Clinical and experimental role of ring finger protein 180 on lymph node metastasis and survival in gastric cancer

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1 Original article Clinical and experimental role of ring finger protein 180 on lymph node metastasis and survival in gastric cancer J. Deng 1,H.Liang 1, R. Zhang 1,Y.Hou 1,Y.Liu 1, G. Ying 2,Y.Pan 3 and X. Hao 1 1 Department of Gastroenterology, 2 Central Laboratory and 3 Department of Pathology, Tianjin Medical University Cancer Hospital, City Key Laboratory of Tianjin Cancer Centre and National Clinical Research Centre for Cancer, Tianjin, China Correspondence to: Dr J. Deng, Department of Gastroenterology, Tianjin Medical University Cancer Hospital, City Key Laboratory of Tianjin Cancer Centre and National Clinical Research Centre for Cancer, Tianjin , China ( dengery@126.com) Background: The hypermethylation of ring finger protein (RNF) 180 DNA promoter is significantly associated with lymph node metastasis of gastric cancer. The present study explored the potential mechanism of RNF180-regulated lymph node metastasis of gastric cancer. Methods: Associations between clinicopathological and survival data and RNF180 expression in gastric cancer tissues were analysed. The effects of RNF180 re-expression on gastric cancer cells were determined by means of proliferation, invasion, growth and lymphangiogenesis assays. A genome microarray was used to find potential target genes associated with lymphatic metastasis of gastric cancer cells regulated by RNF180. Results: RNF180 was silenced or downregulated in 99 (73 9 per cent) of 134 gastric cancer tissues compared with 41 8 per cent of paired non-tumour tissues from patients. As an independent prognostic indicator of gastric cancer, RNF180 expression in gastric cancer tissues was negatively related to the number of metastatic lymph nodes. RNF180 was also downregulated in all seven gastric cancer cell lines examined. The re-expression of RNF180 in gastric cancer cells inhibited colony formation, proliferation, migration and invasion in vitro; re-expression of RNF180 also suppressed tumour growth and lymphangiogenesis in mice. Furthermore, re-expression of RNF180 downregulated the expression of hepatocyte growth factor, matrix metalloproteinase (MMP) 2, MMP-14, vascular endothelial growth factor C/D and chemokine receptor 7 in gastric cancer cells; it also downregulated the expression of podoplanin in tumour tissue of nude mice. Conclusion: RNF180 appears to act as a suppressor gene that inhibits lymph node metastasis in gastric cancer. Surgical relevance Biological mechanisms that lead to lymph node metastasis in gastric cancer have not been clarified. Ring finger protein (RNF) 180 has been shown to participate in the processes of lymph node metastasis in several human malignancies. In this study, silencing or downregulation of RNF180 expression was significantly associated with lymph node metastasis of gastric cancer. In vitro, RNF180 expression suppressed the common biological characteristics of gastric cancer cells (HGC-27), including proliferation, invasion, lymphangiogenesis and chemotaxis. RNF180 expression also inhibited tumour growth and tumour lymphangiogenesis in vivo. These results show that RNF180 is capable of inhibiting lymph node metastasis of gastric cancer by suppressing the intracellular activation of malignant molecular signals. Therefore, RNF180 could be considered as a promising biomarker for evaluation of the tumour aggressiveness and may be a target for future therapeutic intervention. Paper accepted 27 October 2015 Published online 25 January 2016 in Wiley Online Library ( DOI: /bjs Introduction Primary tumour invasion and lymph node metastasis are considered the strongest prognostic predictors among various clinicopathological variables, but the accuracy of the TNM classification remains debatable for clinical application in gastric cancer worldwide 1. Given the biological characteristics of malignant tumours, novel biomarkers with high specificity have been proposed as 2016 BJS Society Ltd BJS 2016; 103:

2 408 J. Deng, H. Liang, R. Zhang, Y. Hou, Y. Liu, G. Ying et al. Gastric cancer cell cultures RNF180 transfected cancer cells in nude mice Patients with gastric cancer n=134 mrna Tumour growth Lymphangiogenesis Gastric cancer tissue Normal tissue RT-PCR Immunohistochemistry RNF180 expression Target proteins Ubiquitinproteasome complex RNF180 Western blot RNF180 MMP-2, MMP-14 CCR-7 HGF VEGF-C, VEGF-D Gene microarray RNF180 65KD Invasion, chemotaxis, migration Proliferation Lymphangiogenesis Actin 43KD T1 T2 N1 N2 Lymph node metastasis Fig. 1 Summary of experimental procedures to study the effect of ring finger protein (RNF) 180 expression in gastric cancer cell cultures, patients with gastric cancer, and nude mice. RT, reverse transcription; MMP, matrix metalloproteinase; CCR, chemokine receptor; HGF, hepatocyte growth factor; VEGF, vascular endothelial growth factor promising in improving the accuracy of prediction of prognosis of gastric cancer in clinical practice. Ring finger protein (RNF) 180, a novel E3 ubiquitin ligase involved in protein ubiquitination, has been identified as a potential tumour suppressor in the carcinogenesis and progression of gastric cancer 2. The low expression of RNF180 in gastric cancer has been associated with aberrant mrna transcription and protein translation events, caused by RNF180 DNA promoter methylation 2. The present authors have shown previously 3 that hypermethylation of the RNF180 promoter is related directly to the increasing number of metastatic lymph nodes; however, the specific mechanism underlying inhibition of lymph node metastasis in gastric cancer by RNF180 has not been elucidated. The present study aimed to explore how RNF180 inhibits lymph node metastasis in gastric cancer cells using clinical cohort and basic experimental analyses to validate whether RNF180 can be deemed a novel biomarker to enhance the accuracy of prediction of lymph node metastasis in gastric cancer. Methods Experiments were performed in gastric cancer cell lines, human cancer specimens and nude mice (Fig. 1). Patient cohort and tissue samples Fresh tumour and adjacent non-tumour tissue samples were collected from patients with gastric cancer who

3 Role of ring finger protein 180 in lymph node metastasis and survival in gastric cancer 409 underwent curative gastrectomy between April 2003 and December 2007 at the Tianjin Medical University Cancer Hospital. Oral and written informed consent was obtained from all patients, and the study protocol was approved by the Institutional Research Ethics Committee of Tianjin Medical University Cancer Hospital. All surgical specimens were assessed pathologically in accordance with the International Union Against Cancer TNM classification for gastric cancer 1. Patients were followed up regularly in the clinic. Surgical procedure Curative resection was defined as the complete absence of grossly visible tumour tissues and metastatic lymph nodes after resection, with pathologically negative resection margins. Primary tumours were resected en bloc with limited or extended lymphadenectomy. Immunohistochemistry Formalin-fixed paraffin-embedded archive tissues from gastric cancer and paired adjacent non-tumour tissue samples were analysed by immunohistochemical staining to detect RNF180 protein and podoplanin protein expression. Immunohistochemistry was performed using anti-rnf180 antibody (1 : 200) (sc x; Santa Cruz Biotechnology, Dallas, Texas, USA) and anti-podoplanin antibody (1:200) (cat ; Abbiotec Company, Suite A, San Diego, USA). The cytoplasmic expression of RNF180 and podoplanin was assessed according to the average intensity of positive tumour cells (, none; +, weak; ++, intermediate; +++, strong). Intensity scores of ++ and +++ were considered to represent positive staining 4. Experiments using cell lines Cell lines Seven gastric cancer cell lines (AGS, Kato-III, SUN-1, SGC-7901, HGC-27, MGC-803 and BGC-823) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Human normal gastric mucosa cell line GES-1 was purchased from Biowit Technologies Corporation (Shenzhen, China). All cell lines were cultured in RPMI-1640 medium (Thermo Electron Corporation, Beijing, China) supplemented with 10 per cent fetal bovine serum (Life Tech, Mulgrave, Victoria, Australia) and incubated in 5 per cent carbon dioxide at 37 C. Semiquantitative reverse transcription PCR Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, California, USA) and cdna was synthesized using Transcriptor Reverse Transcriptase (Roche Diagnostics, Shanghai, China) followed by semiquantitative reverse transcription (RT) PCR 5. The primers designed for the target genes are shown in Table S1 (supporting information). Western blot analysis Western blotting was used to demonstrate RNF180 protein expression in gastric cancer and paired adjacent non-tumour tissue samples. Goat anti-rnf180 was used as primary antibody at 1 : 1000 dilution (sc x; Santa Cruz Biotechnology). Construction of RNF180 expression vectors and transfection A mammalian expression vector (pcmv6-ac-gfp) (Biovector, Buffalo, New York, USA) was used to generate the RNF180 expression vector (pcmv6-ac- GFP-RNF180) or empty vector (pcmv6-ac-gfp) 2. Lipofectamine TM 2000 transfection reagent (Invitrogen) was used to transfect the vectors into HGC-27 cells 2. Gene microarray analysis The gene expression profiles of RNF180 cells stably transfected with pcmv6-ac-gfp-rnf180 or pcmv6-ac- GFP empty vector were analysed using an RNA microarray system (039494; Agilent Technologies, Santa Clara, California, USA). GeneSpring software version 12.5 (Agilent Technologies) was employed to complete the basic analysis of the raw data, which were normalized by quantile algorithm. Gene expression changes of at least 2 0-fold higher or lower were considered to be of biological significance. Colony formation assay G418 sulphate (Merck, Darmstadt, Germany) was used in colony formation experiments with transfected HGC-27 cells, as described by Yu and colleagues 5. Cell viability assay Dimethylthiazolyl-2-5-diphenyltetrazolium bromide (MTT) dye solution (Sigma, St Louis, Missouri, USA) was applied to transfected HGC-27 cells for assessment of cell viability, as described by Germack and Dickenson 6. Cell-based scratch assay A scratched wound assay was performed on transfected HGC-27 cells for evaluation of cell migration ability, using the method reported by Sung and co-workers 7. Transwell tumour cell invasion assay Matrigel (Sigma) invasion experiments were performed on transfected HGC-27 cells as described by Zhou et al. 8.

4 410 J. Deng, H. Liang, R. Zhang, Y. Hou, Y. Liu, G. Ying et al. Experiments in nude mice HGC-27 cells ( cells in 0 1 ml phosphate-buffered saline) transfected with RNF180 expression vector or empty vector ( and HGC-27- vehicle cells respectively) were injected subcutaneously into the dorsal flank of 3-week-old male nude mice. Tumour diameter was measured every 2 or 3 days for 3 weeks. Tumour size was estimated by measuring the longest diameter of the tumour. Care of animals and all experimental procedures were approved by the Animal Ethics Committee of Tianjin Medical University Cancer Hospital 5. Enzyme histochemistry assay Lymphatic vessel counting was carried out on transplanted tumour tissues from mice, using an enzyme histochemistry assay 9. Statistical analysis The Mann Whitney U test was used to compare differences in expression of RNF180 protein between tumour and adjacent non-tumour tissue samples. Tumour growth rates in different groups of nude mice, and biological variables associated with malignancy in different HGC-27 cell lines, were compared by repeated-measures ANOVA. The χ 2 test was used to determine associations between clinicopathological variables and biomarkers. Multivariable analysis to identify relationships between RNF180 expression and other clinicopathological variables was carried out by means of multinomial logistic regression. Overall survival in relation to RNF180 expression status was evaluated by Kaplan Meier analysis and log rank test. Cox proportional hazards model was used for multivariable analysis of overall survival. Optimal cut-offs for continuous variables were identified by cut-point survival 10. P < was considered statistically significant. All statistical analyses were performed with SPSS version 18.0 software (IBM, Armonk, New York, USA). Results Clinical patient series Fresh tumour and adjacent non-tumour tissue samples were collected from 134 patients. Median follow-up from the time of diagnosis was 41 (range 1 104) months. Table 1 Survival analysis of 134 patients with gastric cancer No. of patients χ 2 test Multivariable analysis* Median overall survival (months) χ 2 P Hazard ratio P Age at surgery (years) > Sex M F Tumour location Other location Lower third Tumour size (cm) < Laurén classification Intestinal Diffuse Mixed 6 15 Tumour invasion (T category) T T T T Lymph node metastasis (N category) < (1 20, 1 66) < N N N N RNF180 protein expression in gastric cancer tissues (immunohistochemical staining) < (1 54, 4 69) < No (, +) Yes (++, +++) Values in parentheses are 95 per cent c.i. RNF, ring finger protein. *Cox proportional hazards model.

5 Role of ring finger protein 180 in lymph node metastasis and survival in gastric cancer 411 Clinical data and patient characteristics are summarized in Table 1. RNF180 protein expression in cancer tissues was detected mainly in the cytoplasm by immunohistochemical staining. The intensity of expression was classified as none ( ) in73patients(54 5 per cent), weak (+) in26(19 4 per cent), intermediate (++) in 23(17 2 per cent) and strong (+++)in12(9 0 per cent). Thus only 35 patients (26 1per cent) were considered to have positive RNF180 protein expression. Meanwhile, RNF180-positive expression was detected in 78 paired adjacent non-tumour tissues (58 2 per cent). RNF180 protein expression was downregulated significantly in gastric cancer tissues compared with paired adjacent non-tumour tissues (χ 2 = 1 783, P = 0 019) (Fig. S1A C, supporting information). Western blot detection showed that the relative expression level of RNF180 protein in 20 gastric cancer tissues was significantly lower than that in paired adjacent non-tumour tissues (mean(s.d.) 0 532(0 285) versus 1 870(0 421); P < 0 001) (Fig. S1D, supporting information). Patient outcomes related to RNF180 The results of univariable and multivariable survival analysis for the 134 patients with gastric cancer are summarized in Table 1. Median overall survival was 22 months for all patients, and 28 were alive at the end of follow-up. Kaplan Meier analysis showed that patients with negative RNF180 protein expression had worse survival than those with positive expression (Fig. 2). RNF180 protein expression in gastric cancer tissues was identified as an independent predictor of outcome (HR 0 32; P < 0 001), as was node status (HR 1 61; P < 0 001). Associations between RNF180 protein expression (immunohistochemical staining) and clinicopathological characteristics of gastric cancer are summarized in Table S2 (supporting information). In multinomial logistic regression analysis, number of metastatic lymph nodes was validated as more relevant to RNF180 expression (Bayesian information criterion value ) than other variables. Patients with gastric cancer and positive RNF180 expression had fewer metastatic lymph nodes than those with negative RNF180 expression. RNF180 in gastric cancer cell lines Relative mrna expression levels of RNF180 in seven gastric cancer cell lines (HGC-27, SGC-7901, MGC-803, BGC-823, AGS, SUN-1 and KATO-III) and the normal gastric mucosal cell line GES-1 were evaluated by RT PCR (Fig. S2A, supporting information). Levels of the RNF180 transcript were significantly lower in the Cumulative survival rate No. at risk RNF180-negative RNF180-positive RNF180-negative RNF180-positive Time after surgery (months) Fig. 2 Kaplan Meier survival curves for patients with gastric cancer in relation to ring finger protein (RNF) 180 positive or negative expression. P < (log rank test) seven gastric cancer cell lines than in GES-1 cells. The relative expression of RNF180 mrna was lowest in HGC-27 cells (mean(s.d.) 0 21(0 15) versus 1 85(0 62) in GES-1; P < 0 001). RNF180 modulates biological characteristic genes of gastric cancer cells RT PCR showed that HGC-27 cells that had been stably transfected with an RNF180 expression vector () had a much higher expression level of RNF180 than HGC-27 cells tranfected with an empty vector () (Fig. S2B, supporting information). An RNA microarray was used to demonstrate differences in expression of the generally characterized genes implicated in human tumorigenesis in HGC-27, and cells, to explore the potential inhibitory mechanisms of RNF180 against the malignant biological characteristics of gastric cancer cells. RNF180 re-expression in cells significantly inhibited the expression of genes involved in proliferation (hepatocyte growth factor, HGF), invasion (matrix metalloproteinase (MMP) 2 and 14), lymphangiogenesis (vascular endothelial growth factor (VEGF) C and D) and chemotaxis (chemotaxis receptor (CCR) 7) in gastric cancer cells (Table S3 and Fig. S2B, supporting information). RNF180 inhibits gastric cancer cell proliferation and viability As a tumour suppressor, RNF180 can theoretically inhibit proliferation and decrease the viability of gastric cancer

6 412 J. Deng, H. Liang, R. Zhang, Y. Hou, Y. Liu, G. Ying et al. 100 * 100 μm 100 μm 100 μm Scratch wound healing rate at 48 h (%) a Scratch assays 0 h 100 μm 100 μm 100 μm 24 h 48 h 0 70 b Transwell assays 50 μm 50 μm Relative cell invasive ability at 4 h Fig. 3 Effect of ring finger protein (RNF) 180 on cell migration and invasion. An RNF180 expression vector and an empty vector were stably transfected into human gastric cancer cell line HGC-27 to produce and cells respectively. These cells were used in: a cell-based scratch assays and b transwell tumour cell invasion assays. Values are mean(s.d.). *P = 0 022, P = (repeated-measures ANOVA) cells. The effects of RNF180 re-expression on the growth and viability characteristics of gastric cancer cells were investigated in colony formation and MTT assays. Significantly fewer colonies were formed by cells than by cells (mean(s.d.) 5 05(2 26) versus 18 35(8 40); P < 0 001) (Fig. S3A, supporting information). Furthermore, the MTT assay showed that cell viability at 72 and 96 h was significantly lower in cells than in cells (at 72 h: mean(s.d.) 0 25(0 07) versus 0 36(0 06), P = 0 041; at 96 h: 0 42(0 11) versus 0 53(0 09), P < 0 001) (Fig. S3B, supporting information). These results suggest that RNF180 transfection effectively inhibited the proliferation and viability of gastric cancer cells. RNF180 inhibits gastric cancer cell migration and invasion Migration and invasion are essential biological behaviours of cancer cells for metastasis to occur. Scratch assays were performed to determine the effect of RNF180 on gastric cancer cell migration. Cell migration was significantly inhibited in cells compared with cells (Fig. 3a). The scratch wound healing rate was 21 7 per cent less for cells than for cells at 48 h (P = 0 022), indicating that RNF180 significantly inhibited gastric cancer cell migration. The effect of RNF180 on the invasiveness of gastric cancer cells was studied using the Matrigel model. The invaded cell count of the cells was significantly less than that of the cells at 4 h (P = 0 017) (Fig. 3b), suggesting that RNF180 inhibited invasion of HGC-27 cells. Experiments in nude mice RNF180 inhibits tumour growth Tumour size, a measurable variable representing tumour growth, is associated with lymph node metastasis of gastric cancer. The ability of RNF180 to inhibit the growth of gastric cancer cells in nude mice in vivo was therefore explored as a means of assessing lymph node metastasis. The tumour growth curves of nude mice subcutaneously inoculated with or transfected cells are shown in Fig. 4a. After 3 weeks, mean tumour size was significantly smaller in mice injected with

7 Role of ring finger protein 180 in lymph node metastasis and survival in gastric cancer Mean tumour size (mm) Time after inoculation of tumour cells (days) a Tumour growth curves * 40 Mean tumour size (mm) b Tumour size after 3 weeks Fig. 4 Effect of ring finger protein (RNF) 180 on tumour growth in nude mice. An RNF180 expression vector and an empty vector were stably transfected into human gastric cancer cell line HGC-27 to produce and cells respectively. These cells were inoculated subcutaneously into nude mice and tumour growth was monitored: a growth curves and b images of tumours and comparison of mean(s.d.) size after 3 weeks. *P = (repeated-measures ANOVA) cells (P = 0 015) (Fig. 4b), providing evidence that RNF180 functions as a tumour suppressor in gastric carcinogenesis and tumour progression. RNF180 inhibits tumour lymphangiogenesis Lymphatic vessel counts in tumour tissues from nude mice inoculated subcutaneously with cells were significantly lower than those from mice that received cells (mean(s.d.) 12 25(4 60)versus 24 50(5 35); P < 0 001), based on the findings of enzyme histochemistry analysis (Fig. S4A, supporting information). Immunohistochemical staining with podoplanin antibody confirmed that tumour lymphatic vessel counts were lower in tumour tissue from mice inoculated with (8 6(3 5) versus 27 2(6 4) in mice treated with ; P < 0 001) (Fig. S4B, supporting information). Moreover, RNF180 suppressed the transcription of lymphangiogenesis factors (VEGF-C and VEGF-D) in the tumour tissues of nude mice inoculated subcutaneously with cells (Fig. S4C, supporting information). Discussion The present study explored the mechanism of action of RNF180, a biomarker related to lymph node metastasis of gastric cancer cells. The findings suggest the potential feasibility of using RNF180 assessment for accurate prediction of lymph node metastasis and indicate that RNF180 might be a promising target for therapeutic intervention in gastric

8 414 J. Deng, H. Liang, R. Zhang, Y. Hou, Y. Liu, G. Ying et al. cancer. RNF180 exerts its antagonistic effects against lymphatic metastasis by inhibiting the malignant characteristics of gastric cancer cells. First, it suppresses activation of HGF, inhibiting proliferation of cancer cells and tumour growth, such that the number of cancer cells is insufficient for invasion and metastasis. Second, by suppressing the activation of MMPs, RNF180 inhibits invasion of peripheral tissues, blocking the potential pathway of tumour spread via the lymphatic network. Third, RNF180 suppresses the activation of CCR-7, and reduces chemotaxis and migration of cancer cells to lymphatic vessels. Finally, RNF180 suppresses the activation of VEGF-C/D, resulting in inhibition of lymphangiogenesis, which may lead to separation of cancer cells from the lymphatic network. Thus, RNF180 may be deemed as a tumour suppressor by impairing lymphatic involvement of gastric cancer cells. The ubiquitin proteasome system, one of the most important regulators of protein metabolism, is associated with oncogenesis and tumour progression 11. Protein ubiquitylation is carried out by a three-step enzymatic cascade that involves an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme and an E3 ubiquitin ligase 12. RNF180, an E3 ubiquitin ligase, is potentially involved in several pathological processes including carcinogenesis 13. It has been shown that RNF180 transcription silencing could alter the malignant biological characteristics of gastric cancer cells, indicating that RNF180 is a tumour suppressor factor of gastric cancer 2,14. In the present study, expression of RNF180 protein, a favourable prognostic factor in gastric cancer, was significantly negatively associated with lymph node metastasis, indicating that RNF180 potentially suppresses lymph node metastasis of gastric cancer cells. Theoretically, cell proliferation, invasion, lymphangiogenesis and chemotaxis should be considered as important molecular events involved in the process of lymph node metastasis from gastric cancer 15. In this study, RNF180 protein expression was found to inhibit gastric cancer cell proliferation in several respects: RNF180 protein expression was negatively associated with tumour size in patients with gastric cancer; transfection of an RNF180 expression vector inhibited tumour growth in nude mice; and RNF180 inhibited proliferation and viability of HGC-27 cells. RNA microarray analysis showed downregulation of HGF expression by RNF180, providing a potential clue to the mechanism by which it inhibited HGC-27 cell proliferation. Expression of MMP-2 has been associated with tumour infiltration depth, lymph node metastasis and degree of tumour differentiation 16. Increased MMP-14 expression has been found to be associated with tumour invasion, metastasis and TNM stage, and restoration of MMP-14 expression can rescue gastric cancer cells from defects in migration, invasion and angiogenesis 17. In the present study, expression of both MMP-2 and MMP-14 was significantly lower in cells than in cells, indicating that RNF180 can potentially inhibit the invasive capability of gastric cancer cells. This ability of RNF180 to inhibit invasion of gastric cancer cells was confirmed by transwell tumour cell invasion assay and cell-based scratch tests. The incidence of lymph node metastasis has been correlated with the presence of CCR-7 on tissue sections from different human cancers 18. Li and colleagues 19 demonstrated that CCR-7 promoted PcI-37B cell adhesion and migration, which correlated positively with lymph node metastasis of squamous cell carcinoma of the head and neck. Recently, the CCR-7 chemokine ligand (CCL)19/CCL21 axis was shown to be significantly associated with progression and metastasis of hepatocellular carcinoma (HCC) by enhancing the nuclear accumulation of β-catenin through the Akt glycogen synthase kinase 3β signalling pathway in HCC 20. In the present study, RNA microarray analysis demonstrated that CCR-7 expression was inhibited by RNF180 in HGC-27 gastric cancer cells. Evidence from mouse models and human patients indicates that the VEGF-C VEGF-D VEGF receptor (VEGFR) 3 signalling axis is the major driving force of lymphangiogenesis 21. VEGF-C-induced VEGFR-3 activation leads to phosphorylation of the serine kinases AKT and extracellular signal-related kinase, which promotes lymphatic endothelial cell proliferation, migration and survival 22. VEGF-D signalling suppresses the expression of a prostaglandin-degrading enzyme in the collecting lymphatic vessels 23. The present authors reported previously 24 that VEGF-D promotes hepatic and lymph node metastases after curative gastrectomy through the VEGF-D VEGFR-3 signalling axis. Furthermore, signal transducer and activator of transcription 3, an important molecule in gastric oncogenesis, can upregulate VEGF-D expression in HGC-27 gastric cancer cells to contribute to lymph node metastasis by promoting lymphangiogenesis 25. In the present study, the number of lymph node metastases (N category), an independent predictor of survival of patients with gastric cancer, was significantly related to RNF180 expression in tumour tissues. Both RNF180 suppression of lymphangiogenic growth factor (VEGF-C and VEGF-D) expression in HGC-27 gastric cancer cells and RNF180 inhibition of lymphangiogenesis in nude mice were demonstrated in this study.

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10 416 J. Deng, H. Liang, R. Zhang, Y. Hou, Y. Liu, G. Ying et al. 24 Deng J, Liang H, Sun D, Pan Y, Wang B, Guo Y. Vascular endothelial growth factor-d is correlated with hepatic metastasis from gastric cancer after radical gastrectomy. Surgery 2009; 146: Deng J, Cui J, Jiang N, Zhang R, Zhang L, Hao X et al. STAT3 regulation the expression of VEGF-D in HGC-27 gastric cancer cell. Am J Transl Res 2014; 6: Supporting information Additional supporting information may be found in the online version of this article: Table S1 Primers used in reverse transcription PCR (Word document) Table S2 Association between RNF180 protein expression and clinicopathological characteristics (Word document) Table S3 Expression differences of generally characterized genes (Word document) Fig. S1 Ringer finger protein 180 protein expression in gastric cancer tissues compared with adjacent non-tumour tissues (Word document) Fig. S2 Ring finger protein 180 mrna expression in gastric cancer cell lines compared with normal mucosal cell lines, and effect of re-expression of RNF180 in gastric cancer cells on expression of genes involved in tumorigenesis (Word document) Fig. S3 Effect of ring finger protein 180 on cell proliferation and viability (Word document) Fig. S4 Effect of ring finger protein 180 on lymphangiogenesis in nude mice (Word document)