Implications of Epithelial–Mesenchymal Plasticity for Heterogeneity in Colorectal Cancer

Colorectal cancer (CRC) is a genetically heterogeneous disease that develops and progresses through several distinct pathways characterized by genomic instability. In recent years, it has emerged that inherent plasticity in some populations of CRC cells can contribute to heterogeneity in differentiation state, metastatic potential, therapeutic response, and disease relapse. Such plasticity is thought to arise through interactions between aberrant signaling events, including persistent activation of the APC/β-catenin and KRAS/BRAF/ERK pathways, and the tumor microenvironment. Here, we highlight key concepts and evidence relating to the role of epithelial-mesenchymal plasticity as a driver of CRC progression and stratification of the disease into distinct molecular and clinicopathological subsets

Implications of epithelial–mesenchymal plasticity for heterogeneity in colorectal cancer

Colorectal cancer (CRC) is a genetically heterogeneous disease that develops and progresses through several distinct pathways characterized by genomic instability. In recent years, it has emerged that inherent plasticity in some populations of CRC cells can contribute to heterogeneity in differentiation state, metastatic potential, therapeutic response, and disease relapse. Such plasticity is thought to arise through interactions between aberrant signaling events, including persistent activation of the APC/β-catenin and KRAS/BRAF/ERK pathways, and the tumor microenvironment. Here, we highlight key concepts and evidence relating to the role of epithelial-mesenchymal plasticity as a driver of CRC progression and stratification of the disease into distinct molecular and clinicopathological subsets.This work was supported by grants from the National Health and Medical Research Council of Australia (to Amardeep Singh Dhillon)

Widespread FRA1-Dependent Control of Mesenchymal Transdifferentiation Programs in Colorectal Cancer Cells

Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs), and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT)-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression.This work was supported by project grants 1026228 and 1044168 (to A.S.D.) and Senior Research Fellowships (to R.D.H., R.B.P. and J.M.M.) from the National Health and Medical Research Council of Australia

Oncogenic B-Raf mutations Crystal clear at last

AbstractThe Raf/MEK/ERK pathway is a conserved signaling module controlling cell growth, proliferation, apoptosis, and differentiation. Constitutive activation of this pathway is involved in malignant transformation by several oncogenes, most notably, Ras. The recent discovery by Davies et al. of somatic mutations in the B-RAF gene in human tumors has generated enormous interest in how Raf kinases are regulated and how mutations in B-RAF lead to transformation. A recent study in Cell by Wan et al. reports the crystal structure of the B-Raf kinase domain, providing important new insights into these questions

Regulation of Raf-1 activation and signalling by dephosphorylation

The Raf-1 kinase is regulated by phosphorylation, and Ser259 has been identified as an inhibitory phosphorylation site. Here we show that the dephosphorylation of Ser259 is an essential part of the Raf-1 activation process, and further reveal the molecular role of Ser259. The fraction of Raf-1 that is phosphorylated on Ser259 is refractory to mitogenic stimulation. Mutating Ser259 elevates kinase activity because of enhanced binding to Ras and constitutive membrane recruitment. This facilitates the phosphorylation of an activating site, Ser338. The mutation of Ser259 also increases the functional coupling to MEK, augmenting the efficiency of MEK activation. Our results suggest that Ser259 regulates the coupling of Raf-1 to upstream activators as well as to its downstream substrate MEK, thus determining the pool of Raf-1 that is competent for signalling. They also suggest a new model for Raf-1 activation where the release of repression through Ser259 dephosphorylation is the pivotal step

Cyclic AMP-Dependent Kinase Regulates Raf-1 Kinase Mainly by Phosphorylation of Serine 259

The Raf-1 kinase activates the ERK (extracellular-signal-regulated kinase) pathway. The cyclic AMP (cAMP)-dependent protein kinase (PKA) can inhibit Raf-1 by direct phosphorylation. We have mapped all cAMP-induced phosphorylation sites in Raf-1, showing that serines 43, 259, and 621 are phosphorylated by PKA in vitro and induced by cAMP in vivo. Serine 43 phosphorylation decreased the binding to Ras in serum-starved but not in mitogen-stimulated cells. However, the kinase activity of a RafS43A mutant was fully inhibited by PKA. Mutation of serine 259 increased the basal Raf-1 activity and rendered it largely resistant to inhibition by PKA. cAMP increased Raf-1 serine 259 phosphorylation in a PKA-dependent manner with kinetics that correlated with ERK deactivation. PKA also decreased Raf-1 serine 338 phosphorylation of Raf-1, previously shown to be required for Raf-1 activation. Serine 338 phosphorylation of a RafS259A mutant was unaffected by PKA. Using RafS259 mutants we also demonstrate that Raf-1 is the sole target for PKA inhibition of ERK and ERK-induced gene expression, and that Raf-1 inhibition is mediated mainly through serine 259 phosphorylation

Phylogenetic and Expression Analysis of Fos Transcription Factors in Zebrafish

Members of the FOS protein family regulate gene expression responses to a multitude of extracellular signals and are dysregulated in several pathological states. Whilst mouse genetic models have provided key insights into the tissue-specific functions of these proteins in vivo, little is known about their roles during early vertebrate embryonic development. This study examined the potential of using zebrafish as a model for such studies and, more broadly, for investigating the mechanisms regulating the functions of Fos proteins in vivo. Through phylogenetic and sequence analysis, we identified six zebrafish FOS orthologues, fosaa, fosab, fosb, fosl1a, fosl1b, and fosl2, which show high conservation in key regulatory domains and post-translational modification sites compared to their equivalent human proteins. During embryogenesis, zebrafish fos genes exhibit both overlapping and distinct spatiotemporal patterns of expression in specific cell types and tissues. Most fos genes are also expressed in a variety of adult zebrafish tissues. As in humans, we also found that expression of zebrafish FOS orthologs is induced by oncogenic BRAF-ERK signalling in zebrafish melanomas. These findings suggest that zebrafish represent an alternate model to mice for investigating the regulation and functions of Fos proteins in vertebrate embryonic and adult tissues, and cancer