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

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-CreER activation in ApcSmad4 mice resulted in homozygote floxed alleles (ApcSmad4) 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 ApcSmad4 adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC 534 pM) compared to ApcSmad4 (IC 24 pM). TGF-β1 (390 pM) altered adenoma bulk mRNA expression most significantly for Id1 and Spp1 in ApcSmad4. Single cell RNAseq of caecal adenoma identified expansion of Lgr5, Pak3 and Id1 progenitor populations in ApcSmad4. Of the 76 Smad4 and TGF-β1 dependent genes identified in ApcSmad4 adenoma organoids, only 7 human equivalent genes were differentially expressed in SMAD4 mutated colorectal cancer (TCGA cohorts), including ID1. SMAD4, ID1 SPP1 and PAK3 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.