Cancer associated fibroblast derived SLIT2 drives gastric cancer cell metastasis by activating NEK9

Abstract The secretory properties of cancer-associated fibroblasts (CAFs) play predominant roles in shaping a pro-metastatic tumor microenvironment. The present study demonstrated that SLIT2, an axon guidance protein, produced by CAFs and promoted gastric cancer (GC) metastasis in two gastric cancer cell lines (AGS and MKN45) by binding to roundabout guidance receptor 1 (ROBO1). Mass-spectrometry analysis revealed that ROBO1 could interact with NEK9, a serine/threonine kinase. And their mutual binding activities were further enhanced by SLIT2. Domain analysis revealed the kinase domain of NEK9 was critical in its interaction with the intracellular domain (ICD) of ROBO1, and it also directly phosphorylated tripartite motif containing 28 (TRIM28) and cortactin (CTTN) in AGS and MKN45 cells. TRIM28 function as a transcriptional elongation factor, which directly facilitate CTTN activation. In addition, Bioinformatics analysis and experimental validation identified transcriptional regulation of STAT3 and NF-κB p100 by TRIM28, and a synergetic transcription of CTTN by STAT3 and NF-κB p100 was also observed in AGS and MKN45. Therefore, CAF-derived SLIT2 increased the expression and phosphorylation levels of CTTN, which induced cytoskeletal reorganization and GC cells metastasis. A simultaneous increase in the expression levels of NEK9, TRIM28 and CTTN was found in metastatic GC lesions compared with paired non-cancerous tissues and primary cancer lesions via IHC and Multiplex IHC. The analysis of the data from a cohort of patients with GC revealed that increased levels of NEK9, TRIM28 and CTTN were associated with a decreased overall survival rate. On the whole, these findings revealed the connections of CAFs and cancer cells through SLIT2/ROBO1 and inflammatory signaling, and the key molecules involved in this process may serve as potential biomarkers and therapeutic targets for GC.