Munoz-Gomez, Miguel

Abstract Colon polyps are the most common neoplastic finding at colonoscopy and have the potential to transform to cancer. Understanding the mechanisms involved in the transformation from normal colon to polyp is central to the identification of interventions to prevent or stop a polyp from forming or, in the worst case, from transforming. Therefore, the clinical conundrum is to predict which polyps will progress. It is crucial to understand if there are molecular features that define premalignant lesions and that may contribute to the final steps of transformation, which could potentially be targets for early detection or more individualized clinical management. Cellular transformation is driven by the interplay of genetic and epigenetic changes, and a large body of research has identified modifications of chromatin and the role of those modifications in regulating genes. Telomeres, telomere maintenance mechanisms, and chromatin remodelers maintain genetic stability, and their disruption is associated with the development of many diseases, including cancer. Organoids are multicellular structures grown from the stem cells of an organ to recapitulate the diverse cell types of the parent organ. This renewable resource of an individual’s tissue provides the opportunity to study colon carcinogenesis in combination with advanced genomic tools such as gene editing. The overall objective of this study is to determine the role of telomere and chromatin remodeler alterations in neoplastic transformation. The functional interaction between hTERT and specific chromatin remodelers will be resolved in two steps: 1) activating telomerase in normal colon and polyp organoids from patients and measuring chromatin remodeler expression, and 2) incorporating mutations in the chromatin remodelers using genome editing and then measuring the expression of hTERT in the normal colon and polyp organoids from patients with cancer-adjacent and cancer-free polyps. Gene editing experiments were performed utilizing transposon-based vectors (PiggyBac), short homology arm-based knockin (GeneWeld), or CRISPR/Cas9, coupled with electroporation for cargo delivery. To assess neoplastic transformation as a result of the gene edits, we used histology and cellular morphology of the organoid specimen by hematoxylin and eosin staining and pathologist review, and the markers by immunofluorescence. These models of neoplastic transformation in organoids from patient tissues provide an accessible tool to visualize the functional changes that correspond to alterations in telomerase and chromatin remodelers and how alterations in these targets affect growth, proliferation, differentiation, and cell turnover. Identification of epigenomic molecular alterations linked with progression of polyps to cancer could lead to modifiable targets for preventive interventions and provide candidate molecular markers in order to better tailor polyp patient screening recommendations. Note: This abstract was not presented at the conference. Citation Format: Brooke R. Druliner, Brandon Simone, Miguel Munoz-Gomez, Kim Kossick, Daniel O'Brien, Alexej Abyzov, Stephen Ekker, Lisa A. Boardman. Characterizing telomere and chromatin dynamics in colorectal cell transformation [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A33.

ABSTRACT High-fat diet (HFD)-fed mouse models have been widely used to study early type 2 diabetes. Decreased β-cell glucokinase (GCK) expression has been observed in HFD-induced diabetes. However, owing to its crucial roles in glucose metabolism in the liver and in islet β-cells, the contribution of decreased GCK expression to the development of HFD-induced diabetes is unclear. Here, we employed a β-cell-targeted gene transfer vector and determined the impact of β-cell-specific increase in GCK expression on β-cell function and glucose handling in vitro and in vivo. Overexpression of GCK enhanced glycolytic flux, ATP-sensitive potassium channel activation and membrane depolarization, and increased proliferation in Min6 cells. β-cell-targeted GCK transduction did not change glucose handling in chow-fed C57BL/6 mice. Although adult mice fed a HFD showed reduced islet GCK expression, impaired glucose tolerance and decreased glucose-stimulated insulin secretion (GSIS), β-cell-targeted GCK transduction improved glucose tolerance and restored GSIS. Islet perifusion experiments verified restored GSIS in isolated HFD islets by GCK transduction. Thus, our data identify impaired β-cell GCK expression as an underlying mechanism for dysregulated β-cell function and glycemic control in HFD-induced diabetes. Our data also imply an etiological role of GCK in diet-induced diabetes. This article has an associated First Person interview with the first author of the paper.

Abstract Glucokinase (GCK) is crucial to regulating glucose metabolism in the liver and in pancreatic β-cells. There are two major GCK isoforms, hepatic and pancreatic GCKs, which differ only in exon 1. However, the functional differences between the two GCK isoforms remain poorly understood. Here, we used a β-cell-targeted gene transfer vector to determine the impact of isoform-specific GCK overexpression on β-cells in vitro and in vivo. We showed that pancreatic GCK had a nuclear localization signal unique to the pancreatic isoform, facilitating its nuclear distribution in β-cells. Despite the difference in subcellular distribution, overexpression of GCK isoforms similarly enhanced glucose uptake and β-cell proliferation in vitro. Overexpression of hepatic or pancreatic GCK also similarly enhanced β-cell proliferation in normal diet mice without affecting fasting glucose and intraperitoneal glucose tolerance tests (IPGTT). Our further study on human GCK sequences identified disproportional GCK amino acid variants in exon 1, while mutations linked to maturity onset diabetes of the young type 2 (MODY2) were disproportionally found in exons 2 through 10. Our results therefore indicate functional conservation between the two major GCK isoforms despite their distinct subcellular distribution.

Summary Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.