Smad4 and TGF-β1 dependent gene expression biomarkers in conditional intestinal adenoma, organoids and colorectal cancer

TGF-β ligand activation suppresses cell growth yet can paradoxically and potently promote cancer invasion and metastasis depending on downstream pathway mutational context. Here, we evaluated the basis of this observation in conditional murine intestinal adenoma models with and without loss of Mothers against decapentaplegic homolog 4 ( Smad4 ), with the aim of identifying TGF-β-BMP-SMAD4 pathway dependent gene expression biomarkers for translational application. Conditional Lgr5 -CreER ^T2 activation in Apc ^fl/fl Smad4 ^fl/fl resulted in adenoma formation with recombined homozygote floxed alleles (Apc ^Δ/Δ Smad4 ^Δ/Δ ). The adenoma phenotype was discordant, with a reduced small intestinal adenoma burden yet development of large non-metastatic caecal adenoma with nuclear localisation of phospho-Smad2/3. Derived Apc ^Δ/Δ Smad4 ^Δ/Δ adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC ₅₀ 534pM) compared to Apc ^Δ/Δ Smad4 ^+/+ (IC 24pM). TGF-β1 (390pM) modified adenoma mRNA expression (bulk RNA-Seq) most significantly for Id1 ^low and Spp1 ^high in Apc ^Δ/Δ Smad4 ^Δ/Δ . Single cell RNAseq of caecal adenoma identified expansion of Lgr5 ^low , Pak3 ^high and Id1 ^low progenitor populations in Apc ^Δ/Δ Smad4 ^Δ/Δ . Of the 76 Smad4 and TGF-β1 dependent genes identified in adenoma organoids, 7 human equivalent genes were also significantly differentially expressed in colorectal cancer, including ID1 ^low , SPP1 ^high and PAK3 ^high that also correlated with poorer survival (TCGA cohorts). Murine conditional models identified Smad4 loss of function mRNA expression biomarkers that require further evaluation as functional classifiers of colorectal cancer subtypes.