The B7 family, and their receptors, the CD28 family, are major immune checkpoints that regulate T-cell activation and function. In the present study, we explore the role of two B7 immune-checkpoints: HERV-H LTR-Associating Protein 2 (HHLA2) and B7 Family Member, H4 (B7x), in the progression of gastrointestinal and pancreatic neuroendocrine tumors (GINETs and PNETs). We demonstrated that both HHLA2 and B7x were expressed to a high degree in human GINETs and PNETs. We determined that the expression of B7x and HHLA2 correlates with higher grade and higher incidence of nodal and distant spread. Furthermore, we confirmed that HIF-1 overexpression is associated with the upregulation of B7x both in our in vivo (animal model) and in vitro (cell culture) models. When grown in vitro, islet tumor β-cells lack B7x expression, unless cultured under hypoxic conditions, which results in both hypoxia inducible factor 1 subunit alpha (HIF-1α) and B7x upregulation. In vivo, we demonstrated that Men1/B7x double knockout (KO) mice (with loss of B7x expression) exhibited decreased islet β-cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single knockout mice. We have also shown that systemic administration of a B7x mAb to our Men1 KO mice with PNETs promotes an antitumor response mediated by increased T-cell infiltration. These findings suggest that B7x may be a critical mediator of tumor immunity in the tumor microenvironment of NETs. Therefore, targeting B7x offers an attractive strategy for the immunotherapy of patients suffering from NETs.
We reported that inactivation of menin (the protein product of MEN1) increases activity of Dnmt1 and mediates DNA hypermethylation in the development of multiple endocrine neoplasia type 1 (MEN1) syndrome. We have developed a RCAS-TVA-based somatic gene transfer system that enables tissue-specific delivery of Dnmt1 to individual β-cells of the pancreas in a RIP-TVA mouse model. In the present study, we mediated Dnmt1 expression in islet β-cells in RIP-TVA mice by utilizing the RCAS-TVA system to test if the upregulation of Dnmt1 can promote β-cell proliferation. In vitro, we demonstrated that upregulation of Dnmt1 increased β-cell proliferation. In vivo, our results showed that the levels of serum insulin were increased in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Furthermore, we confirmed that mRNA and protein expression of Dnmt1 as well as Dnmt1 enzyme activity were upregulated in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Finally, we demonstrated that upregulation of Dnmt1 resulted in hyperplasia through β-cell proliferation. We conclude that the upregulation of Dnmt1 promotes islet β-cell proliferation and targeting Dnmt1 may be a promising therapy for patients suffering from pancreatic neuroendocrine tumors.
Multiple endocrine neoplasia type 1 (MEN1) is an inherited cancer syndrome caused by a germline mutation in the MEN1 gene. While a germline heterozygous mutation in the MEN1 gene predisposes tumor formation in specific tissues such as the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in mouse pancreatic exocrine tissue does not result in tumor formation, suggesting a tissue-specific mechanism. Loss of menin activates a menin-interacting protein retinoblastoma-binding-protein 5 (RBBP5). Since RBBP5 transcriptionally regulates DNA methyltransferase 1 (DNMT1), this causes global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. We hypothesize that while RBBP5 is ubiquitously expressed, it exclusively binds to the DNMT1 promoter in MEN1-target-tissues through its recruitment by tissue-specific factors. Using ChIP-PCR, we demonstrated that Rbbp5 is bound to the Dnmt1 promoter in MEN1-target-tissues, while not in non-target tissues. In the present study, we set out to identify and validate putative endocrine-specific factors involved in Rbbp5 binding to the Dnmt1 promoter. To investigate this tissue-specific occupancy of Rbbp5 at the Dnmt1 promoter, a ChIP-Seq analysis was carried out on DNA purified from pancreatic endocrine islet cells and pancreatic exocrine cells from wild type (WT) mice. These DNA samples were analyzed to identify differentially occupied DNA binding sites of menin and Rbbp5 by a high-throughput tissue-specific ChIP-seq assay. This analysis yielded a list of endocrine-specific genes regulated by both menin and Rbbp5. In order to further identify the tissue-specific factors that may alter menin-Rbbp5 binding to the Dnmt1 promoter, we performed tissue-specific RNA-Seq on endocrine and exocrine pancreas tissues from WT mice. Integrating tissue-specific ChIP-Seq and RNA-seq data, we identified 20 endocrine tissue-specific factors that may alter the interaction of the menin-Rbbp5 complex with the Dnmt1 promoter. By carrying out this unbiased genome-wide screen, we identified endocrine-specific candidate factors that may interact with the menin-Rbbp5 complex at the Dnmt1 promoter. After screening all candidates, the stand-out candidates were identified for further validation. Criteria to be met to establish stand-out candidates included: MEN1-target-tissue-specific expression through immunohistochemical staining and high mRNA expression by qRT-PCR analysis in WT mouse pancreatic islets on samples extracted by laser capture microscopy. In conclusion, we have identified candidates for Rbbp5 recruitment to the Dnmt1 promoter that must be tested further to determine their role in the observed tissue specificity of MEN1-related tumorigenesis. Citation Format: Juliet C. Gardiner. Tissue-specific tumorigenesis in multiple endocrine neoplasia type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 728.
While a germline heterozygous mutation in the MEN1 gene predisposes tumor formation in the endocrine pancreas, parathyroid glands and anterior pituitary, this tissue-specific tumorigenesis is not dependent on MEN1 mutations alone. In fact, a homozygous deletion of Men1 in the entire pancreas of a mouse results in tumor formation only in the endocrine pancreas, not in the exocrine pancreas, suggesting an endocrine tissue-specific mechanism. The MEN1 gene encodes the menin protein, which interacts with chromatin associated protein complexes, therefore engaging in epigenetic control mechanisms. Recognizing menin’s participation in epigenetic regulation led to an investigation of whether the pathogenesis of MEN1 syndrome may be related to epigenetic changes in the affected endocrine tissues. Indeed, MEN1-associated endocrine cell types exhibit various menin-dependent epigenetic mechanisms. In fact, a significant increase in methylated DNA loci was observed in MEN1 human parathyroid tumors when compared to human parathyroid adenomas and carcinomas without known MEN1 mutations. Subsequent studies revealed that loss of menin results in increased activity of DNA methyltransferase 1 (Dnmt1). Our studies have shown that Dnmt1 is transcriptionally regulated by the menin-interacting protein Rbbp5. While menin normally functions to suppress Rbbp5 activity, loss of menin activates Rbbp5, thus upregulating Dnmt1 expression, causing global DNA hypermethylation and subsequent tumorigenesis in MEN1-target endocrine tissues. In order to assess the behavior of Rbbp5 in both MEN1-target tissues and non-target tissues, Rbbp5 protein expression was analyzed in both MEN1-target tissues (endocrine pancreas, anterior pituitary, parathyroid) and non-MEN1-target tissues (kidney, lung, liver, brain, heart) of wild-type (WT) mice. We confirmed that Rbbp5 protein expression is ubiquitous throughout all of these WT mouse tissues. Since Rbbp5 is a transcriptional activator responsible for enhanced Dnmt1 gene expression, and the loss of menin causes Dnmt1 overexpression solely in MEN1-target tissues, we assessed whether Rbbp5 binds preferentially in a tissue-specific manner to the Dnmt1 promoter. We determined the presence of Rbbp5 on the Dnmt1 promoter in MEN1-target tissues (WT mouse endocrine pancreas, normal human parathyroid, WT mouse pituitary) and the absence of Rbbp5 on the Dnmt1 promoter in non-MEN1-target tissues (WT mouse liver, WT mouse kidney, WT mouse lung). These results confirmed that Rbbp5 does exhibit MEN1-target-tissue-specific occupancy at the Dnmt1 promoter. This endocrine-specific localization of Rbbp5 to the Dnmt1 promoter suggests the presence of additional tissue-specific factors (with tissue-specific expression or interactions/activity) that must be validated and tested further.
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