Abstract
Transforming growth factor beta (TGFβ) superfamily signaling is a prime inducer of epithelial-mesenchymal transitions (EMT) that foster cancer cell invasion and metastasis, a major cause of cancer-related deaths. Yet, TGFβ signaling is frequently inactivated in human tumor entities including colorectal cancer (CRC) and pancreatic adenocarcinoma (PAAD) with a high proportion of mutations incapacitating SMAD4, which codes for a transcription factor (TF) central to canonical TGFβ and bone morphogenetic protein (BMP) signaling. Beyond its role in initiating EMT, SMAD4 was reported to crucially contribute to subsequent gene regulatory events during EMT execution. It is therefore widely assumed that SMAD4-mutant (SMAD4mut) cancer cells are unable to undergo EMT. Here, we scrutinized this notion and probed for potential SMAD4-independent EMT execution using SMAD4mut CRC cell lines. We show that SMAD4mut cells exhibit morphological changes, become invasive, and regulate EMT marker genes upon induction of the EMT-TF SNAIL1. Furthermore, SNAIL1-induced EMT in SMAD4mut cells was found to be entirely independent of TGFβ/BMP receptor activity. Global assessment of the SNAIL1-dependent transcriptome confirmed the manifestation of an EMT gene regulatory program in SMAD4mut cells highly related to established EMT signatures. Finally, analyses of human tumor transcriptomes showed that SMAD4 mutations are not underrepresented in mesenchymal tumor samples and that expression patterns of EMT-associated genes are similar in SMAD4mut and SMAD4 wild-type (SMAD4wt) cases. Altogether, our findings suggest that alternative TFs take over the gene regulatory functions of SMAD4 downstream of EMT-TFs, arguing for considerable plasticity of gene regulatory networks operating in EMT execution. Further, they establish that EMT is not categorically precluded in SMAD4mut tumors, which is relevant for their diagnostic and therapeutic evaluation.
Highlights
The TGFβ superfamily comprises the TGFβ and bone morphogenetic protein (BMP) signaling pathways, whose activities have profound effects in developmental processes, adult tissue homeostasis, injury and wound healing, and in disease [1, 2]
No SMAD4 protein expression could be observed in HT29 and SW403 cells in Western blot experiments using a SMAD4 antibody that recognizes an epitope encoded by SMAD4 exon 5 and would have allowed detection of shorter protein forms which might have potentially been generated from the mutant alleles [26] (Fig. 1c)
Cancer Genome Atlas (TCGA) database of colon adenocarcinoma (COAD) transcriptomes, we found that SMAD4mut tumors are not underrepresented among consensus molecular subtype 4 (CMS4) samples when compared to other subtypes or all tumors in the database (Fig. 7b), raising the possibility that SMAD4 is not obligatory for epithelial-mesenchymal transitions (EMT) in vivo
Summary
The TGFβ superfamily comprises the TGFβ and BMP signaling pathways, whose activities have profound effects in developmental processes, adult tissue homeostasis, injury and wound healing, and in disease [1, 2]. TGFβ/BMP signaling processes are initiated upon ligand-induced complex formation by two pairs of type I and type II transmembrane receptor proteins with intrinsic serine/ threonine kinase activity [1, 2]. Following tetramerization and phosphorylation by type II subunits, type I receptors recognize and phosphorylate receptor-regulated or R-SMAD proteins. These are SMAD2 and SMAD3 (SMAD2/3) in case of TGFβ receptors, and SMAD1, SMAD5, and SMAD8 (SMAD1/5/8) for BMP receptors. Aside from canonical, SMAD-dependent processes, TGFβ and BMP receptors can trigger SMAD-independent cellular responses by signaling via mitogen-activated protein kinases and other signal transducers [1,2,3]
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