State Key Laboratory of Cancer Biology and National Clinical Research Center for Digestive Diseases, Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi'an, 710032, China.
Department of Physiology and Pathophysiology, National Key Discipline of Cell Biology, Fourth Military Medical University, Xi'an, 710032, China.
Cell Death Dis. 2023 Jul 13;14(7):421. doi: 10.1038/s41419-023-05965-z.
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.
癌相关成纤维细胞 (CAFs) 的分泌特性在塑造有利于转移的肿瘤微环境中起着主要作用。本研究表明，CAFs 产生的轴突导向蛋白 SLIT2 通过与 ROBO1 结合促进了两种胃癌细胞系 (AGS 和 MKN45) 的胃癌转移。质谱分析显示，ROBO1 可以与丝氨酸/苏氨酸激酶 NEK9 相互作用。并且 SLIT2 进一步增强了它们的相互结合活性。结构域分析表明，NEK9 的激酶结构域对于其与 ROBO1 的细胞内结构域 (ICD) 的相互作用至关重要，并且它还直接在 AGS 和 MKN45 细胞中磷酸化包含三部分基序的 28 (TRIM28) 和 cortactin (CTTN)。TRIM28 作为转录延伸因子起作用，其直接促进 CTTN 的激活。此外，生物信息学分析和实验验证确定了 STAT3 和 NF-κB p100 对 TRIM28 的转录调控，并且还观察到 STAT3 和 NF-κB p100 对 CTTN 的协同转录。因此，CAF 衍生的 SLIT2 增加了 CTTN 的表达和磷酸化水平，这诱导了细胞骨架重排和 GC 细胞转移。通过 IHC 和多重 IHC，与配对的非癌组织和原发性癌病变相比，在转移性 GC 病变中发现 NEK9、TRIM28 和 CTTN 的表达水平同时增加。对 GC 患者队列数据的分析表明，NEK9、TRIM28 和 CTTN 的水平升高与总生存率降低相关。总的来说，这些发现揭示了 CAFs 和癌细胞之间通过 SLIT2/ROBO1 和炎症信号的联系，并且该过程中涉及的关键分子可以作为 GC 的潜在生物标志物和治疗靶标。
