METTL3-Associated FOXD1 Regulation Promotes Lung Squamous Cell Carcinoma Progression: A Multimodel Experimental Approach.

Abstract

Lung squamous cell carcinoma (LUSC) has limited treatment options, motivating investigation of molecular pathways that support tumor-cell growth. Forkhead box D1 (FOXD1) expression was assessed using gene expression profiling interactive analysis (GEPIA), quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry in paired tissues and cell lines. FOXD1 or methyltransferase-like 3 (METTL3) was depleted in LUSC cells, and cell viability, apoptosis, migration, global N6-methyladenosine (m6A), transcript stability, and protein stability were evaluated. A FOXD1-knockdown xenograft model tested the in vivo role of FOXD1; METTL3 was not manipulated in animals. FOXD1 expression was higher in LUSC tissues and cells than in corresponding controls. FOXD1 knockdown reduced viability and migration, increased apoptosis, and suppressed xenograft growth. METTL3 expression correlated with FOXD1 in paired LUSC tissues. In cultured cells, METTL3 knockdown lowered global m6A and FOXD1 abundance, whereas FOXD1 re-expression partially rescued the associated phenotypes. Actinomycin D, cycloheximide, and MG132 assays were consistent with METTL3-associated changes in FOXD1 RNA and protein stability. These data support a functional association between METTL3 and FOXD1 in LUSC cells. In vivo, FOXD1 knockdown suppressed xenograft growth and reduced Ki-67 staining, establishing FOXD1 as a promoter of xenograft growth. Direct METTL3-FOXD1 binding, site-specific m6A modification, reader dependence, and in vivo validation of the complete METTL3-FOXD1 pathway remain to be established.