BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells

To decrease bowel cancer incidence and improve survival, we need to understand the mechanisms that drive tumorigenesis. Recently, B-cell lymphoma 3 (BCL-3; a key regulator of NF-κB signalling) has been recognised as an important oncogenic player in solid tumours. Although reported to be overexpressed in a subset of colorectal cancers (CRCs), the role of BCL-3 expression in colorectal tumorigenesis remains poorly understood. Despite evidence in the literature that BCL-3 may interact with β-catenin, it is perhaps surprising, given the importance of deregulated Wnt/β-catenin/T-cell factor (TCF) signalling in colorectal carcinogenesis, that the functional significance of this interaction is not known. Here, we show for the first time that BCL-3 acts as a co-activator of β-catenin/TCF-mediated transcriptional activity in CRC cell lines and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. We demonstrate that targeting BCL-3 expression (using RNA interference) reduced β-catenin/TCF-dependent transcription and the expression of intestinal stem cell genes LGR5 and ASCL2. In contrast, the expression of canonical Wnt targets Myc and cyclin D1 remained unchanged. Furthermore, we show that BCL-3 increases the functional stem cell phenotype, as shown by colorectal spheroid and tumoursphere formation in 3D culture conditions. We propose that BCL-3 acts as a driver of the stem cell phenotype in CRC cells, potentially promoting tumour cell plasticity and therapeutic resistance. As recent reports highlight the limitations of directly targeting cancer stem cells (CSCs), we believe that identifying and targeting drivers of stem cell plasticity have significant potential as new therapeutic targets. This article has an associated First Person interview with the first author of the paper

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of β-catenin we carried out the first nuclear/cytoplasmic proteomic analysis of the β-catenin interactome in myeloid leukemia cells and identified putative novel β-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear β-catenin) versus Wnt-unresponsive cells (low nuclear β-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of β-catenin. The relative levels of nuclear LEF-1 and β-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed β-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and β-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first β-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear β-catenin level human myeloid leukemia

p16INK4a polymorphism: Associations with tumour progression in patients with sporadic colorectal cancer

Deregulated tumour expression of p16INK4a has previously been described in association with clinical progression in sporadic colorectal cancer patients (CRC). Furthermore, p16INK4a promoter hypermethylation leading to gene silencing has been shown to occur in advanced colorectal tumours and has been associated with patient survival. p16INK4a is polymorphic, with variant alleles being associated with tumour progression in melanoma. In this study we have examined p16INK4a polymorphism as a marker of tumour progression in sporadic CRC. Polymorphic sites G/A(442), C/G(500), and C/T(540), were studied, these alleles obeyed Hardy Weinberg equilibrium in a control group, but not in the CRC cases. G/A(442) and CG(500) alleles were in linkage disequilibrium in both cases and controls. In controls the C/T(540) alleles demonstrated no linkage with either other site, whilst an association was demonstrated between C/G(500) and C/T(540) alleles in the cases (p=0.011). Furthermore, the distribution of C/T(540) genotypes was different between the groups (p=0.002). Within the CRC cases, patients with the GG(442) genotype were more commonly associated with decreased tumour differentiation (p=0.018), advancing Dukes' stage (p=0.006) and T-stage (p=0.007) than patients with the GA(442) and AA(442) genotypes. Patients with the CC(500) genotype were more commonly associated with decreased tumour differentiation (p=0.012), advancing Dukes' stage (p=0.015), and N-stage (p=0.031). No associations between patient C/T(540) genotype and clinical prognostic parameters were found. An analysis of patient tumour expression with p16INK4a genotype revealed patients with the CC(500) genotype were more commonly associated with reduced tumour p16 expression (p=0.046). In summary our data indicate that p16INK4a polymorphism is associated with tumour progression in patients with sporadic CRC

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of β-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the β-catenin interactome in myeloid leukemia cells and identified putative novel β-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear β-catenin) versus Wnt-unresponsive cells (low nuclear β-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of β-catenin. The relative levels of nuclear LEF-1 and β-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed β-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and β-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first β-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear β- catenin level in human myeloid leukemia

LEF-1 drives aberrant ß-catenin nuclear localization in myeloid leukemia cells

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß-catenin level human myeloid leukemia

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field