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Updated: May 14, 2025
Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications
Published on: March 8, 2024
Smad4 and TGFβ1 dependent gene expression signatures in conditional intestinal adenoma, organoids and colorectal
Mirvat Surakhy1, Julia Matheson1, David J Barnes1,2
1Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK.
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 Apcfl/flSmad4fl/fl mice resulted in homozygote floxed alleles (ApcΔ/ΔSmad4Δ/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 ApcΔ/ΔSmad4Δ/Δ adenoma organoids resisted TGF-β1 dose dependent growth arrest and cell death (IC50 534 pM) compared to ApcΔ/ΔSmad4+/+ (IC50 24 pM). TGF-β1 (390 pM) altered adenoma bulk mRNA expression most significantly for Id1low and Spp1high in ApcΔ/ΔSmad4Δ/Δ. Single cell RNAseq of caecal adenoma identified expansion of Lgr5low, Pak3high and Id1low progenitor populations in ApcΔ/ΔSmad4Δ/Δ. Of the 76 Smad4 and TGF-β1 dependent genes identified in Apcfl/flSmad4fl/fl adenoma organoids, only 7 human equivalent genes were differentially expressed in SMAD4 mutated colorectal cancer (TCGA cohorts), including ID1low. SMAD4low, ID1low SPP1high and PAK3high 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.
Insights
Loss of Mothers against decapentaplegic homolog 4 (Smad4) in mice altered TGF-β1 response in intestinal adenomas. Smad4 loss created distinct gene expression signatures linked to poorer patient survival in colorectal cancer.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Transforming growth factor-beta (TGF-β) signaling plays a dual role in cancer, suppressing growth but promoting invasion and metastasis under specific mutational contexts.
- Loss of Mothers against decapentaplegic homolog 4 (Smad4) is a key factor influencing TGF-β's paradoxical effects in cancer progression.
Purpose of the Study:
- To characterize the phenotypic and gene expression differences in murine intestinal adenomas with and without Smad4.
- To investigate the impact of Smad4 loss on TGF-β1 response in adenoma organoids.
- To identify potential biomarkers for SMAD4-mutated colorectal cancer.
Main Methods:
- Conditional knockout of Apc and Smad4 in murine intestinal epithelium using Lgr5-CreERT2.
- Generation and characterization of ApcΔ/ΔSmad4Δ/Δ and ApcΔ/ΔSmad4+/+ adenoma organoids.
- TGF-β1 dose-response assays on organoids.
- Bulk and single-cell RNA sequencing (scRNAseq) of adenoma tissues.
- Analysis of The Cancer Genome Atlas (TCGA) cohorts for human colorectal cancer data.
Main Results:
- ApcΔ/ΔSmad4Δ/Δ mice exhibited reduced small intestinal adenoma burden but developed large, non-metastatic caecal adenomas.
- Smad4-deficient adenoma organoids showed significantly increased resistance to TGF-β1-induced growth arrest and cell death compared to Smad4-wildtype organoids.
- TGF-β1 treatment in Smad4-deficient adenomas led to decreased Id1 and increased Spp1 expression.
- scRNAseq identified an expansion of Lgr5low, Pak3high, and Id1low progenitor populations in Smad4-deficient adenomas.
- Only 7 of 76 Smad4 and TGF-β1-dependent genes in murine models were differentially expressed in human SMAD4-mutated colorectal cancer, including ID1.
- Lower SMAD4, ID1 expression, and higher SPP1 and PAK3 expression correlated with poorer patient survival.
Conclusions:
- Loss of Smad4 alters TGF-β1 signaling in intestinal adenomas, leading to distinct cellular phenotypes and gene expression profiles.
- Specific gene expression signatures, including ID1low, SPP1high, and PAK3high, associated with Smad4 deficiency in mice, are also linked to poor prognosis in human colorectal cancer.
- These findings highlight the potential of Smad4-dependent gene expression signatures as functional biomarkers for classifying SMAD4-mutated colorectal cancer subtypes.
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