Matjaz Rokavec

14-3-3σ is a p53-inducible gene with tumor suppressive properties and SMAD4 is a transcription factor encoded by a tumor suppressor gene, which is commonly inactivated in colorectal cancer (CRC). Here, 14-3-3σ was characterized as direct transcriptional target of SMAD4. TGF-β treatment of tumoroids derived from CRC patients and mouse models resulted in a SMAD4 -dependent induction of 14-3-3σ . In murine, intestinal epithelia, the apical expression of 14-3-3σ was dependent on Smad4 . Ectopic SMAD4 or 14-3-3σ promoted mesenchymal-to-epithelial transition (MET) and suppressed invasion, migration, and autophagy of CRC cells. As experimental inactivation of 14-3-3σ abolished these tumor-suppressive functions of SMAD4, 14-3-3σ mediates these effects of SMAD4. Inhibition of autophagy and promotion of MET by SMAD4 was mediated by inhibition of TFEB via binding and sequestration of TFEB by 14-3-3σ. The association of 14-3-3σ and TFEB was dependent on phosphorylation of the TFEB serine 211 residue, which is a target of mTORC1. Taken together, the TGF-β/SMAD4/14-3-3σ/TFEB axes characterized here antagonizes epithelial plasticity and autophagy. Thereby, it may ultimately suppress the progression of CRC and other types of cancer.

Down-regulation of the RNA-binding motif protein 47 (RBM47) frequently occurs in colorectal cancer (CRC) and is associated with poor prognosis. However, the downstream effectors of RBM47 have remained unknown. Therefore, we performed a comprehensive RNA-Seq analysis after inactivation or ectopic expression of RBM47. Gasdermin A/GSDMA, a poorly characterized member of the gasdermin family highly expressed in gastrointestinal epithelium, was identified as the most differentially regulated transcript. RBM47 directly bound to the 3′-untranslated region of GSDMA mRNA and stabilized it. Consistently, ectopic RBM47 increased GSDMA mRNA and protein expression, whereas RBM47 knockdown had the opposite effect. GSDMA was necessary for the RBM47-induced mesenchymal-to-epithelial transition (MET) and suppression of migration and invasion by RBM47 in CRC cells. Moreover, activation of the RBM47/GSDMA axis triggered pyroptosis, a form of cell death characterized by cell swelling, plasma membrane rupture, and, therefore, immunogenic effects. Both RBM47 and GSDMA enhanced sensitivity to Oxaliplatin through the induction of MET and pyroptosis. Knockdown of GSDMA abolished RBM47-mediated pyroptosis and chemo-sensitization. Analysis of CRC patient cohorts revealed that RBM47 expression correlates with response to FOLFOX chemotherapy. Therefore, our results establish GSDMA as a critical downstream mediator of RBM47-induced tumor suppression that links epithelial differentiation and pyroptosis to chemotherapy sensitivity. Finally, these findings identify the RBM47/GSDMA axis as a potential predictive biomarker for the response to Oxaliplatin in CRC patients.

Upregulated genes in SMAD4 mutant tumor organoid lines and DepMAP Project dependency scores.

Characteristics of tumor organoid lines used for CRISPR/Cas9-mediated SMAD4 deletion.

Supplementary Figures S1-5 S1: Effect of SMAD4 loss on intestinal stem cell, c-MYC, and NLE1 gene expression in CRC organoids S2: Effects of NLE1 ablation on growth and clonogenicity of CRC cell lines S3: Effects of NLE1 ablation on growth and clonogenicity of CRC PDTOs S4: Effects of NLE1 over-expression in human colonic epithelial cells and CRC organoids S5: NLE1 loss-mediated cell cycle arrest and apoptosis in HCT116 CRC cells and HCEC-1CT normal human colonic epithelial cells

Supplementary Materials Oligonucleotides, qRT-PCR primer pairs, and Taqman probe assays used in this study.

Endoscopy-guided orthotopic injection of colorectal cancer organoids into the colonic wall of immune-deficient mice.

Differentially expressed genes in PDTOS4 versus PDTO cells in the presence of recombinant TGF-beta1