Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is methylated in cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. ADAMTS9 was silenced in 6 out of 8 gastric cancer cell lines. The loss of ADAMTS9 expression was regulated by promoter hypermethylation and could be restored by demethylation agent. Ectopic expression of ADAMTS9 in gastric cancer cell lines (AGS, BGC823) inhibited cell growth curve in both the cell lines (P<0.0001), suppressed colony formation (P<0.01) and induced apoptosis (P<0.001 in AGS, P<0.01 in BGC823). Moreover, conditioned culture medium from ADAMTS9-transfected cell lines significantly disrupted the human umbilical vein endothelial cell tube formation capacity on Matrigel (P<0.01 in AGS, P<0.001 in BGC823). The in vivo growth of ADAMTS9 cells in nude mice was also markedly diminished after stable expression of ADAMTS9 (P<0.001). On the other hand, ADAMTS9 knockdown promoted cell proliferation (P<0.001). We further revealed that ADAMTS9 inhibited tumor growth by blocking activation of Akt and its downstream target the mammalian target of rapamycin (mTOR). ADAMTS9 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase, eIF4E-binding protein and downregulated hypoxia-inducible factor-1α. Therefore, this is the first demonstration that ADAMTS9 is a critical tumor suppressor of gastric cancer progression at least in part through suppression of oncogenic AKT/mTOR signaling. Moreover, promoter methylation of ADAMTS9 was detected in 29.2% (21/72) of primary gastric tumors. Multivariate analysis showed that patients with ADAMTS9 methylation had a poorer overall survival (relative risk (RR)=2.788; 95% confidence interval, 1.474-5.274; P=0.002). Kaplan-Meier survival curves showed that ADAMTS9 methylation was significantly associated with shortened survival in gastric cancer patients (P=0.001, log-rank test). In conclusion, ADAMTS9 acts as a functional tumor suppressor in gastric cancer through inhibiting oncogenic AKT/mTOR signaling pathway. Methylation of ADAMTS9 is an independent prognostic factor of gastric cancer.

Zinc-finger protein 331 (ZNF331), a Kruppel-associated box zinc-finger protein gene, was identified as a putative tumor suppressor in our previous study. However, the role of ZNF331 in tumorigenesis remains elusive. We aimed to clarify its epigenetic regulation and biological functions in gastric cancer. ZNF331 was silenced or downregulated in 71% (12/17) gastric cancer cell lines. A significant downregulation was also detected in paired gastric tumors compared with adjacent non-cancer tissues. In contrast, ZNF331 was readily expressed in various normal adult tissues. The downregulation of ZNF331 was closely linked to the promoter hypermethylation as evidenced by methylation-specific PCR, bisulfite genomic sequencing and reexpression by demethylation agent treatment. DNA sequencing showed no genetic mutation/deletion of ZNF331 in gastric cancer cell lines. Ectopic expression of ZNF331 in the silenced cancer cell lines MKN28 and HCT116 significantly reduced colony formation and cell viability, induced cell cycle arrests and repressed cell migration and invasive ability. Concordantly, knockdown of ZNF331 increased cell viability and colony formation ability of gastric cancer cell line MKN45. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomic approach were applied to analyze the molecular basis of the biological functions of ZNF331. In all, 10 downstream targets of ZNF331 were identified to be associated with regulation of cell growth and metastasis. The tumor-suppressive effect of ZNF331 is mediated at least by downregulation of genes involved in cell growth promotion (DSTN, EIF5A, GARS, DDX5, STAM, UQCRFS1 and SET) and migration/invasion (DSTN and ACTR3), and upregulation of genome-stability gene (SSBP1) and cellular senescence gene (PNPT1). A novel target of ZNF331 (DSTN) was functionally validated. Overexpression of DSTN in BGC-823 cells increased colony formation and migration ability. In conclusion, our results suggest that ZNF331 possesses important functions for the suppression of gastric carcinogenesis as a novel functional tumor-suppressor gene.

Legal wildlife trade creates opportunities for the sale of illegally procured animals and their derivatives, since it is difficult to differentiate legal from laundered items. This problem is common across many wildlife trade areasexotic pets, ornaments, seafoodand involves a variety of taxa. Here, we tested the ability of bulk and compound-specific stable isotope analysis to help monitor and regulate trade of the yellow-crested cockatoo Cacatua sulphurea, a critically endangered species threatened by overexploitation for the pet trade. Global trade in wild-caught yellowcrested cockatoos was banned in 2002; sale of captive-bred individuals, however, is still permitted. Our surveys in Hong Kong markets revealed more yellow-crested cockatoos for sale in 2017-2018 than the total number recorded as legally imported over the previous 13 years, emphasizing the need for a forensic tool to identify the source of the individuals for sale in the markets. Stable isotope analysis was successful at distinguishing between captive and wild cockatoos; we found significant differences between wild and captive cockatoos in both stable carbon (P < 0.001) and stable nitrogen (P < 0.001) isotope values. Linear discriminant analysis allocated samples to the correct group with high reliability (Accuracy = 0.91, Kappa = 0.81), although reliability was lower for some individuals with values on the edge of the distribution. In cases where the bulk isotope analysis was ambiguous, compound-specific stable isotope analysis, which provides carbon isotope values in specific amino acids, can be applied. We found six amino acids that differed significantly between captive and wild samples, with valine (P = 0.009) being the most informative. Together, stable and compound-specific isotope analysis represents an important potential forensic tool to help combat illegal trade of cockatoos and could be expanded to other species threatened by wildlife trade.