The Epigenetic Basis of Phenotypic Plasticity in Colon Cancer Metastasis
Carcinomas of the breast, prostate, pancreas, and colon are thought to arise from normal tissues along a multistep progression from precursor lesions to increasingly more invasive stages. Along this sequence of events, a stepwise accumulation of genetic alterations in specific tumor suppressors and oncogenes is regarded as the driving force in tumor initiation and progression towards malignancy. These alterations reflect well-defined cellular changes, the hallmarks of cancer, which provide growth advantage to the developing tumor cell and are thought to represent essential requirements for cancer onset and progression towards malignancy. However, with regard to the capacity to invade the tumor microenvironment and form distant metastases, phenotypic plasticity is possibly the most clinically relevant hallmark of cancer cells. It is now well-established that malignant cells and in particular those responsible for local dissemination and distant metastasis are endowed with plasticity, i.e. the capacity to undergo transient and reversible morphological and functional changes. In particular, epithelial to mesenchymal transition (EMT), i.e. the loss of epithelial identity and acquisition of a migratory and more mesenchymal phenotype, is regarded as the crucial event in invasion and dissemination. Of note, EMT endows cancer cells with stem-like features which underlie their phenotypic plasticity and capacity not only to acquire mesenchymal morphology at the invasive front of the primary tumor but also to reverse back to more epithelial states (MET, mesenchymal to epithelial transition) at the metastasis site. Epigenetic alterations at EMT-inducing transcription factors (EMT-TFs) underlie the transient nature of these cellular modifications.
This migrating cancer stem cell (mCSC) model has been first proposed for colorectal cancer. It was proposed that secreted factors from the tumor microenvironment are likely to elicit full blown Wnt signaling, earmarked by nuclear b-catenin, and EMT in cells located at the invasive front. However, the identity of these secreted stromal factors and the molecular and cellular mechanisms underlying Wnt and EMT activation at the invasive front of colon cancers are yet largely unknown also due to a lack of robust in vitro an in vivo models.
In this project proposal, by taking advantage of the results obtained in the Dutch Digestive Foundation (MLDS) project FP 15-08 entitled “EpCAMlow cancer stem cells: the culprit of liver metastasis in colon cancer?” we aim to elucidate the epigenetic alterations likely to characterize the CD44highEpCAMlow cells when compared with their more committed and epithelial counterpart (CD44highEpCAMhigh). This will be initially implemented by ChIP-Seq of the sorted CD44highEpCAMlow and CD44highEpCAMhigh subpopulations followed by bioinformatics analysis and integration with the already available RNAseq data from the same cell subpopulations.
As a second and more in-depth approach, we will combine ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) and RNA-seq analysis at the single cell level to couple specific epigenetic alterations (likely to occur at chromatin remodeling genes) and the corresponding gene expression (RNA) changes, and the EMT-state of the individual CD44highEpCAMlowcells and their metastatic potential.
This approach is expected to result in the identification of not only key therapeutic and diagnostic gene targets in colon cancer but also of specific cellular identities to be searched in patient-derived CTCs. In view of the EMT-like, invasive, and metastatic features of CD44highEpCAMlow cells, our analyses will likely identify new upstream regulatory sequences and their corresponding genes. Follow-up experiments will include the use of in vitro (cell lines, organoids) and in vivo preclinical mouse models to assess these validity of these targets. Last, CTCs directly isolated from the portal blood of late-stage patients undergoing colon cancer surgery will be employed to assess the validity of the newly identified target genes and CTC markers and their diagnostic, prognostic, and therapeutic potential.