Id1, Spp1 and Pak3 are biomarkers of Smad4 and TGF-β1 dependency in conditional intestinal adenoma, organoids and colorectal cancer

The evolutionarily conserved TGF-β signalling pathway suppresses cell growth, yet loss of function results in cancer phenotypes. Here, we evaluate loss of the TGF-β pathway signal transduction regulator, Mothers against decapentaplegic homolog 4 ( Smad4 ) with respect to intestinal adenoma phenotypes and specific TGF-β dependent gene expression. Conditional Lgr5 - Cre activation ( Apc ^fl/fl Smad4 ^fl/fl Lgr5 CreER ^T2 ) resulted in loss of function of Apc and Smad4 , reduced small intestinal adenoma burden, yet discordant development of large caecal adenoma with nuclear localisation of phospho-Smad2/3. Apc ^Δ/Δ Smad4 ^Δ/Δ adenoma organoids resisted TGF-β1 induced cell death and EMT (IC ₅₀ 534pM) compared to Apc ^Δ/Δ Smad4 ^+/+ (IC ₅₀ 24pM). TGF-β1 (390pM) modified adenoma bulk mRNA gene expression (RNA-Seq), with decreased Id1 and high Spp1 in Apc ^Δ/Δ Smad4 ^Δ/Δ . Single cell RNAseq of caecal adenoma identified Lgr5 ^low , Pak3 ^high and Id1 ^low progenitor populations in Apc ^Δ/Δ Smad4 ^Δ/Δ , that also correlated with poor prognosis in colorectal cancer (TCGA). Smad4 loss of function-TGF-β1 dependent Id1 ^low , Spp1 ^high and Pak3 ^high in intestinal epithelial progenitor cells are specific gene-pathway biomarkers for further evaluation in intestinal cancers.