m5C and m6A cooperatively stabilize EPHB4 to drive lymphatic metastasis in gastric cancer

Gastric cancer remains a major cause of cancer-related mortality worldwide, and lymph node metastasis is a major determinant of recurrence and poor prognosis. However, the molecular basis of lymphatic dissemination, particularly the epitranscriptomic regulation of metastasis-related genes, remains unclear. We investigated whether RNA modifications promote gastric cancer lymphatic metastasis by regulating EPHB4, a receptor tyrosine kinase implicated in lymphangiogenesis and nodal spread.

We integrated transcriptome sequencing of gastric cancer tissues, bioinformatic database analyses, and immunohistochemical validation to identify EPHB4 as a metastasis-associated gene. In vitro gain- and loss-of-function assays and an in vivo hindfoot lymphatic metastasis mouse model were used to assess the role of EPHB4 in metastatic behavior. Mechanistic studies, including RNA stability assays, RIP, MeRIP-qPCR, dual-luciferase reporter assays, RIPiT, co-immunoprecipitation, and confocal microscopy, were performed to determine how NSUN2/YBX1-mediated m⁵C regulation and IGF2BP1-associated m⁶A recognition affect EPHB4 mRNA.

Transcriptome sequencing identified EPHB4 as significantly upregulated in lymph node-positive gastric cancer. Database analyses and immunohistochemistry further supported the association of high EPHB4 expression with poor clinical outcome. Functionally, EPHB4 promoted lymphatic metastasis in vivo and enhanced migration and invasion of gastric cancer cells in vitro. Mechanistically, NSUN2, YBX1, and IGF2BP1 each increased EPHB4 expression by stabilizing its mRNA. NSUN2 deposited m⁵C marks on EPHB4 transcripts that were recognized by YBX1, whereas IGF2BP1 preferentially bound m⁶A-modified EPHB4 RNA and supported its stability. Mutational mapping and dual-luciferase assays identified functional m⁵C- and m⁶A-associated sites within EPHB4 mRNA. YBX1 and IGF2BP1 also physically interacted, co-occupied EPHB4 transcripts, and reciprocally enhanced m5C- and m6A-associated EPHB4 RNA enrichment.

These findings reveal a cooperative epitranscriptomic mechanism in which NSUN2/YBX1-mediated m⁵C signaling and IGF2BP1-associated m⁶A recognition converge on EPHB4 mRNA to promote gastric cancer lymphatic metastasis, highlighting the NSUN2-YBX1-IGF2BP1-EPHB4 axis as a potential therapeutic target.