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^T2 activation in Apc^fl/flSmad4^fl/fl mice resulted in homozygote floxed alleles (Apc^Δ/ΔSmad4^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/ΔSmad4^Δ/Δ adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC₅₀ 534 pM) compared to Apc^Δ/ΔSmad4^+/+ (IC₅₀ 24 pM). TGF-β1 (390 pM) altered adenoma bulk mRNA expression most significantly for Id1^low and Spp1^high in Apc^Δ/ΔSmad4^Δ/Δ. Single cell RNAseq of caecal adenoma identified expansion of Lgr5^low, Pak3^high and Id1^low progenitor populations in Apc^Δ/ΔSmad4^Δ/Δ. Of the 76 Smad4 and TGF-β1 dependent genes identified in Apc^fl/flSmad4^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.