BackgroundTriple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis, and there are no targeted treatments available. TNBC patients are more likely to develop metastases and relapse than patients with other breast cancer subtypes. Lymph node metastasis is the first sign of metastatic spread. We aimed to characterize the mechanism of lymph node metastasis in TNBC to provide a new strategy for the treatment of TNBC.MethodsGene Expression Omnibus (GEO) TNBC database was utilized to screen for genes related to N staging. Screening the downstream target of Melanophilin (MLPH) in TNBC through RNA sequencing (RNA seq) analysis. Protein mass spectrometry was utilized to analyze the protein which interacts with MLPH, and RNA binding protein immunoprecipitation and quantitative real-time PCR (RIP qPCR) were utilized to verify the regulation of sphingosine kinase 1 (SPHK1) expression by MLPH through Non-POU domain-containing octamer-binding protein (NONO). Cell functional assays and in vivo models experiments further confirmed the effects of MLPH on proliferation and lymph node metastasis of TNBC through the SPHK1-S1P axis.ResultsMLPH is downregulated in TNBC and inhibits tumor growth and lymph node metastasis though the MLPH-NONO-SPHK1-S1P pathway. NONO was identified as an essential factor involved in SPHK1 mRNA splicing. MLPH interacts with NONO to inhibit SPHK1 mRNA splicing of SPHK1, which reduces the content of S1P, thereby inhibiting growth and lymph node metastasis in TNBC.ConclusionsThis study preliminarily elucidated a mechanism underlying lymph node metastasis in TNBC and identified the role of the MLPH-NONO-SPHK1-S1P axis in regulating proliferation and lymph node metastasis in TNBC. These findings may help design strategies for predicting and treating metastasis in TNBC.