A sphingolipid metabolite regulator, sphingosine kinase 1 (SPHK1), plays a critical role in the development of colorectal cancer (CRC). Studies have demonstrated that invasion and metastasis of CRC are promoted by SPHK1-driven autophagy. However, the exact mechanism of SPHK1 drives autophagy to promote tumor progression remains unclear. Here, immunohistochemical detection showed the expression of SPHK1 and tumor necrosis factor receptor-associated factor-6 (TRAF6) in human CRC tissues was stronger than in adjacent normal tissues, they were both associated with distance metastasis. It was discovered that knockdown of SPHK1 reduced the expression of TRAF6, inhibited autophagy, and inhibited the growth and metastasis of CRC cells in vitro. Moreover, the effects of SPHK1-downregulating were reversed by overexpression of TRAF6 in CRC cells transfected by double-gene. Overexpression of SPHK1 and TRAF6 promoted the expression of autophagy protein LC3 and Vimentin, while downregulated the expression of autophagy protein P62 and E-cadherin. The expression of autophagy-related ubiquitination protein ULK1 and Ubiquitin protein were significantly upregulated in TRAF6-overexpressed CRC cells. In addition, autophagy inhibitor 3-methyladenine (3MA) significantly inhibited the metastasis-promoting effect of SPHK1 and TRAF6, suppressed the expression of LC3 and Vimentin, and promoted the expression of P62 and E-cadherin, in CRC cells. Immunofluorescence staining showed SPHK1 and TRAF6 were co-localized in HT29 CRC cell membrane and cytoplasm. Immunoprecipitation detection showed SPHK1 was efficiently combined with the endogenous TRAF6, and the interaction was also detected reciprocally. Additionally, proteasome inhibitor MG132 treatment upregulated the expression of TRAF6 and the level of Ubiquitin protein, in SPHK1-downregulating CRC cells. These results reveal that SPHK1 potentiates CRC progression and metastasis via regulating autophagy mediated by TRAF6-induced ULK1 ubiquitination. SPHK1-TRAF6-ULK1 signaling axis is critical to the progression of CRC and provides a new strategy for the therapeutic control of CRC.

鞘脂代谢调节剂鞘氨醇激酶 1 (SPHK1) 在结直肠癌 (CRC) 的发展中发挥着关键作用。研究表明，SPHK1驱动的自噬促进CRC的侵袭和转移。然而，SPHK1驱动自噬促进肿瘤进展的确切机制仍不清楚。免疫组化检测显示，人结直肠癌组织中SPHK1和肿瘤坏死因子受体相关因子6（TRAF6）的表达强于癌旁正常组织，两者均与远处转移相关。体外研究发现，敲低SPHK1可降低TRAF6的表达，抑制自噬，抑制CRC细胞的生长和转移。此外，在双基因转染的CRC细胞中，TRAF6的过表达可以逆转SPHK1下调的影响。SPHK1和TRAF6的过表达促进自噬蛋白LC3和Vimentin的表达，同时下调自噬蛋白P62和E-cadherin的表达。TRAF6过表达的CRC细胞中自噬相关泛素化蛋白ULK1和泛素蛋白的表达显着上调。此外，自噬抑制剂3-甲基腺嘌呤（3MA）显着抑制CRC细胞中SPHK1和TRAF6的促转移作用，抑制LC3和Vimentin的表达，并促进P62和E-cadherin的表达。免疫荧光染色显示SPHK1和TRAF6共定位于HT29 CRC细胞膜和细胞质中。免疫沉淀检测显示SPHK1与内源TRAF6有效结合，并且也检测到了相互作用。此外，在 SPHK1 下调的 CRC 细胞中，蛋白酶体抑制剂 MG132 处理上调了 TRAF6 的表达和泛素蛋白的水平。这些结果表明，SPHK1 通过调节 TRAF6 诱导的 ULK1 泛素化介导的自噬来增强 CRC 进展和转移。SPHK1-TRAF6-ULK1信号轴对于CRC的进展至关重要，为CRC的治疗控制提供了新策略。