Fig. 5: Deprivation of pSMAD2L signalling altered TGFβ1-induced gene expression in A549 cells and contributed to cell plasticity.

a Prior to transcriptome analysis, A549^WT and A549L^sub cells were stimulated with human TGFβ1 (5 ng/ml) or respective controls for 48 h (n = 3). Differential gene expression between TGFβ1-stimulated and non-stimulated cells was determined by RM one-way ANOVA and Benjamini–Hochberg correction (p ≤ 0.05). A Venn diagram was used to identify exclusive. TGFβ1-induced signatures for each cell type. The absence of pSMAD2L expression resulted in 2414 differentially expressed genes in A549L^sub and 3,991 genes in A549^WT. Gene lists were subjected to Gene Set Enrichment Analysis (GSEA) using the Molecular Signature Database (MSigDB). b–e The 500 most upregulated (↑) and downregulated (↓) TGFβ1-dependent expressed genes (FC ≥ 2) in A549L^sub and A549^WT cells were analysed using Gene Ontology Biological Process (GOBP; FDR = 0.05). Genes in the overlap of the top ten GOBP terms are shown as percentages in bar plots (q ≤ 0.0205). f C-terminal TGFβ receptor-mediated phosphorylation of SMAD2 (pSMAD2C) exerts an anti-proliferative effect, whereas phosphorylation of the SMAD2 linker domain (pSMAD2L) promotes proliferation and migration, achieved by non-TGFβ signalling pathways via intracellular kinases (e.g., MAPK, CDK2, CDK4/6). This will result in higher protein abundance of the full-length SMAD2 splice variant (SMAD2^FL), while short SMAD2 (SMAD2^ΔE3) is underrepresented. g Loss of pSMAD2L signalling by linker-serine substitution or treatment with the CDK4/6 inhibitor Palbociclib causes an increase in short SMAD2, resulting in approximately equal abundance of both SMAD2 splice variants. This leads to lower proliferation and migration, but induction of stem cell factors (e.g. POU5F1/OCT4 and NANOG), whereby invasion capability remained comparable to wild-type cells. Thus, we hypothesised that pSMAD2L enables trans-differentiation of NSCLC and contributes to cancer cell plasticity by transitioning cancer cells from a highly proliferative (EMT/MET = pSMAD2L↑) to a cancer stem cell-like (CSC = pSMAD2L↓) phenotype.