SMAD4 mutations do not preclude epithelial–mesenchymal transition in colorectal cancer

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 (SMAD4(mut)) cancer cells are unable to undergo EMT. Here, we scrutinized this notion and probed for potential SMAD4-independent EMT execution using SMAD4(mut) CRC cell lines. We show that SMAD4(mut) cells exhibit morphological changes, become invasive, and regulate EMT marker genes upon induction of the EMT-TF SNAIL1. Furthermore, SNAIL1-induced EMT in SMAD4(mut) 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 SMAD4(mut) 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 SMAD4(mut) and SMAD4 wild-type (SMAD4(wt)) 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 SMAD4(mut) tumors, which is relevant for their diagnostic and therapeutic evaluation.