Synaptotagmin 11 scaffolds MKK7-JNK signaling process to promote stem-like molecular subtype gastric cancer oncogenesis.

BACKGROUND: Identifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC. METHODS: In vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient's survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation. SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis. RESULTS: SYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover, SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC. SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells. CONCLUSION: SYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.

TNF-α Secreted from Macrophages Increases the Expression of Prometastatic Integrin αV in Gastric Cancer.

The tumor microenvironment comprising blood vessels, fibroblasts, immune cells, and the extracellular matrix surrounding cancer cells, has recently been targeted for research in cancer therapy. We aimed to investigate the effect of macrophages on the invasive ability of gastric cancer cells, and studied their potential mechanism. In transcriptome analysis, integrin αV was identified as a gene increased in AGS cells cocultured with RAW264.7 cells. AGS cells cocultured with RAW264.7 cells displayed increased adhesion to the extracellular matrix and greater invasiveness compared with AGS cells cultured alone. This increased invasion of AGS cells cocultured with RAW264.7 cells was inhibited by integrin αV knockdown. In addition, the increase in integrin αV expression induced by tumor necrosis factor-α (TNF-α) or by coculture with RAW264.7 cells was inhibited by TNF receptor 1 (TNFR1) knockdown. The increase in integrin αV expression induced by TNF-α was inhibited by both Mitogen-activated protein kinase (MEK) inhibitor and VGLL1 S84 peptide treatment. Finally, transcription of integrin αV was shown to be regulated through the binding of VGLL1 and TEAD4 to the promoter of integrin αV. In conclusion, our study demonstrated that TNFR1-ERK-VGLL1 signaling activated by TNF-α secreted from RAW264.7 cells increased integrin αV expression, thereby increasing the adhesion and invasive ability of gastric cancer cells.