Discoveries

Drivers of Intestinal Tumorigenesis

December 6, 2017

Drivers of Intestinal Tumorigenesis

​A hallmark of all epithelia is the presence of adherens junctions that connect adjacent cells to each other. The junctions are formed through the interaction of the extracellular domains of E-cadherin on the neighboring cells. In turn, the intracellular domain of E-cadherin forms a complex with other proteins, including p120-catenin (p120), α-catenin, and β-catenin, that provides a link to actin filaments in the underlying cytoskeleton. Considerable evidence supports a role for E-cadherin and other adherens junction proteins as tumor suppressors.  Now, work from Basic Sciences investigator Albert Reynolds, along with his collaborators, Robert Coffey (Department of Medicine), Nancy Jenkins and Neal Copeland (Methodist Hospital Research Institute, Houston, TX), and Michael Payne (Chulalongkorn University, Bangkok), provides important new insight in the role of p120 in tumor suppression. Their work focused on mouse models of intestinal tumorigenesis driven by mutations in the gene encoding the adenomatous polyposis coli (APC) protein. The investigators created a new model in these mice in which the gene for p120 could be selectively knocked out in the intestine to varying degrees. They discovered that knockout of p120 led to a substantial increase in tumors in the mice. However, the tumors that were generated consistently contained one wild-type and one knocked out p120 gene; there were no tumors in which p120 expression was totally ablated. Further work showed some regions in the intestines that exhibited a total loss of p120, but all of these were normal epithelium. Time course studies revealed the presence of regions of total p120 loss in early tumor formation, but these were lost over a period of several weeks. When the researchers established APC-driven tumor organoids in culture, they discovered that total knockout of p120 led to a marked increase in proliferation and a change to a branched morphology, but the organoids exhibited rapid death by apoptosis unless an inhibitor of Rho kinase was also present in the culture medium. The results indicate that reduction of p120 levels due to loss of one gene for the protein promotes APC-driven tumorigenesis, but complete loss is fatal to tumor cells. Further work using the Sleeping Beauty transposon to detect gene derangements that drive intestinal tumorigenesis indicated that deficiencies of p120, E-cadherin, and α-catenin are all very strong tumor drivers. The findings provide important new information for the design of therapies directed towards mechanisms that specifically lead to cancer in the intestinal tract. The work is published in the Journal of Clinical Investigation (S. P. Short et al. J. Clin. Invest., 2017, doi:10.1172/JCI77217).

Tracing Cell Origins in the Gut

December 5, 2017

Tracing Cell Origins in the Gut

The organs of multicellular animals comprise highly organized aggregates of many cell types, each of which has differentiated from a multi-potent stem cell. Although we have learned much about the process of differentiation and organogenesis through studies of tissues such as bone marrow, tracing the lineages of cells in solid organs remains a challenge. New techniques of single-cell analysis that enable the simultaneous characterization of large numbers of cells should lead to substantial progress in identification of transitional cell populations. However, these techniques generate huge datasets that require appropriate computational approaches for their analysis. Thus far, most such approaches have been limited to the identification of single branch points in a differentiation pathway, and statistical methods for the comparative evaluation of multiple pathways have been lacking. This led Vanderbilt Basic Sciences investigator Ken Lau, in collaboration with Robert Coffey (Department of Medicine) and Michael Gerdes (GE Global Research) to develop p-Creode, a computational method for the evaluation of large datasets obtained from single cell analysis. Major advantages to p-Creode are that no prior assumptions about the nature, number, or arrangements of branch points in a differentiation pathway are necessary, multiple branch points can be identified, and robust statistical analysis enables comparison of multiple pathways. To test their approach, the investigators first used p-Creode to analyze publicly available single cell mass-cytometry data from human bone marrow. Hematopoiesis, the differentiation of blood cells in bone marrow is very well-defined, and p-Creode generated a differentiation hierarchy that matched the known process. The researchers then turned to a single cell multiplex immunofluorescence (MxIF) data set of intestinal and colonic epithelial cells. The results identified known mature cells in each case, as well as the expected lineages for the differentiation of those cells. However, results suggested that tuft cells were derived from different lineages in the intestine and the colon. This unexpected finding led to follow-up studies that confirmed that tuft cells arise from absorptive cell lineages, rather than secretory lineages as previously thought in the intestine, whereas in the colon, they branch off very close to progenitor cells. Analysis of a single cell RNA-seq dataset from mouse colon revealed additional new cell transition relationships. These findings support p-Creode as a robust and versatile approach for the elucidation of cell lineages in complex populations from single cell datasets. We look forward to the results of further applications of p-Creode in the future. The work is published in the journal Cell Systems [C.A. Herring, et al. Cell Systems, (2017) published online November 15, DOI: 10.1016/j.cels.2017.10.012].

The Road to Ubiquitin’s Destruction

December 5, 2017

The Road to Ubiquitin’s Destruction

Ubiquitin is a small (8.5 kDa) protein that is attached singly, or in chains, to lysine residues of other proteins via a complex, three step mechanism. The pattern of ubiquitin addition, referred to as ubiquitination, marks the protein for degradation, alters its function and interactions with other proteins, or modulates its transit via endocytosis. Recent research suggests that abnormalities in the ubiquitin pathway play a role in diseases that involve aberrant accumulation of proteins as is seen in Alzheimer’s, Parkinson’s, and Huntington’s diseases. To better understand the role that ubiquitination may play in such diseases requires a full understanding of the dynamics of the ubiquitin protein itself, leading Vanderbilt Basic Sciences investigators Jason MacGurn and Walter Chazin along with their laboratories to explore mechanisms of ubiquitin turnover. The studies began with the discovery of two phosphatase enzymes that affect the turnover of ion channels in yeast. Phosphoproteomics analysis of yeast mutants lacking these enzymes revealed an increased level of phosphorylation of ubiquitin at Ser-57. Further studies in yeast expressing a Ser57Ala mutant ubiquitin, which cannot be phosphorylated, or a Ser57Asp mutant ubiquitin, which mimics permanent phosphorylation, showed that phosphorylation at Ser-57 increases the turnover rate of the protein. Phosphorylation at Ser-57 also increased the rate of turnover of membrane-associated ion channels through the endocytic pathway. To explain the link between endocytic turnover of membrane proteins and ubiquitin, the investigators showed that phosphorylation of Ser-57 prevents the enzyme Doa4 from removing ubiquitin from proteins in endosomes. Prior research had shown that addition of a single ubiquitin molecule to a membrane protein tags it for uptake and degradation via the endocytic pathway. Removal of the ubiquitin by Doa4 just before protein degradation preserves the ubiquitin for reuse; however, phosphorylation at Ser-57 prevents this removal, leading to destruction of the ubiquitin along with the tagged protein. Follow up studies demonstrated that the endocytic pathway is the primary mechanism of ubiquitin turnover in yeast. These findings provide important new information on ubiquitin dynamics and their relationship to the turnover of other cellular proteins. Further research is needed to determine how this pathway may relate to abnormalities in the ubiquitin pathway in various disease states. The work is published in the journal eLife [S. Lee, et al., (2017) eLife, 6, e29176].

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