By Andrew Stewart, MD, and Priya Vellanki, MD
Genetic and genomic assessment and confirmation experiments identified novel genes involved in the regulation of growth of beta cells,1 according to findings published in Nature Communications.
Insulinomas, pancreatic tumors originating in the beta cells, are rare neuroendocrine growths that secrete insulin and are usually benign, though there are instances in which they metastasize.
“There is no easily available model system for obtaining replicating beta cells. The only cells we have to work with are cadaveric beta cells, which by definition don’t replicate," said lead author Andrew Stewart, MD, director of the Diabetes Obesity and Metabolism Institute and professor of medicine, endocrinology, diabetes and bone disease at the Mount Sinai and Icahn School of Medicine in New York City,
"The new spin here is that when insulinomas, which are very rare, replicate, their beta-cell mass increases significantly. The usual insulinoma is about 1-2 cm in diameter," said Dr. Stewart, "That is enough insulin to make a healthy person hypoglycemic."
The novelty is comparing insulinomas, which have the replicative machinery, and demonstrably do increase functional beta-cell mass, to normal beta cells that don’t replicate,” Dr. Stewart told EndocrineWeb.
Beta cells, located in the islet of Langerhans in the pancreas, release post-prandial insulin. Developing treatments that will effectively increase the number of functional beta cells has become an exciting priority for diabetes management.
There is an opportunity to improve insulin function in both type 1 diabetes (T1D), an autoimmune-driven loss of beta cells, while a lack of adequately functioning beta cells also forms part of the etiology of type 2 diabetes (T2D). Inducing restoration of beta cells function offers a promising opportunity for diabetes treatment for these patients.
Since insulinomas are proliferative beta cells, researchers examined 38 human insulinomas to elucidate potential therapeutic pathways that might lead to regeneration of beta cells.1 Through the use of whole-exome sequencing (WES) and RNA-sequencing data, this study characterized the genomic and molecular landscape of insulinomas when compared to normal beta cells. WES sequences the DNA of genes, and RNA sequencing assessed the expression of genes by sequencing the RNA.
Multiple Endocrine Neoplasia type 1 (MEN1) is a gene with a typically autosomal recessive variant that causes familial tumors of parathyroid glands, pancreatic islets, and/or pituitary glands.
“We intentionally excluded samples from people known to be members of MEN1 kindreds because we wanted to find new genes that have mutations,” said Dr. Stewart.
Of the 38 insulinomas, 26 were paired whole blood and tumor samples that underwent WES. Twenty-five of 38 insulinomas underwent RNA sequencing, with 13 of those overlapping with WES (12 did not undergo WES given a lack of paired blood samples). This study used enrichment analysis to assess which families of genes and pathways might be enriched for variants and differences in expression between insulinomas and healthy beta cells.1
The researchers interrogated DNA methylation patterns on 11p15, a stretch of the chromosome with known involvement in the proliferation of beta cells.1 Additionally, researchers executed functional validation tests on top candidate genes, using adenovirus to silence and to over-express genes in the cell culture of human islets. Finally, chromatin immunoprecipitation (ChIP) was conducted to examine interactions of two genes involved in the regulation of the cell cycle.
Analyses of exome-sequencing revealed a total of 258 somatic (tumor-specific) single nucleotide polymorphisms (SNPs) and 20 non-SNP variants. An SNP is a variation in a single nucleotide occurring at a specific location within a genome. The non-SNP variants included indels and multiple-nucleotide polymorphisms. An average of 10.7 variants occurred per insulinoma.1
Unexpectedly, recurrent variants were rare in insulinomas, with only four of 26 tumors harboring the previously identified T372R SNP in the YY1 gene. Though MEN1 kindreds had been excluded, two insulinomas had tumors in MEN1, suggesting the importance of the gene in beta cell growth.1
“I was completely surprised,” Dr. Stewart told EndocrineWeb, “I thought we would find two or three other genes that were frequently mutated or lost or amplified in these insulinomas that didn’t come from MEN1 kindred but that is not at all what we found. What we found was almost every insulinoma had a different mutational profile. In 26 insulinomas, there were 278 different genes that harbored variants. That was confusing but informatically, it became obvious that they were all epigenetically modifying genes. That included MEN1, which is also an epigenetic modifying gene.”
The findings indicated that most insulinomas had mutations, copy number variants, and/or dysregulation of genes that modify epigenetics1. These variants were largely in the polycomb and trithorax gene families. Genes in the polycomb family code for proteins that act in chromatin- and epigenetic-remodeling. Similarly, genes in the polycomb family code for proteins that affect gene expression by modifying histones, remodeling chromatin, or binding DNA.
Enrichment analysis of transcriptomics was similarly associated with epigenetic dysregulation.1 CpG methylome sequencing revealed DNA methylation patterns on 11p15 in insulinomas characterized by widespread hypomethylation as well as select regions of hypermethylation in regions known to be involved in imprinting regulation.
Functional validation experiments tested the effects of over-expression of EZH2, CCDN1, and YY1 and the silencing of MEN1 and CDKN1C. Neither silencing of MEN1 nor over-expression of mutant or wild-type YY1 resulted in proliferation, suggesting a requirement for additional mitogenic events or a longer time for necessary epigenetic modifications to occur.1
Over-expression of CCND1 or EZH2 or silencing of CDKN1C (alone and in select combinations) induced proliferation of beta cells at rates similar to those of insulinomas. The researchers found that over-expression of EZH2 coupled with silencing of CDKN1C resulted in efficacious increases in beta-cell proliferation.
“Through genomics of families with the MEN1 mutation, the MEN1 gene was positionally cloned. Though we knew MEN1 is associated with insulinoma, nobody really knew how or why this was so we were able to test the hypothesis that MEN1 and other epigenetic modifying genes could lead to activation of cell cycle activity. In fact, we showed that that is the case,” said Dr. Stewart.
The KDM6A gene, responsible for sending instructions for synthesis of lysine demethylase 6A, was recurrently mutated and had decreased expression in insulinomas. Its biology had not previously been examined in beta cells. Researchers selected CDKN1C, a gene that codes for cell cycle inhibitor p57, as the target and found expression of CDKN1C was reduced in insulinomas. Pharmacological and adenoviral inhibition of KDM6A resulted in large decreases of CDKN1C in islet cells.
ChIP analysis revealed that KDM6A directly interacts with CDKN1C at its upstream enhancer. These results suggested the trithorax genes support CDKN1C expression in human beta cells and inhibition of trithorax genes may allow beta cells to more readily enter into the cell cycle due to decreased CDKN1C/p57.
Functional validation of key predictions from genetic and genomic analyses support the approach of examining the molecular complexity of insulinomas to understand beta cell proliferation and to develop pharmacological approaches to treat diabetes.1
“We validated several of the central conclusions of the manuscript,” said Dr. Stewart, which was that polycomb and trithorax and related families absolutely directly and indirectly modulate the beta cell cycle.”
“What I find surprising is that the same genes that are repressed in normal beta cells are not repressed in insulinomas. This suggests that if there is way to unrepress these genes in normal beta cells, then the beta cells could replicate,” said Priyathama Vellanki, MD, assistant professor of endocrinology, metabolism, and lipids at Emory University School of Medicine in Atlanta, Georgia, who was not involved in the study.
“Since this study did not compare beta cells of patients with diabetes who have insulinomas, ideally, a forthcoming experiment will examine the repressed gene pathways to see if bête cells of patients with diabetes can be made to regenerate,” said Dr. Vellanki. She also would like for future studies to look more closely at beta cell function in patients with type 1 versus type 2 diabetes, to know what if any differences may present.
“The takeaway message is that we can potentially regenerate beta cells in the future and delay or even reverse diabetes or possibly develop a cure for type 1 diabetes,” Dr. Vellanki told EndocrineWeb.
In spite of these advances, access to insulinomas is challenging.
“When I give talks, I make an appeal to clinicians to try to help us by preserving and sharing fresh tumors,” said Dr. Stewart.
Neither Dr. Stewart nor Dr. Vellanki has any competing financial interests.