p53 tumor suppressor maintains genomic stability, typically acting through cell-cycle arrest, senescence, and apoptosis. We discovered a function of p53 in preventing conflicts between transcription and replication, independent of its canonical roles. p53 deficiency sensitizes cells to Topoisomerase (Topo) II inhibitors, resulting in DNA damage arising spontaneously during replication. Topoisomerase IIα (TOP2A)-DNA complexes preferentially accumulate in isogenic p53 mutant or knockout cells, reflecting an increased recruitment of TOP2A to regulate DNA topology. We propose that p53 acts to prevent DNA topological stress originating from transcription during the S phase and, therefore, promotes normal replication fork progression. Consequently, replication fork progression is impaired in the absence of p53, which is reversed by transcription inhibition. Pharmacologic inhibition of transcription also attenuates DNA damage and decreases Topo-II-DNA complexes, restoring cell viability in p53-deficient cells. Together, our results demonstrate a function of p53 that may underlie its role in tumor suppression.

Loss of primary cilia is a key feature of von Hippel-Lindau tumor suppressor (VHL)-associated pathology. Although VHL-deficiency predisposes cells to precipitous cilia disassembly in response to growth factor cues, it does not affect ciliogenesis. Here, using a siRNA-based screen to find genes that are essential for ciliogenesis only in the presence of the VHL tumor suppressor gene product pVHL, we identify ubiquitin-specific protease (USP)8. The pVHLdependency of USP8 for ciliogenesis is directly linked to its function as a HIF1a deubiquitinating enzyme. By counteracting pVHL-mediated ubiquitination of HIF1a, USP8 maintains a basal expression of HIF1a and HIF transcriptional output in normoxia, including the repression of Rabaptin5, which is essential for endosome trafficking-mediated ciliogenesis.

CitationTumor-derived circulating endothelial cell clusters in colorectal cancer. 2016, 8 (345) * Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumorderived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

Tissue engineering is a promising approach to implement endothelial cells as a cellular delivery therapy for vascular disease. We and others previously demonstrated that endothelial cells embedded in three-dimensional collagen-based matrices retain their full biosecretory spectrum, enabling them to serve as powerful regulators of vascular diseases. Fascinatingly, matrix embedding of endothelial cells not only allows for their implantation but also seems to provide protection from allo-and xenogeneic-triggered host immune responses. This is not an effect of simple physical shielding but a more fundamental influence of cell-matrix interconnectivity on the cellular immune phenotype. Reduced cytokine-induced levels of costimulatory and adhesion molecules associated with significantly lower expression levels of major histocompatibility class II expression on matrix-embedded human aortic endothelial cells when compared to the same cells cultured on two-dimensional polystyrene coated-tissue culture plates. Strikingly, the entire interferon-γ-dependent signaling cascade resulting in MHC class II molecule expression is markedly suppressed in endothelial cells grown to confluence within three-dimensional scaffolds. These findings might be of pivotal importance for designing endothelial cell-based therapies in general and might enhance our understanding of the underlying pathophysiology in a broad range of cardiovascular diseases (e.g., atherosclerosis, vasculitis, chronic allograft vasculopathy).