Smad4 and TGFβ1 dependent gene expression signatures in conditional intestinal adenoma, organoids and colorectal cancer

Abstract

Abstract TGF-β ligands suppress growth yet can paradoxically and potently promote cancer invasion and metastasis depending on downstream pathway mutational context, such as loss of Mothers against decapentaplegic homolog 4 (Smad4). Here, we characterised phenotypes and associated gene expression signatures in conditional murine intestinal adenoma with and without Smad4. Conditional Lgr5-CreERT2 activation in Apc fl/fl Smad4 fl/fl mice resulted in homozygote floxed alleles (Apc Δ/Δ Smad4 Δ/Δ) and adenoma formation. The adenoma phenotype was discordant, with 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 (IC50 534 pM) compared to Apc Δ/Δ Smad4 +/+ (IC50 24 pM). TGF-β1 (390 pM) altered adenoma bulk mRNA expression 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 Apc fl/fl Smad4 fl/fl adenoma organoids, only 7 human equivalent genes were differentially expressed in SMAD4 mutated colorectal cancer (TCGA cohorts), including ID1 low . SMAD4 low , ID1 low SPP1 high and PAK3 high all correlated with poorer survival. Murine adenoma identified Smad4 dependent gene expression signatures that require further evaluation as functional biomarker classifiers of SMAD4 mutated cancer subtypes.