RNF180 Suppresses Methylation of ADAMTS9 DNA Promotor by Ubiquitinating DNMT3A Inhibiting Metastasis in Gastric Cancer

BACKGROUND: A disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is hypermethylated and inhibits the proliferation of various cancers. In this study, we demonstrated that the expression of ADAMTS9 was associated with the lymphatic metastasis of gastric cancer (GC) and elucidated the down- and upstream molecular pathways in GC progression. METHODS: The study explored the expression level, biological function, clinical application, and involved molecular mechanism of ADAMTS9 in GC. RESULTS: In 135 GC tissue samples, ADAMTS9 expression level was signicantly correlated with the pN stage, the number of metastatic lymph nodes (LNs), and the overall survival of patients with GC. The in vitro and in vivo experiments showed that ADAMTS9 attenuated the viability and motile capacity of GC cells. Mechanistic investigations revealed that ADAMTS9 signicantly inhibited the transcription of C–C motif chemokine ligand 5 (CCL5)/C–X–C motif chemokine ligand 11 (CXCL11). This effect impaired the migration and invasion in GC cells. This study revealed that the hypermethylation level in the promotor of ADAMTS9 gene was mainly mediated by DNA-methyltransferase(DNMT) 3A, which reduces ADAMTS9 expression in GC. Ring nger protein (RNF) 180 could promote DNMT3Aubiquitination and degradation, thereby restoring the ADAMTS9 expression in GC cells. CONCLUSIONS: ADAMTS9 expression is restored by the RNF180 via suppressing the promotor methylation of the ADAMTS9 gene. ADAMTS9 inhibits metastasis and improves the prognosis of patients with GC via CCL5/CXCL11-dependent pathway. Thus, ADAMTS9 should be considered as a predictor of LN metastasis and a therapeutic target in GC.

cancers 11 , and GC 12 . However, the correlation between ADAMTS9 and LN metastases in GC has been rarely reported, and the downstream molecular pathway remains unclear.
The promotor methylation of tumor-suppressor genes or tumor-related genes is usually involved in the multistep progression of tumor carcinogenesis and the development of cancer 13 . The hypermethylation of the promotor of ADAMTS9 gene is the most important cause that decreases or silences the expression of ADAMTS9 12 . The mechanism of modulating the promotor methylation of ADAMTS9 gene remains unclear. The novel E3 ubiquitin ligase involved in protein ubiquitination process, ring nger protein (RNF) 180 is a tumor suppressor in GC 14 . Ubiquitination plays an essential role in protein post-translation modi cation and is involved in various processes of tumorigenesis and tumor development 15 . Identifying the substrate of E3 ubiquitin ligase and improving the understanding of the ubiquitination process bene t clinicians in tumor treatment. Through the correlation analysis from the GEPIA database (http://gepia.cancer-pku.cn/), we found that the mRNA expression level of RNF180 might have a positive correlation with ADAMTS9. In addition, the immunohistochemistry (IHC) staining of tissue microarrays (TMAs) with 135 GC tissue samples also showed a signi cant positive correlation between RNF180 and ADAMTS9 expression. Thus, we hypothesized that RNF180 may suppress the methylation of ADAMTS9 DNA promotor to restore the ADAMTS9 expression in GC. In this study, we initially explored the interactional substrate of RNF180 and revealed the potential molecular mechanism of mediating ADAMTS9 expression in GC.
In present study, we demonstrated that the low expression of ADAMTS9 was closely associated with advanced pN stage and poor survival outcome in GC. ADAMTS9 attenuated the viability and motile capacity of GC cells. We uncovered that ADAMTS9 waspartly located in the cell nucleus region and inhibited the transcription of chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 11 (CXCL11). This effect impaired the migration and invasion behavior of GC. ADAMTS9 expression was mainly regulated by the promotor methylation level via modifying the expressions of DNAmethyltransferase (DNMT)3A. RNF180 promotes DNMT3Aubiquitination and degradation, thereby restoring the expression of ADAMTS9 in GC. Thus, our study identi ed ADAMTS9 as the potential target for the prediction of LN metastasis and prognosis and elucidated the potential up-and downstream molecular pathways in GC.

Methods:
Patients and tissue samples After curative gastrectomy, 135 pairs of GC tissues and matched adjacent non-tumor tissues were retrieved from the Department of Gastroenterology, Tianjin Medical University Cancer Institute and Hospital (Tianjin, China) between August 2004 and December 2007. These matched tissues were then sent to Shanghai Outdo Biotech Company (Shanghai China) for TMAs (Cat No. T14-501 TMA1-3). Tissue total RNA was extracted from 16 pairs of randomly selected GC tissue and matched adjacent non-tumor tissues after gastrectomy between July 2018 and December 2018. All tumor and adjacent non-tumor tissue samples were histologically veri ed. The patients in this study were not subjected to radiation, chemical, or biological treatment before potential curative gastrectomy. Adjuvant chemotherapy or radiotherapy was not routinely administered to the patients. The clinicopathological characteristics of the two cohorts are summarized in Table 1 and Supplementary Table S1, respectively. Patient consent was obtained for the use of the tissue samples. The study protocol and permission for the use of the clinical data were given by the Institutional Research Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China).

Ubiquitination assay
To evaluate the ubiquitination of DNMT3A, we transiently transfected empty vector, pCMV-DNMT3A-ag plasmid, pCMV-RNF180-myc plasmid or pCMV-HA-UB plasmid the HEK293T cells. After transfection for 36 h, HEK293T cells were incubated with MG132 (10 µM) for 12 h. The correlated proteins were extracted and analyzed by the co-immunoprecipitation assay.
Genome-wide mRNA sequencing analysis Other methods and statistical analysis are available in the Supplementary dataset.

ADAMTS9 was associated with LN metastases and survival outcome in patients with GC
We examined the mRNA levels of ADAMTS9 in the 16 pairs of tumor and adjacent non-tumor tissues. A total of 9/16 of tumor tissues showed lower mRNA expression level compared with that of adjacent nontumor tissues ( Figure 1A), which were classi ed into low expression group. The correlation between the clinicopathological characteristics and mRNA expression of ADAMTS9 was analyzed. ADAMTS9 mRNA expression was signi cantly associated with pN stage (P=0.044) and lauren type (P=0.019) (Supplementary Table S1). Patients with low ADAMTS9 mRNA expression were at high risk with increased metastatic LNs (12.89±3.49 vs 2.571±1.020, P=0.024, Figure 1B).
We further examined the protein expression of ADAMTS9 in GC tissues. With the IHC staining, ADAMTS9 protein expression was found in the cytoplasm and nucleus of the gastric tissue cells (Figures 1C and  3A). H-score was used to evaluate the ADAMTS9 protein expression. A total of 111 pairs of GC tissues and adjacent non-tumor tissues and additional 18 GC tissues were completely saved and well stained. Compared with that of adjacent non-tumor tissue, the H-Score of GC tissues was signi cantly decreased (P=0.008, Figure 1D). Exactly 129 GC tissues were then divided into the low and high expression groups according to the median of H-score as the cut-off value. The clinicopathological characteristics are shown inTable 1. Univariate analysis suggested that patients with low ADAMTS9 expression was associated with advanced pN stage (P=0.018). We further examined the number of LNs involvement between the two groups, and the number of metastatic LNs of high expression group was signi cantly decreased (11.38±11.64 vs 6.73±7.63, P=0.007; Figure 1E). ADAMTS9 expression was closely correlated to the LN metastasis.
To evaluate the prognostic value of ADAMTS9 expression in GC, we employed the Kaplan-Meier analysis. Figure 1F shows that patients with GC with low ADAMTS9 expression had a signi cantly poorer 5-year survival rate than those with high expression (P=0.007). To remove the various interference factors, we also acquired the multivariate Cox regression analysis. In multivariate Cox regression analysis, ADAMTS9 expression level (HR 1.602, 95%CI 1.069-2.399, P=0.022) was an independent predictor of the prognosis of patients with GC with the lowest Akaike information criterion (AIC, AIC=71.555) and Bayesian information criterion (BIC, BIC=88.714, Table 2). We demonstrated the crucial effect on the survival outcomes through strati cation by pN stage. In the subgroup of patients with pN3 stage, ADAMTS9 expression was signi cantly associated with survival outcomes (P=0.011, Figure 1F).

Low-expression of ADAMTS9 in GC cell lines
The ADAMTS9 mRNA expression in 10 types of human GC cell lines and normal GES-1 was determined.
Excluding SNU-1 and HGC-27, the mRNA expression of ADAMTS9 in 8/10 GC cell lines was lower than that in GES-1 (Figure 2A). To con rm whether the protein expression follows its mRNA expression, we tested seven GC cell lines and GES-1. Western blot analysis results showed that ADAMTS9 protein was silenced or down-regulated in 6/7 GC cell lines ( Figure 2B), which was consistent with their mRNA expression. The low ADAMTS9 expression in most GC cell lines might be a tumor suppressor.

ADAMTS9 suppressed GC cell proliferation and viability
To explore the biological function of ADAMTS9 in GC cells, we overexpressed ADAMTS9 in AGS, BGC-823, and SGC-7901 cells through the pCNDA3.1-ADAMTS9 plasmid. The mRNA and protein expression of ADAMTS9 were proven by RT-PCR and Western blot analysis (Figure 2c and Supplementary Figure S1A).
CCK8 assay and cell growth curve results then showed that ADAMTS9 suppressed cell proliferation and viability ( Figure 2D and Supplementary Figure S1B). The results of colony formation assays were consistent with the CCK8 assay results ( Figure 2E and Supplementary Figure S1C). Thus, ADAMTS9 inhibited GC cell proliferation and viability.

ADAMTS9 inhibited tumor growth in nude mouse
We investigated the effects of ADAMTS9 in vivo. The size of subcutaneous tumor transfected with ADAMTS9 was signi cantly decreased than that in tumor transfected with empty vector. The growth curve and tumor weight gure of the subcutaneous tumor are shown in Figure 2F. ADAMTS9 inhibited tumor growth in vivo.

ADAMTS9 inhibited GC cells migration and invasion
The effects of ADAMTS9 expression on AGS, BGC823, and SGC-7901 cells on migration and invasion were also assessed. Wound healing assay result showed that the migration distances of over-expressed ADAMTS9 group were signi cantly reduced compared with that of the control group (AGS, 48H, P<0.001; BGC-823, 48H, P=0.009; and SGC-7901 36H P<0.001, Figure 2G and Supplementary Figure S1D). To con rm the effect on migration and invasion, we further examined motile ability by using Transwell chambers. Similar results were obtained, thereby indicating that the number of migratory and invaded AGS and BGC-823 cells was signi cantly decreased in the over-expressed ADAMTS9 group (AGS, BGC-823, and SGC-7901; migration: P=0.003, P=0.010, and P=0.010; invasion: P=0.035, P=0.010, and P=0.004; Figure 2H and Supplementary Figure S1E). ADAMTS9 inhibited GC cell migration and invasion.
ADAMTS9 inhibited GC metastasis through CCL5 and CXCL11 pathways With IHC results for ADAMTS9, we found ADAMTS9 was partly located in the nucleus of the gastric tissue cells ( Figure 3A). To elucidate the downstream molecular mechanism of ADAMTS9, we performed the mRNA sequencing analysis of BGC-823 cells transfected with pCDNA3.1-ADAMTS9 plasmid or pCDNA3.1 empty plasmid. Among the 20031 genes screened by the mRNA sequencing, 183 genes showed signi cant decrease or increase greater than 1.5-fold change ( Figure 3B). Several DEGs were signi cantly up-or down-regulated in AGS and BGC-823 cells by real-time PCR analysis ( Figure 3F). The gene information and potential biological functions of these DEGs are shown in the Figure 3D and Figure   3E. To further examine the key roles of ADAMTS9 on GC metastasis, we performed the KEGG pathway analysis ( Figure 3C). The chemokine signaling pathway and the cytokine-cytokine receptor interaction pathway were included in the top 20 enrichment pathways (Supplementary Figure S2). Two of the signi cantly down-regulated genes, namely, CCL5 and CXCL11, played a crucial role in metastasis. To further validate this nding, we detected the down-regulated mRNA and protein expression of CCL5 and CXCL11 in the four GC cell lines (AGS, BGC-823, SGC-7901, and NCI-N87; Figures 3G and 3H). The relationships between ADAMTS9 and CCL5 and between ADAMTS9 and CXCL11 in human GC tissues were further explored. We performed immunohistochemical staining to examine the correlation between the protein levels of CCL5, CXCL11, and ADAMTS9 in the GC tissue specimens on TMAs (Supplementary Figure S3). Figure 3I shows  Figure 4A). We also validated the hypermethylation of ADAMTS9 promotor by using the 5-Aza (2 µM) treatment in the AGS and BGC-823 cell lines. Figure 4B shows that the mRNA expression of ADAMTS9 signi cantly increased after 5-Aza (2 µM) treatment. MassARRAY analysis was adopted to examine the methylation of ADAMTS9 DNA promotor. Figure 4C and Supplementary Figure S4A show that aberrant methylation was detected in BGC823 cells treated with 5-Aza (2 µM). In human tissues, DNMT1, DNMT3A, and DNMT3B are the most common methyltransferases that play key roles in various biological functions. We then explored the mechanism underlying the regulation of promotor methylation of ADAMTS9 DNA. AGS and BGC-823 cell lines were transfected with shDNMT1, shDNMT3A, or shDNMT3B (Supplementary Figure S5). PCR and Western blot analysis results showed that the DNMT3A should be the main methyltransferase that regulate ADAMTS9 expression ( Figure 4D). DNA methylation analysis through MassARRAY platform also con rmed that downregulated DNMT1 and DNMT3A expressions signi cantly increased the methylation level of ADAMTS9 gene in the BGC823 cell, especially for DNMT3A (Figure 4Eand Supplementary Figures S4B and S4C).

RNF180 restores ADAMTS9 expression by promoting DNMT3A ubiquitination and degradation
Our previous studies reported RNF180 plays the tumor suppressive roles on patients with GC. In this study, the IHC results of TMAs showed a signi cant positive correlation between RNF180 and ADAMTS9 expression.(N=126, Pearson r=0.454, P<0.001, Figure 5A and Supplementary Figure S6). In addition, with the correlation analysis from the GEPIA database (http://gepia.cancer-pku.cn/), we found that the mRNA expression level of RNF180 might be positively correlated with that ADAMTS9 (Spearman r=0.28, P=5e-09, Pearson r=0.18, P=0.00024, Figure 5B)Therefore, we hypothesized that the RNF180 restores ADAMTS9expression by decreasing ADAMTS9 methylation. Figure 5C shows the results of PCR and Western blot analysis, which indicated that RNF180 upregulated ADAMTS9 expression through the epigenetic pathway. We then performed the methylation analysis of ADAMTS9 in the BGC823 cells transfected with RNF180, and the signi cant downregulated methylation of ADAMTS9 DNA was detected ( Figure 5Dand Supplementary Figure S4D). These results con rmed our previous hypothesis.
Considering that RNF180 restores the expression of ADAMTS9 by decreasing the methylation of ADAMTS9 promotor and that the methylation of ADAMTS9 promotor is mainly regulated by DNMT3A, we hypothesized that RNF180 ubiquitinated DNMT3A, which promoted their degradation via proteasome pathway. The CHX pulse-chase assay was performed to examine the function of RNF180 on DNMT3A stability. We observed that RNF180 promoted the degradation of DNMT3A protein and reduced their halflife. (Figure 5E). AGS and BGC823 cells were then incubated with MG132 (10 µM) to inhibit protein degradation via proteasome pathway. Figure 5F shows that RNF180 promoted the accumulation of DNMT3A after treatment with MG132. Hence, we speculated that DNMT3A might be the substrate of RNF180. To explore the interaction between RNF180 and DNMT3A, we performed the coimmunoprecipitation assay. The reciprocal co-immunoprecipitation con rmed the interaction between DNMT3A and RNF180 ( Figure 5G). Ubiquitination assay was also performed to validate that the ubiquitination level of DNMT3A was greatly promoted by RNF180 ( Figure 5H). In conclusion, ADAMTS9 was restored by RNF180 via promoting DNMT3A ubiquitination and degradation.

Discussion:
LN metastasis is a crucial factor to predict the survival outcomes, and the great understanding of the LN metastasis bene ts how clinicians treat patients with GC. According to previous studies, ADAMTS9 is a tumor suppressor that inhibits the proliferation of several types of malignant tumors, such as esophageal 8 , and breast cancers 10 , GC 12 , and nasopharyngeal carcinoma 9 . However, the involvement of ADAMTS9 in LN metastasis and as the predictive factor in GC have been rarely reported. In this study, we provided experimental and clinical evidences to support the suppressive effect of ADAMTS9 on LN metastasis in GC. Our study demonstrated that ADAMTS9 inhibited migration and invasion in GC cells.
Clinical data suggested that the low expression of ADAMTS9 was closely associated with advanced pN stage and poor survival outcomes, which was identi ed as an independent risk factor. ADAMTS9 might be a potential biomarker to predict the risk of LN metastasis and improve the understanding of LN metastasis mechanism in GC.
The hypermethylation of CpG islands in the gene promotor region of ADAMTS9, which was detected by the MassARRAY analysis, was closely associated with the absent or down-regulated ADAMTS9 expression. And ADAMTS9 expression was largely regulated by the promotor methylation.These ndings were consistent with those obtained in previous study 12 . Silencing or promoting some tumor-related genes through hypermethylation or hypomethylation plays a crucial role in the occurrence and development of various cancer 17 . Thus, the potent molecular mechanism of the promotor methylation of ADAMTS9 must be investigated. In this study, ADAMTS9 expression was mostly inhibited by DNMT3A by enhancing the promotor methylation. DNMT1, DNMT3A, and DNMT3B are the three catalytic active DNMTs in mammals, which participate in a diverse range of biological processes 18 . DNMT3A and DNMT3B are responsible for the maintenance of DNA methylation through de novo methylation activity in the early stage of embryo or during cell differentiation, whereas DNMT1 is mostly responsible for the maintenance of DNA methylation during replication 19 . The disorder in the expression of DNMTs can elicit the hypo-or hyper-methylation of several tumor-related genes through epigenetic changes [20][21][22] . Through the online GEPIA database and IHC staining in this study, we found a signi cant positive correlation between the expression of RNF180 and ADAMTS9. According to our previous studies, RNF180, which is an E3 ubiquitin ligase, is a suppressor gene that inhibits LN metastasis 14 . RNF180 belongs to the ubiquitin-proteasome system that plays an important role in several oncogenesis and tumor progression processes 23 , whereas the substrate of RNF180 remains unclear. In present study, we uncovered that RNF180 could reduce the stability of DNMT3A through the ubiquitination process, and that DNMT3A was identi ed as the substrate of RNF180. In such a process, RNF180, as a tumor suppressor, could restore some tumor-related genes, including ADAMTS9, and played a crucial role in impairing the lymphatic involvement of GC cells. In conclusion, ADAMTS9 was restored by RNF180-mediated ubiquitination and degradation of DNMT3A.
We further investigated the effect of ADAMTS9 on GC metastasis and its downstream molecular an important role in in ammation and also in cancer development 27  Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.