[J]. Cell Mol Life Sci,2023,80(2):56. Yu G, Klionsky D J. In vitro and in vivo reconstitution systems reveal the membrane remodeling ability of LC3B and ATG16L1 to form phagophore-like membrane cups[J]. Autophagy,2024,20(11):2359-2360. Mehta P, Shende P. Dual role of autophagy for advancements from conventional to new delivery systems in cancer[J]. Biochim Biophys Acta Gen Subj,2023,1867(10):130430. Kwantwi L B. The dual role of autophagy in the regulation of cancer treatment[J]. Amino Acids,2024,56(1):7. Zhao H, Yang M, Zhao J, et al. High expression of LC3B is associated with progression and poor outcome in triple-negative breast cancer[J]. Med Oncol,2013,30(1):475. Han C, Sun B, Wang W, et al. Overexpression of microtubule-associated protein-1 light chain 3 is associated with melanoma metastasis and vasculogenic mimicry[J]. Tohoku J Exp Med,2011,223(4):243-251. Dong Y, Xu W, Qi D, et al. CLDN6 inhibits colorectal cancer proliferation dependent on restraining p53 ubiquitination via ZO-1/PTEN axis[J]. Cell Signal,2023,112:110930. Luong A M, Koestel J, Bhati K K, et al. Cargo receptors and adaptors for selective autophagy in plant cells[J]. FEBS Lett,2022,596(17):2104-2132. Additional Declarations No competing interests reported. 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00:32:20","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1223217,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-6357129/v1/d10f7e1e4763b766b847d946.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"VRK2 promotes HCC metastasis through stabilizing ATG5 to activate autophagy-mediated degradation of ZO-1","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDisruption of tight junctions cause the looseness of cell-to-cell contact and provide a space for the motility of cancer cells, which are essential events in the invasion and metastasis process\u0026nbsp;[1]. Studies in many experimental models have confirmed that tight junction proteins play an important role in cancer metastasis\u0026nbsp;[2]. Zonula occluden-1(ZO-1), the first identified tight junction protein with a molecular weight of 195 kDa, acts as a peripheral scaffolding protein in tight junction complex, and alters the assembly, maintenance, and barrier function of the tight junction complex\u0026nbsp;[3]. Depletion of ZO-1 leads to tight junction disruption\u0026nbsp;[4]. ZO-1 is down-regulated in various metastatic cancers\u0026nbsp;[5, 6], previous studies have shown that low expression of ZO-1 is associated with metastasis in HCC\u0026nbsp;[7, 8], but the regulatory mechanism is largely unknown. Thereby, its paramount important to understand the molecular mechanism leading to the abnormal low expression of ZO-1 in HCC, as it may be the development of new therapies for metastatic HCC.\u003c/p\u003e\n\u003cp\u003eAutophagy is a highly conserved process which cells use to deliver damaged or macromolecular protein to the lysosome for degradation, dysregulation of autophagy is implicated in various cancers include HCC\u0026nbsp;[9]. The process of autophagy is controlled by a highly regulated set of signaling events, studies of autophagy mainly focus on the regulative kinases and their phosphorylation initially\u0026nbsp;[10]. VRK2, as a serine-threonine kinase, has been regarded as a central regulator of autophagy\u0026nbsp;[11]. It is worth noting that the key of autophagy pathway is the\u0026nbsp;lipidation of LC3\u0026nbsp;proteins and their anchoring to autophagosome membranes\u0026nbsp;[12]. This lipidation system include E1 activating enzyme ATG7, E2 conjugating enzyme ATG3 and E3 ligases ATG12-ATG5/ATG16L1complex\u0026nbsp;[13]. However, it is not clear whether and how the process of the LC3 lipidation might be modified by VRK2.\u003c/p\u003e\n\u003cp\u003eVRK2 is involved in various cellular processes, including immune response, cell cycle, apoptosis and chromatin instability\u0026nbsp;[14]. In recent decade, the role of VRK2 in promoting cancer progression has attracted wide attention, a lot of studies have confirmed that VRK2 is highly expressed in tumors and promotes proliferation, invasion and metastasis of cancer cells\u0026nbsp;[15, 16]. Previous studies have identified that VRK2 can interact with Akt1 and Akt2 in mammalian cells, VRK2 and phosphorylated Akt accumulated in the lysosomes after autophagy induction, downregulation of VRK2 eliminated the lysosomal accumulation of phosphorylated Akt and impaired nuclear localization of TFEB, ultimately inhibition of autophagy induction\u0026nbsp;[11]. Besides, Chen has revealed that, VRK2 promotes the phosphorylation of Bcl-2 by activating JNK1/MAPK8, enhancing the dissociation of Bcl-2 from Beclin-1 and promoting the formation of the Beclin-1-Atg14-Vps34 complex, which facilitates autophagy, and imbalance between apoptosis and autophagy leading to sorafenib chemoresistance of HCC\u0026nbsp;[17]. These studies bear out that VRK2 a specific and important oncoprotein in HCC. There is still a lack of more experimental data to clarify its impact and regulatory mechanism on the metastasis of HCC. Another interesting question is whether there are other pathways involved in the regulation of autophagy by VRK2 and what\u0026rsquo;s the mechanism. According to this, we speculate whether VRK2 through participates in regulating the autophagy by other pathways, destroying the tight junction of HCC cells, thus promotes the metastasis of HCC.\u003c/p\u003e\n\u003cp\u003eIn this study, we sought to clarify the impact of VRK2 on HCC and its molecular mechanism. We found that over expression of VRK2 was associated with metastasis and poor prognosis of HCC. Moreover, our functional studies provided the first evidence that VRK2 promotes HCC invasion and metastasis by promote the degradation of ZO-1. VRK2 regulated the degradation of ZO-1 through the autophagy instead of known classical proteasome pathway. Further investigations indicated that VRK2 could directly phosphorylates ATG5 at serine 106 and stabilize ATG5 through enhancing the interaction with USP13 to activate autophagy mediated degradation of ZO-1.\u003c/p\u003e\n"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eClinical specimens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBetween January 2017 and January 2020, human HCC specimens and paired adjacent normal tissues were collected from 102 patients at the Second Affiliated Hospital School of Medicine Zhejiang University. Informed consent was obtained from all patients with approval by the Medical Research Ethics Committee of Second Affiliated Hospital School of Medicine Zhejiang University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture, plasmids, and reagents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human HCC cell lines Li-7, HuH-7, Hep 3B, Hep G2, SK-HEP-1, MHCC97H and normal liver cell line L-O2 were purchased from the Shanghai Institute of Cell Biology, China. All cells were cultured in the recommended DMEM(Gibco) supplemented with 10% fetal bovine serum and were exposed to 100 U/mL penicillin and streptomycin at 37\u0026deg;C in 5% CO2. VRK2 short hairpin RNA (shVRK2) plasmid, ligated in pGV248 vector, VRK2 overexpressed plasmid, ZO-1 overexpressed plasmid, ATG5 overexpressed plasmid, were purchased from Shanghai GeneChem Technologies (Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWesternblot\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and Co-immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor westernblot analysis, equal amounts of protein were isolated and fractionated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins were transferred to PVDF membranes, blocked with 5% nonfat milk. The membranes were then incubated with primary antibodies according to the recommended concentration at 4 \u0026deg;C overnight, followed by HRP-conjugated secondary antibodies, washed three times with 1x TBST, then, The results were obtained via chemiluminescence using Quantity-One software(Bio-Rad, Hercules, CA, USA).\u003c/p\u003e\n\u003cp\u003eFor co-immunoprecipitation, cells were harvested in western and IP lysis buffer, rotated in rotator at 4℃ for 2h, centrifuged at10,000rpmfor10min to get rid of cellular debris, then, the supernatant was incubated with primary antibody at 4 \u0026ordm;C for 2h and mixed with protein A/GPLUS-Agarose overnight, the immuno-complexes and collected and washed five times by western and IP lysis buffer after centrifugation, the pellet was mixed with SDS-PAGE sample buffer and boiling for 10 min following by western blot and autoradiography.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsobaric tags for relative and absolute quantitation (iTRAQ)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were collected and extracted the proteins using extraction buffer (50 mM phosphate-buffered saline, 100 mM NaCl, 1 mM PMSF, and 1 mM EDTA), Groups N1, N2 and N3 (N = controls) and groups Over1, Over2 andOver3 (Over VRK2) were formed. After precipitated with acetone, the protein samples were dissolved in TEAB buffer, and then reduced, alkylated, trypsin-digested, and labeled according to the manufacturer s instructions. Samples from N1, N2 and N3 were labeled with iTRAQ tags 113,114 and 115, samples from Over1, Over2 and Over3were labeled with iTRAQ tags 116, 117 and 118 respectively. After the combination of six labeled samples, the iTRAQ-labeled peptides were fractionated by high-performance liquid chromatography (HPLC), and analyzed by LC-MS/MS. The MS raw data were analyzed with Proteome Discoverer Software version 2.1 (Thermo Fisher Scientific).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative RT-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA of liver cancer cells was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Quantitative RT-PCR for mRNA detection was performed using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa,RR047A) and TB GREEN Premix Ex Taq (TaKaRa,RR420A). Relative quantification of the mRNA levels was performed using the comparative Ct method, and GAPDH was used as the reference gene for mRNA expression, each experiment was run in triplicate and a mean value was used for the comparison of each group. Primer sequences are listed in Supporting Data section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro migration and invasion assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the migration assay, the HCC cells were washed with PBS and resuspended in serum-free medium, then, counting 1\u0026times;105 HCC cells add into the upper chambers (pore size of 8 \u0026mu;m, BD Biosciences); for the invasion assay, the cells were seeded in a Matrigel-coated chamber (BD Biosciences, Bedford, MA, USA). The, incubated in the 24 plates, which filled with DMEM(Gibco) supplemented with 10% fetal bovine serum. 24h later, the cells passed through the polycarbonate membrane and adhered to the lower chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, the images were extracted with a light microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUbiquitination assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCC cells were exposed to MG132 (15 mmol/L) for 12 h, and then the cell lysate was immunoprecipitated with an anti-ATG5 antibody. The ubiquitination of ATG5 was detected by an anti-ubiquitin antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry (IHC) and Hematoxylin \u0026amp; eosin (H\u0026amp;E)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHCC and adjacent normal tissues were fixed with formalin, then, dehydration with xylene and graded alcohol and embedded in paraffin. A slide was subjected to antigen retrieval in 0.01 M citrate buffer, blocked in goat serum for 30 min and incubated with antibody overnight at 4 \u0026deg;C in optimal dilution, then, slides were further incubated with biotin-conjugated secondary antibody for 30 minutes in 37℃and developed by 3,3-diaminobenzidine(DAB) following by hematoxylin staining. The stained IHC slides were scanned and the scores are as follows: 0, 1, 2, and 3 points indicated no, minimal staining, moderate staining and strong staining, respectively. For HE, the slides were stained with hematoxylin \u0026amp; eosin (H\u0026amp;E) after deparaffinized to detect morphologic changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were collected and washed twice with phosphate buffer(pH 7.4), fixed with 2.5% glutaraldehyde for 2h at 37℃and stored at 4℃until embedding. Then, the cells were fixed with 1% osmic acid for 2h at room temperature, washed with PBS three times for fifteen minutes，dehydrated with discontinuous density gradient ethyl alcohol(50％,70％,80％,90％,95％,100％) and acetone, samples were then embedded in embedding medium for 12h in 37℃. Ultrathinsections (60\u0026ndash;80 nm) were cut using an ultramicrotome (Leica UC7), stained with both uranyl acetate and lead citrate, finally, images were examined with transmission electron microscope (HT7700).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLive-cell fluorescence for autophagic flux\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mRFP-GFP-LC3 lentivirus particles(NM_022818) were purchased from Shanghai Gene chem. Co., LTD (Shanghai, China). Liver cancer cells were infected with mRFP-GFP-LC3 lentivirus particles for 12h and cultured for another 24 h before observation. Images were obtained using a confocal laser scanning microscope(Zeiss LSM 900).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence(IF)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded on a confocal dish for 36h, fixed with 4% paraformaldehyde for 30 min and washed three times with PBS, using 0.5%TritonX-100 to penetrate the nucleus before blocking with goat serum. Then, cells were incubated with primary antibody according to the recommended concentration at 4 \u0026deg;C overnight, washed with PBS and followed by incubated again with FITC-conjugated anti-goat IgG and TRITC-conjugated anti-rabbit IgG and DAPI in dark environment, then, washed three times and the fluorescent change of the cells were observed using a confocal laser scanning microscope(Zeiss LSM 900).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePulmonary metastasis assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor tail vein injection assay, eight-week-old athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd and fed in specific pathogen-free conditions, All the animal experiments was approved by the Ethics Committee for Animal Experiments of the Second Affiliated Hospital School of Medicine Zhejiang University (No. [2024] 393). 1*10\u003csup\u003e6\u003c/sup\u003e cells with GFP fluorescence resuspended in100 ml of phosphate-buffered saline On the super-clean table, the mice were fixed in the mouse fixator, the root of the tail was pressed, the tail was wiped with an alcohol cotton ball to disinfect and expand the blood vessels, the cell suspension was absorbed with a 1 mL syringe, and the needle was injected into the middle third of the tail, and the speed was controlled to prevent the embolization of the mice. 0.1 mL per mice. A month later, Vivo imaging system was used to observe the lung metastasis of tumor, and the lungs were harvested after the nude mice were anesthetized, and the specimen was fixed with formalin for the further experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eContinuous variables are presented as mean \u0026plusmn; standard error and categorical variables are presented as frequency. Two-tailed unpaired Student\u0026apos;s t-test was used for the comparison of continuous variable and and one-way ANOVA was used for analyzing the comparison among multiple groups. Chi-square test was used for the comparison of categorical variable, survival plots were generated using the Kaplan\u0026ndash;Meier method. All date were analyzed by GraphPad Prism 9.5.0 (GraphPad Software, USA) from at least three independent experiments. P \u0026lt; 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eVRK2 is highly expressed in HCC and is closely related to poor prognosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the relationship between VRK2 and HCC, we initially examined the expression of VRK2 in HCC. Data from Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) showed that，VRK2 expression was upregulated in HCC tissues (T) compared with non-tumour tissues (N) (Fig. 1A). Notably, VRK2 expression was negatively associated with both Overall Survival (OS) and Disease Free Survival (DFS) (Fig. 1B and C). In addition, we examine the VRK2 expression in 102 pairs tissue specimens as well as the corresponding adjacent non-tumour tissues, IHC results showed that the protein expression of VRK2 was upregulated in 81.37% (83 of 102) HCC tissues (Fig. 1D and E). Furthermore, western blotting results exhibited that VRK2 protein expression in 10 fresh HCC tissues was consistent with the IHC results (Fig. 1 F). These results indicated that the expression of VRK2 was upregulated in HCC.\u003c/p\u003e\n\u003cp\u003eNext, to determine whether VRK2 might been effective target for predicting HCC patient\u0026rsquo;s survival in 102 patients. A Kaplan\u0026ndash;Meier analysis showed that the OS and DFS of patients with high protein expression of VRK2 was significantly poorer than low expression group (Fig. 1 G and H). Taken together, these data demonstrated that VRK2 was overexpressed and associated with poor prognosis in HCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVRK2 could regulate the invasion and metastasis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eHCC cells\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e\u0026nbsp;and \u003cem\u003ein vivo\u003c/em\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore whether VRK2 could regulate the invasion and metastasis of HCC. We first explore the levels of VRK2 in a variety of HCC cells by Western blotting. The results suggested that the expression of VRK2 in HCC cells was higher than that in normal liver cell (Supplementary Fig. S1A).\u003c/p\u003e\n\u003cp\u003eNext, we investigate whether VRK2 regulated the migration and invasion abilities of HCC cells \u003cem\u003ein vitro\u003c/em\u003e. We transfected two VRK2-specific short hairpin RNA (shVRK2-1, shVRK2-2) into Hep 3B and HuH-7 cells to construct cells stably knocking down VRK2 (Fig. 2A). The real-time cellular analysis, transwell migration and invasion assays revealed that down-regulation of VRK2 caused a decrease of Hep 3B and HuH-7 cells\u0026rsquo; migration and invasion abilities (Fig. 2B-D). And then, we further examined the effects of VRK2 on HCC lung metastasis \u003cem\u003ein vivo\u003c/em\u003e. In the tail vein injection model of Hep 3B cell, VRK2 inhibition can significantly reduce the probability of lung metastasis, the number of metastatic nodules and the area of metastatic foci (Fig.2E-H). In contrast, ectopic expression plasmid (Flag-VRK2)-mediated up-regulation of VRK2 markedly increased probability of lung metastasis, the number of metastatic nodules and the area of metastatic foci in Hep G2 cancer cell (Supplementary Fig. S1B-I). In summary, these data got from \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments demonstrated that VRK2 promoted the metastasis of HCC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZO-1 is a key factor for VRK2 promoting invasion in HCC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThen, we want to explore the molecular event of VRK2 regulating HCC metastasis. We applied iTRAQ (isobaric tags for relative and absolute quantification）to detect proteomic changes in VRK2 overexpressed Hep G2 cell. Biological pathway analyses of the differentially expressed proteins revealed that top dysregulated protein set in VRK2 overexpressed cells was related to aspect of tight junctions (Fig. 3A). Among the tight junction proteins, we pay attention to ZO-1, because ZO-1 act as a peripheral scaffolding protein in tight junction complex, the change of ZO-1 was the second (Fig. 3B), and researches have also showed that ZO-1 played a crucial role in the metastatic process of HCC \u0026nbsp;[18-20], Therefore, we speculated that the pro-metastasis function of VRK2 might be related to ZO-1. To test this hypothesis, we firstly investigated the correlation between the expression of VRK2 and ZO-1 in HCC cells. Western blotting analysis showed that VRK2 was highly expressed while ZO-1 was lowly expressed in HCC cells, and there is a negative correlation between them (Fig. 3C). Besides, the down-regulation of VRK2 significantly increased the protein expression of ZO-1 in Hep 3B and HuH-7 cells (Fig. 3D), whereas up-regulation of VRK2 decreased the protein expression of ZO-1 in Hep G2 cell (Supplementary Fig. S2A). But, qRT-PCR analysis showed that VRK2 had no influence on mRNA expression of ZO-1(Fig. 3E and Supplementary Fig. S2B). Furthermore, down-regulation of ZO-1 inhibited the increase of ZO-1 protein expression, and then rescuing the decrease of migration and invasion abilities in VRK2-silencing Hep 3B cell (Fig. 3F-J). In addition, re-expression of ZO-1 rescued the decrease of ZO-1 protein expression by VRK2, and then attenuating the increase of HCC cells\u0026rsquo; migration and invasion abilities induced by VKR2 overexpression (Supplementary Fig. S2C-G). Collectively, our results confirmed that ZO-1 was the key factor for the pro-metastasis function in HCC of VRK2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVRK2 promotes autophagy-mediated degradation of ZO-1 in HCC cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe previous study has reported that ZO-1 is degraded by autolysosome, we next explore whether VRK2 accelerate the degradation of ZO-1 through autophagy. Firstly, we first observed the expression of ZO-1 protein in HCC cell, which was added with autophagy inhibitor 3-methyladenine(3-MA) or chloroquine (CQ). Our results show that treatment with the lysosome inhibitor for the indicated time caused significant accumulation of the endogenous ZO-1 protein in Hep 3B cell (Fig. 4A). \u0026nbsp;We also observed that Hep G2 cell was treated with Rapamycin or hungry to induce autophagy, both of the autophagy inducers increased LC3-II expression and decreased the protein expression of ZO-1 in a time-dependent manner (Fig. 4B and C). To further prove whether ZO-1 is degraded by autolysosome. Co-IP and immunofluorescence result suggested that ZO-1 and LC-3B can combine with each other (Fig. 4 D and E). Together, these data demonstrated that ZO-1 is degraded by autophagy-lysosomal pathway.\u003c/p\u003e\n\u003cp\u003eNext, to determine whether VRK2 regulate the degradation of ZO-1 through autophagy-lysosomal pathway, we transfected the shVRK2 and Flag-VRK2 plasmids into Hep G2 cell and detected the effects of variable VRK2 on ZO-1 expression, the degradation dynamics assay showed that the half-life of the ectopically expressed ZO-1 was significantly decreased in the VRK2-overexpressing Hep G2 cell compared with that in the control cells, whereas the half-life of the ectopically expressed ZO-1 was significantly increased in the shVRK2 HCC cells (Fig. 4F). These results suggest that VRK2 was involved in the degradation of ZO-1. To further confirm VRK2 regulate the degradation of ZO-1 through autophagy-lysosomal pathway, we transfected Flag-VRK2 plasmids into Hep G2 cell to detect the effect of VRK2 on the degradation of ZO-1, either with or without the autophagy inhibitor 3-MA or Bafilomycin A1. Our results also showed that the reduction or increase of VRK2 had no effect on the degradation rate of ZO-1 after treating Hep G2 cell with autophagy inhibitors 3-MA or Bafilomycin A1 (Fig. 4G and H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVRK2\u0026ndash;mediated ATG5 Ser106 phosphorylation is necessary for the stabilization of ATG5 and activate autophagy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe further explored whether VRK2 activates autophagy and the regulatory axis involving VRK2, autophagy and ZO-1 in HCC. Studies have confirmed that autophagy deliver proteins and organelles to the lysosome for degradation, it is the key for autophagy-lysosomal degradation pathway, and enhanced autophagy can promote autophagy-lysosomal degradation. Thereby, we investigated the effects of VRK2 on the regulation of autophagy in HCC cells. Knockdown the expression of VRK2 decreased the ratio of LC3-II/LC3-I and increased the expression of SQSTM1 in Hep 3B cell (Fig. 5A). Furthermore, live-cell fluorescence for autophagic flux assays also showed that down-regulation of VRK2 expression caused a blockage of autophagy flux (Fig. 5B). Electron microscopy revealed that the number of autophagosomes was decreased in VRK2-silencing cells (Fig. 5C). Moreover, overexpression of VRK2 decreased the expression of SQSTM1, increased the number of autophagosomes and the level of LC-3BII, and promoted autophagy flux (Supplementary Fig. S3A - C). These results showed that VRK2 increased autophagy activity.\u003c/p\u003e\n\u003cp\u003eAs VRK2 is a serine-threonine protein kinase that reportedly stabilization of proteins through its catalytic activity. We conducted further investigations to determine if the phosphorylation of autophagy-related genes was influenced by VRK2. The result of phosphorylation mass spectrometry showed that, ATG5, one of the autophagy-related genes, could be phosphorylated at a novel site of ser106(Fig. 5D). To determine whether the catalytic activity of VRK2 is necessary for ATG5 stability, a VRK2 kinase-dead mutant plasmid was constructed by generating the point mutation of Lys61 (K61) to K61A, which eliminated the enzymatic activity and effectively prevented autophosphorylation of the mutant. Interestingly, we found that WT VRK2, but not VRK2 (K61A), had the ability to increase the expression of ATG5 (Fig. 5E). Subsequently, to determine whether VRK2 directly phosphorylates ATG5, we performed an in vivo kinase assay and found that exogenous expression of WT VRK2 but not VRK2 (K61A) increased the phosphorylation level of ATG5 at the S106 site (Fig. 5F). In addition, by employing an in vitro kinase reaction, we also observed a similar result (Fig. 5G). A previous report demonstrated that USP13, an deubiquitinating enzymes (DUBs), has the capability to deubiquitinate and decrease the degradation of ATG5 by integrate with M1 region (amino acids 1\u0026ndash;184) of ATG5, and PAK1-mediated phosphorylation at residue T101 is critical for the binding and deubiquitination of ATG5 with USP13 [21]. Ser106 site of ATG5 also located in M1 region, so we hypothesize that, VRK2-mediated ATG5 ser106 phosphorylation may have played an important role in the regulation of the ATG5-USP13 protein interaction, ultimately protects ATG5 from ubiquitination-dependent degradation and promote autophagy. We use Co-IP to confirm our hypothesis, and the result revealed that the interaction of ATG5 and USP13 was dramatically increased in VRK2 overexpressing cells but was greatly reduced inVRK2 downregulated cells (Fig. 5H and I). Finally, we find that, WT-VRK2 but not VRK2 K61A could decreased the ubiquitination of ATG5(Fig. 5J). Together, our results confirmed for the first time that VRK2 affected autophagy degradation of ZO-1 by phosphorylating ATG5 at serine 106.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe regulatory axis involving VRK2, ATG5, autophagy and ZO-1 was confirmed to exist in HCC tissue \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo verify whether the above regulatory axis also exists in HCC tissues, we detected the expression of VRK2, ATG5, LC3-Ⅱ/LC3-Ⅰ ratio and ZO-1, and analyzed their correlations in 53 pairs of fresh HCC tissues. We found that VRK2 and ATG5 were highly expressed, and LC3-Ⅱ/LC3-Ⅰ ratio was high while ZO-1 was lowly expressed in tumor tissue specimens analyzed by western blotting (Fig. 6A-E). Further analysis revealed that the expression level of VRK2 was positively correlated with ATG5 but was negatively correlated with ZO-1(Fig. 6F-G). In addition, the expression level of ZO-1 was also negatively correlated with LC3-Ⅱ/LC3-Ⅰ ratio (Fig. 6H). Furthermore, continuous section IHC staining revealed that VRK2, ATG5 and LC3 expression were high, but ZO-1 expression level was low in tumor tissue as compared to the adjacent non-tumor tissue (Fig. 6I). Our results indicated that the regulatory axis which VRK2 activated autophagic degradation of ZO-1 by up-regulating ATG5 expression also existed in HCC tissues.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHCC is one of the most common malignant tumors. According to Global Cancer Statistics 2020, there are approximately 1 million new cases of HCC worldwide each year, and causing about 830,000 people die of HCC\u0026nbsp;[22]. Liver cancer ranks fifth in terms of global incidence and second in terms of mortality for men\u0026nbsp;[23]. Due to early detection and systemic therapy of surgery combined with adjuvant chemotherapy, targeted treatment or immunotherapy, the mortality rate of HCC has declined in the last three decades\u0026nbsp;[24, 25]. However, the 5-year survival rate of patients with advanced HCC is still low, which is mainly due to tumor metastasis\u0026nbsp;[26]. Therefore, it is important to understand the molecular mechanisms underlying HCC\u0026rsquo;s metastasis and identify an effective target for the prevention and treatment. Recent studies have focus on the relationship of HCC and autophagy. For example, a lot of studies have confirmed that autophagy promotes cancer cells\u0026rsquo; proliferation, tumor progression, chemotherapy resistance and inhibits apoptosis\u0026nbsp;[27-29], and the autophagy process were regulated by the process of phosphorylation\u0026nbsp;[30, 31]. Here, we also find that, VRK2, as a serine-threonine kinase, promotes HCC metastasis through autophagy-mediated degradation of ZO-1.\u003c/p\u003e\n\u003cp\u003eVaccinia-related kinase 2 (VRK2) is one of the serine-threonine kinase members, previous studies have demonstrated that VRK2 can stabilize substrates through phosphorylating proteins and antagonizing proteasome degradation pathway\u0026nbsp;[17, 32], VRK2 could disturb the apoptosis-autophagy balance leading to the resistance of sorafenib in HCC, but the role of VRK2 in autophagy-lysosomal degradation pathway is unclear. In the present study, we have found that high expression of VRK2 accompanied with increased autophagic activity was correlated with HCC metastasis and poor survival. Our results revealed the important role of VRK2 in regulating autophagy and tumor metastasis in vitro and in vivo. Mechanistically, VRK2 can directly phosphorylate ATG5 and stabilized its expression through antagonizing ubiquitination by enhancing the interaction with USP13, then further promote autophagic flux. The fluent autophagic flux relies on the lipidation of ATG8-family (LC3- and GABARAP-family in higher eukaryotes, here we called ATG8-family collectively), which serve as bridges of ATG8- family transformed from the cytoplasm to autophagy membranes, the crucial step of ATG8 lipidation cascade is the E3 conjugating enzyme ATG12-ATG5/ATG16L1complex\u0026nbsp;[33], ATG3 can form a covalent intermediate with ATG8, which received from E1 activating enzyme ATG7, the ATG3-ATG8 covalent intermediate was linked by the thioester bond between the catalytic Cys of ATG3 and the C-terminus of ATG8, ultimately, under the mediate of ATG3, Atg8 finally transferred to PE to form a ATG8 lipidation (also known as LC3-II) catalyzed by E3 enzyme ATG12-ATG5/ATG16L1complex\u0026nbsp;[34]. In the present study, increased expression of ATG5 stabilized by VRK2 can accelerate the transferred of LC3-Ⅰ to LC3-II, further promote autophagic flux indicated by Western blot, live-cell fluorescence and electron microscope. On the contrary, down-regulation of VRK2 inhibit the autophagy through decrease the transformation of LC3-Ⅰ to LC3-II mediated by ATG5.\u003c/p\u003e\n\u003cp\u003eAutophagy is process of type II programmed cell death, which plays a pivotal impact in the initiation and development of cancers, the dual role of autophagy in whether promoting tumor cell survival or inhibiting tumor cell initiation remains controversial\u0026nbsp;[27, 35]. Hitherto a lot of studies support the view that, autophagy plays a dynamic tumor-suppressive and tumor-promoting role in different stages and contexts of cancer development\u0026nbsp;[36]. Before the tumorigenesis forms, autophagy act as quality-control role to maintaining the normal cell physiology metabolism through recycling damaged or dysfunctional cellular components, sustaining the genomic stability and preventing the tumor initiation. Once the tumor initiated or progress to late stage, autophagy contributes to be essential in the survival and growth of the established tumor cell under the nutritional deficiency and hypoxia environment. In addition, autophagic activities were found to be upregulated during the metastasis of various tumors. Zhao\u0026nbsp;[37] has demonstrated that increased autophagic flux was associated with lymph node and distant metastasis in triple-negative breast cancer (TNBC). As for melanoma\u0026nbsp;[38], the high expression of LC3 and beclin-1, which indicated the enhanced autophagy activities, was significantly associated with lymph and distant metastasis and poor clinical prognosis, the mechanism is related to vasculogenic mimicry (VM). In the present study, our results suggest that, overexpressed VRK2 was associated with HCC metastasis both in vitro and vivo, mechanistically, VRK2 promotes autophagy-mediated degradation of ZO-1, a protein related to tight junctions of cell-to-cell contact, decreased expression of ZO-1 contribute HCC cells detach from the solid tumor, ultimately result in the metastasis of HCC.\u003c/p\u003e\n\u003cp\u003eZonula occluden-1(ZO-1), is the first identified protein relative to tight junction between cells, it contains various domains (a guanylate kinase domain, a proline-rich region, three PDZ domains and an Src homology 3 domain) that allow its connection with other proteins as well as specialized sites in the plasma membrane, acts as crucial role in tight junction complex to prevent cancer cell migration and invasion. Previous studies have been verified ZO-1 act as metastasis suppressor in various tumor. Quan [39] has demonstrated that ZO-1 is overexpressed in human colorectal cancer examined by colorectal cancer cell lines, colorectal cancer tissues, xenograft tumor model and genetically engineered mouse model, increased levels of ZO-1 inhibit colorectal tumor metastasis both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e through targeting their downstream protein PTEN and AKT/MDM2. Kim [6] has also revealed that, in gastric cancer, down regulation of ZO-1 promotes the progress of gastric cancer cells. Our study also demonstrated ZO-1 was decreased in HCC regulated by VRK2, VRK2 degrades ZO-1 expression and promote HCC metastasis. Notably, as a membrane protein of approximately 195 kDa, our results surprise to revealed that VRK2 degrades such macromolecular protein of ZO-1 through autophagy-lysosome degradation pathway instead of known ubiquitin proteasome pathway, as autophagy refers to degrade protein mainly include macromolecules, organelles, misfolded proteins and pathogenic bacteria\u0026nbsp;[40].\u003c/p\u003e\n\u003cp\u003eIn summary, our results reveal a novel autophagy regulation mechanism for HCC metastasis, involving the antagonism between VRK2 and ubiquitin in regulating the expression of ATG5, alter the autophagy mediated degradation of ZO-1. VRK2 expression was positive correlation with poor prognosis, and promote HCC metastasis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003evivo\u003c/em\u003e. ZO-1 was the key regulator which mediated VRK2 influence the biological behavior of HCC. VRK2 promote autophagy mediated degradation of ZO-1 through antagonizing AGT5 ubiquitination by phosphorylating ATG5 at serine 106 to stabilized its expression. Our study suggested that VRK2 may become a novel target for HCC therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors disclose no conflicts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003cstrong\u003e：\u003c/strong\u003eThis study was supported by the National Natural Science Foundation of China\u0026nbsp;(82203075),\u0026nbsp;the Medical and Health Science and Technology Project of Zhejiang Province (No.2023RC169).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThis study was approved by the Ethics Committee of Second Affiliated Hospital School of Medicine, Zhejiang University.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJiang X, Peng M, Liu Q, et al. Circular RNA hsa_circ_0000467 promotes colorectal cancer progression by promoting eIF4A3-mediated c-Myc translation[J]. Mol Cancer,2024,23(1):151.\u003c/li\u003e\n\u003cli\u003eSantos L, Tomatis F, Ferreira H, et al. ENPP1 induces blood-brain barrier dysfunction and promotes brain metastasis formation in human epidermal growth factor receptor 2-positive breast cancer[J]. Neuro Oncol,2025,27(1):167-183.\u003c/li\u003e\n\u003cli\u003eEl B Y, Chidiac R, Delisle C, et al. ZO-1 interacts with YB-1 in endothelial cells to regulate stress granule formation during angiogenesis[J]. Nat Commun,2024,15(1):4405.\u003c/li\u003e\n\u003cli\u003eChen Y, Fang H, Chen H, et al. Bifidobacterium inhibits the progression of colorectal tumorigenesis in mice through fatty acid isomerization and gut microbiota modulation[J]. Gut Microbes,2025,17(1):2464945.\u003c/li\u003e\n\u003cli\u003eCampbell A C, Baik J E, Sarker A, et al. Breast Cancer-Related Lymphedema Results in Impaired Epidermal Differentiation and Tight Junction Dysfunction[J]. J Invest Dermatol,2025,145(1):85-97.\u003c/li\u003e\n\u003cli\u003eChoi K M, Kim B, Lee S M, et al. Characterization of gastric cancer-stimulated signaling pathways and function of CTGF in cancer-associated fibroblasts[J]. Cell Commun Signal,2024,22(1):8.\u003c/li\u003e\n\u003cli\u003eLiu Y, Mo W, Sun W, et al. NFAT2 Induces Tumor Cell Proliferation and Metastasis by Acting as a Transcriptional Co-activator of the TGF-beta1/SMAD Signaling Pathway and Inducing the Epithelial-Mesenchymal Transition in Liver Cancer[J]. Dig Dis Sci,2025.\u003c/li\u003e\n\u003cli\u003eMao D, Wang H, Guo H, et al. Tanshinone IIA normalized hepatocellular carcinoma vessels and enhanced PD-1 inhibitor efficacy by inhibiting ELTD1[J]. Phytomedicine,2024,123:155191.\u003c/li\u003e\n\u003cli\u003eYu X, Feng M, Guo J, et al. MLKL promotes hepatocarcinogenesis through inhibition of AMPK-mediated autophagy[J]. Cell Death Differ,2024,31(8):1085-1098.\u003c/li\u003e\n\u003cli\u003eZhang R, Wang J, Du Y, et al. CDK5 destabilizes PD-L1 via chaperon-mediated autophagy to control cancer immune surveillance in hepatocellular carcinoma[J]. J Immunother Cancer,2023,11(11).\u003c/li\u003e\n\u003cli\u003eHirata N, Suizu F, Matsuda-Lennikov M, et al. Functional characterization of lysosomal interaction of Akt with VRK2[J]. Oncogene,2018,37(40):5367-5386.\u003c/li\u003e\n\u003cli\u003eWei F, Wang Y, Yao J, et al. ZDHHC7-mediated S-palmitoylation of ATG16L1 facilitates LC3 lipidation and autophagosome formation[J]. Autophagy,2024,20(12):2719-2737.\u003c/li\u003e\n\u003cli\u003eKillackey S A, Bi Y, Soares F, et al. Mitochondrial protein import stress regulates the LC3 lipidation step of mitophagy through NLRX1 and RRBP1[J]. Mol Cell,2022,82(15):2815-2831.\u003c/li\u003e\n\u003cli\u003eShields J A, Meier S R, Bandi M, et al. VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma[J]. Cancer Res,2022,82(21):4044-4057.\u003c/li\u003e\n\u003cli\u003eChen J, Qiao K, Zhang C, et al. VRK2 activates TNFalpha/NF-kappaB signaling by phosphorylating IKKbeta in pancreatic cancer[J]. Int J Biol Sci,2022,18(3):1288-1302.\u003c/li\u003e\n\u003cli\u003eZhang J, Lin X T, Yu H Q, et al. Elevated FBXL6 expression in hepatocytes activates VRK2-transketolase-ROS-mTOR-mediated immune evasion and liver cancer metastasis in mice[J]. Exp Mol Med,2023,55(10):2162-2176.\u003c/li\u003e\n\u003cli\u003eChen S, Du Y, Xu B, et al. Vaccinia-related kinase 2 blunts sorafenib\u0026apos;s efficacy against hepatocellular carcinoma by disturbing the apoptosis-autophagy balance[J]. Oncogene,2021,40(19):3378-3393.\u003c/li\u003e\n\u003cli\u003eKaihara T, Kusaka T, Nishi M, et al. Dedifferentiation and decreased expression of adhesion molecules, E-cadherin and ZO-1, in colorectal cancer are closely related to liver metastasis[J]. J Exp Clin Cancer Res,2003,22(1):117-123.\u003c/li\u003e\n\u003cli\u003eTornavaca O, Chia M, Dufton N, et al. ZO-1 controls endothelial adherens junctions, cell-cell tension, angiogenesis, and barrier formation[J]. J Cell Biol,2015,208(6):821-838.\u003c/li\u003e\n\u003cli\u003eMartin T A. The role of tight junctions in cancer metastasis[J]. Semin Cell Dev Biol,2014,36:224-231.\u003c/li\u003e\n\u003cli\u003eGao Z, Li C, Sun H, et al. N(6)-methyladenosine-modified USP13 induces pro-survival autophagy and imatinib resistance via regulating the stabilization of autophagy-related protein 5 in gastrointestinal stromal tumors[J]. Cell Death Differ,2023,30(2):544-559.\u003c/li\u003e\n\u003cli\u003eSung H, Ferlay J, Siegel R L, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries[J]. CA Cancer J Clin,2021,71(3):209-249.\u003c/li\u003e\n\u003cli\u003eCao W, Chen H D, Yu Y W, et al. Changing profiles of cancer burden worldwide and in China: a secondary analysis of the global cancer statistics 2020[J]. Chin Med J (Engl),2021,134(7):783-791.\u003c/li\u003e\n\u003cli\u003eQiu S, Cai J, Yang Z, et al. Trends in Hepatocellular Carcinoma Mortality Rates in the US and Projections Through 2040[J]. JAMA Netw Open,2024,7(11):e2445525.\u003c/li\u003e\n\u003cli\u003eCavalcante D F, Garcia E M, Farias N, et al. Mortality due to hepatocellular carcinoma associated with hepatitis B and C viruses in the state of Sao Paulo, Brazil[J]. Rev Bras Epidemiol,2022,25:e220004.\u003c/li\u003e\n\u003cli\u003eChen C, Wang Z, Ding Y, et al. Tumor microenvironment-mediated immune evasion in hepatocellular carcinoma[J]. Front Immunol,2023,14:1133308.\u003c/li\u003e\n\u003cli\u003eNiu X, You Q, Hou K, et al. Autophagy in cancer development, immune evasion, and drug resistance[J]. Drug Resist Updat,2025,78:101170.\u003c/li\u003e\n\u003cli\u003eWen W, Ertas Y N, Erdem A, et al. Dysregulation of autophagy in gastric carcinoma: Pathways to tumor progression and resistance to therapy[J]. Cancer Lett,2024,591:216857.\u003c/li\u003e\n\u003cli\u003eWang F, Liao Q, Qin Z, et al. Autophagy: a critical mechanism of N(6)-methyladenosine modification involved in tumor progression and therapy resistance[J]. Cell Death Dis,2024,15(10):783.\u003c/li\u003e\n\u003cli\u003eWhite J, Choi Y B, Zhang J, et al. Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes ATG8-family protein-dependent clearance of MAVS aggregates[J]. Autophagy,2025,21(1):160-177.\u003c/li\u003e\n\u003cli\u003eZhang J, Tian Y, Xu X, et al. PLD1 promotes spindle assembly and migration through regulating autophagy in mouse oocyte meiosis[J]. Autophagy,2024,20(7):1616-1638.\u003c/li\u003e\n\u003cli\u003eZhu H, Li Q, Zhao Y, et al. Vaccinia-related kinase 2 drives pancreatic cancer progression by protecting Plk1 from Chfr-mediated degradation[J]. Oncogene,2021,40(28):4663-4674.\u003c/li\u003e\n\u003cli\u003eIriondo M N, Etxaniz A, Varela Y R, et al. Effect of ATG12-ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion[J]. Cell Mol Life Sci,2023,80(2):56.\u003c/li\u003e\n\u003cli\u003eYu G, Klionsky D J. In vitro and in vivo reconstitution systems reveal the membrane remodeling ability of LC3B and ATG16L1 to form phagophore-like membrane cups[J]. Autophagy,2024,20(11):2359-2360.\u003c/li\u003e\n\u003cli\u003eMehta P, Shende P. Dual role of autophagy for advancements from conventional to new delivery systems in cancer[J]. Biochim Biophys Acta Gen Subj,2023,1867(10):130430.\u003c/li\u003e\n\u003cli\u003eKwantwi L B. The dual role of autophagy in the regulation of cancer treatment[J]. Amino Acids,2024,56(1):7.\u003c/li\u003e\n\u003cli\u003eZhao H, Yang M, Zhao J, et al. High expression of LC3B is associated with progression and poor outcome in triple-negative breast cancer[J]. Med Oncol,2013,30(1):475.\u003c/li\u003e\n\u003cli\u003eHan C, Sun B, Wang W, et al. Overexpression of microtubule-associated protein-1 light chain 3 is associated with melanoma metastasis and vasculogenic mimicry[J]. Tohoku J Exp Med,2011,223(4):243-251.\u003c/li\u003e\n\u003cli\u003eDong Y, Xu W, Qi D, et al. CLDN6 inhibits colorectal cancer proliferation dependent on restraining p53 ubiquitination via ZO-1/PTEN axis[J]. Cell Signal,2023,112:110930.\u003c/li\u003e\n\u003cli\u003eLuong A M, Koestel J, Bhati K K, et al. Cargo receptors and adaptors for selective autophagy in plant cells[J]. FEBS Lett,2022,596(17):2104-2132.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"VRK2, Tight junctions, Autophagy, ZO-1, HCC","lastPublishedDoi":"10.21203/rs.3.rs-6357129/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6357129/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Vaccinia-related kinase 2 (VRK2) is involved in the process of multiple cancers, but its role in the metastasis of hepatocellular carcinoma (HCC) is largely unknown. Here, we have found that VRK2 was markedly overexpressed in HCC, high expression of VRK2 was positively correlated with poor prognosis. Functionally, silencing VRK2 caused a decrease of HCC cell’s invasion and metastasis in vitro and vivo, whereas VRK2 overexpression increased the invasion and metastasis abilities of HCC cells. What’s more, we revealed that the pro-metastatic function of VRK2 was mediated by tight junction protein ZO-1 (Zonula occluden-1), ZO-1 overexpression attenuated the increase of HCC cell’s invasion and metastasis abilities induced by VRK2 overexpression. Notably, we surprised to find that VRK2 promoted the degradation of ZO-1 protein through autophagic degradation pathway instead of known ubiquitin proteasome pathway. Mechanically, phosphorylation mass spectrometry identified ATG5, a E3 conjugating enzyme which catalyze the lipidation of LC3, as a new substrate of VRK2. VRK2 phosphorylates ATG5 at serine 106 and protects it from ubiquitin-dependent proteasomal degradation by enhancing the interaction of ATG5 with USP13, promote autophagy-mediated degradation of ZO-1. The regulatory axis involving VRK2, ATG5, autophagy and ZO-1 also existed in HCC tissue further proved that VRK2 might be a promising therapeutic target for HCC. In summary, our research shows that the crosslink between VRK2 and autophagy plays an important role in HCC metastasis, thus providing a new theoretical basis for treatment of HCC by targeting VRK2.","manuscriptTitle":"VRK2 promotes HCC metastasis through stabilizing ATG5 to activate autophagy-mediated degradation of ZO-1","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-06 00:32:15","doi":"10.21203/rs.3.rs-6357129/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"edae1851-029c-4cf2-93e8-35d01c97119c","owner":[],"postedDate":"June 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-19T14:24:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-06 00:32:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6357129","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6357129","identity":"rs-6357129","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}