Disturbed epigenetic pathways emerged as important contributors to tumor occurrence and to tumor progression, which was only recently uncovered through cancer-sequencing projects. These projects also revealed a high degree of tissue- and tumor-specificity in the growth factor signaling and chromatin regulatory pathways affected. TGF-β signaling is one of the major signal transduction pathways affected in colorectal cancers (CRC). Activation of the TGF-β pathway is controlling epithelial-to-mesenchymal transition (EMT) of the colonic epithelial cells and thus, this pathway is highly relevant for the invasive and metastatic behavior of CRC cells. TGF-β signaling culminates in transcription control of target genes through activation of the SMAD2/SMAD3/SMAD4 transcription factor complex.
Mutations in the SMAD2, SMAD3 and SMAD4 genes occur in ~15% of CRCs. In contrast, the BMP signaling pathway acts downstream in the EMT process and BMP signaling is not disturbed by gene mutations in CRC. TGF-β SMAD mutations are almost exclusively missense and clustered in hotspot regions in their MH2 domains known to mediate protein-protein interactions. We hypothesize that many of TGF-β SMAD mutations in CRC are gain-of-function. This will be investigated in different CRC cell systems to examine the interactomes of hotspot SMAD2, SMAD3 and SMAD4 mutations using our quantitative proteomics pipeline. CRC-specific SMAD2, SMAD3 and SMAD4 interactors will be examined in functional assays for cell proliferation, motility, invasion and other EMT-related parameters.
Our recent work linked the MLL4 histone methyltransferase complex to TGF-β mediated activation of gene transcription via the SMAD2, SMAD3 and SMAD4 proteins. These studies will be expanded to CRC cell lines in order to examine: i) the effect of CRC-mutations in SMAD2, SMAD3 and SMAD4 on MLL4 complex subunits interactions, ii) the contributions of SET1/MLL complexes and, in particular MLL4, to TGF-β mediated transcriptional regulation and TGF-β dependent alterations in (global) H3K4 and H3K27 modification patterns, iii) genome-wide binding patterns of SMAD2, SMAD3 and SMAD4 (CRC-mutant) proteins during TGF-β activation by implementing the innovative ChECseq technology, and iv) involvement of H3K4 and H3K27 methylation in TGF-β mediated EMT and cell motility.
Overall, these investigations will provide an integrative model for epigenetic regulation of TGF-β signaling and of SMAD function through histone H3K4 and H3K27 methylation control in CRC cell systems. Our work will determine the therapeutic potential of rebalancing histone methylation pathways in colorectal cancer.