Human Pancreatic Neuroendocrine Tumors Research Articles

Metabolic intervention by low carbohydrate diet suppresses the onset and progression of neuroendocrine tumors

Insulin signaling often plays a role in the regulation of cancer, including tumor initiation, progression, and response to treatment. In addition, the insulin-regulated PI3K-Akt-mTOR pathway plays an important role in the regulation of islet cell proliferation, and this pathway is hyperactivated in human non-functional pancreatic neuroendocrine tumors (PanNETs). We, therefore, investigated the effect of a very low carbohydrate diet (ketogenic diet) on a mouse model that develops non-functional PanNETs to ask how reduced PI3K-Akt-mTOR signaling might affect the development and progression of non-functional PanNET. We found that this dietary intervention resulted in lower PI3K-Akt-mTOR signaling in islet cells and a significant reduction in PanNET formation and progression. We also found that this treatment had a significant effect on the suppression of pituitary NET development. Furthermore, we found that non-functional PanNET patients with lower blood glucose levels tend to have a better prognosis than patients with higher blood glucose levels. This preclinical study shows that a dietary intervention that results in lower serum insulin levels leads to lower insulin signals within the neuroendocrine cells and has a striking suppressive effect on the development and progression of both pancreatic and pituitary NETs.

ATP6AP2 is robustly expressed in pancreatic β cells and neuroendocrine tumors, and plays a role in maintaining cellular viability

ATP6AP2, also known as (pro)renin receptor, has been shown to be expressed in several tissues including pancreatic β cells. Whereas ATP6AP2 plays an important role in regulating insulin secretion in mouse pancreatic β cells, the expression profiles and roles of ATP6AP2 in human pancreatic endocrine cells and neuroendocrine tumor cells remain unclear. Here in this study, we investigated the expression profiles of ATP6AP2 in pancreatic endocrine cells, and found that ATP6AP2 is robustly expressed in pancreatic insulinoma cells as well as in normal β cells. Although ATP6AP2 was also expressed in low-grade neuroendocrine tumors, it was not or faintly detected in intermediate- and high-grade neuroendocrine tumors. Knockdown experiments of the Atp6ap2 gene in rat insulinoma-derived INS-1 cells demonstrated decreased cell viability accompanied by a significant increase in apoptotic cells. Taken together, these findings suggest that ATP6AP2 plays a role in maintaining cellular homeostasis in insulinoma cells, which could lead to possible therapeutic approaches for endocrine tumors.

Abstract 2733: TYMS as a novel target for AAV-based therapy

Abstract The purpose of this study is to develop a strategy for treating pancreatic neuroendocrine tumor (PanNET) through blocking Thymidylate Synthase (TYMS or TS), a DNA metabolic enzyme. Elevated TYMS levels are frequently observed in human PanNET and are associated with worse prognosis and resistance to cytotoxic agents such as 5FU. Elevated TYMS levels directly promote tumorigenesis in vitro and ectopic expression of human TS (hTS) in transgenic mice promotes adenoma development in the endocrine pancreas in vivo. Pancreatic islet tumor formation in hTS transgenic mice occurred with a long latency period, suggesting that additional somatic events are required for PanNET formation and progression. Men1 was shown to be a frequent target for somatic mutation in PanNETs (44% have MEN1 mutations). Therefore, we established a new mouse model designated hTS/Men1-/-, where hTS is overexpressed in pancreatic islet cells carrying conditional Men1 null alleles. We observed that ectopic hTS cooperates with Men1 inactivation to significantly accelerate PanNET progression and shorten survival. Since traditional TYMS inhibitors, such as 5-FU, consistently induce tumor resistance and cannot cure advanced disease, we have now tested the ability of TS-shRNA delivered using adeno-associated viral (AAV) vectors as a pharmacological safe and effective anti-tumor strategy. We packaged TS shRNA under the mouse insulin promoter (mIP) for islet-cell specific expression using capsid optimized Y to F double mutant AAV (AAV8-2M) to generate viral particles of scAAV8-2M-mIP-GFP-hTS-shRNA. We treated hTS/Men1-/- mice with intraperitoneal (IP) injection (1x1011 viral genomes/mice). Treated and control mice were analyzed for PanNET progression, TYMS expression and survival. In addition, we tested a human PanNET tumor cell line (BON) that was isogenic with or without hTS expression for tumor formation in xenograft model. We confirmed sustained reduction of TYMS levels 3 weeks after IP delivery of AA8-2M-mIP-GFP-hTS-shRNA into PanNET tissues of hTS/Men1-/- mice. In addition, AA8-2M-mIP-GFP-hTS-shRNA delivery blocked PanNET tumor progression and enhanced the survival of treated mice as compared to vector controls. We also confirmed that TYMS knock-down in BON cells by hTS-shRNA reduced xenograft tumor growth as compared to non-specific shRNA controls. In conclusion, the ability of AAV-mediated TS shRNA to block PanNET tumor progression directly demonstrates the importance of elevated TYMS in tumorigenesis and offers a novel anti-cancer strategy. Citation Format: Vinod Vijayakurup, Benjamin Meyer, Akbar Nawab, Frederic J. Kaye, Maria V. Guijarro, Maria Zajac-Kaye. TYMS as a novel target for AAV-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2733.

Novel treatment strategy of targeting epigenetic dysregulation in pancreatic neuroendocrine tumors.

Epigenetic dysregulation is an integral step in the progression of pancreatic neuroendocrine tumors. We hypothesized that tumor suppressor repression by DNA methyltransferase 1 in pancreatic neuroendocrine tumors could be targeted with epigenetic treatment. Resected pancreatic neuroendocrine tumors from 32 patients were stained for DNA methyltransferase 1 and scored. Human (BON1) and murine (STC) pancreatic neuroendocrine tumor cells were treated with DNA methyltransferase 1 inhibitor 5-azacytidine and chemotherapeutic agents 5-fluorouracil and temozolomide. Cell proliferation assay and tumor suppressor gene analysis were performed with qRT-PCR and Clarion S microarray. Tumor measurements were compared in a murine treatment model. High DNA methyltransferase scores were associated with high Ki-67 (6.7% vs 70.6% P < .01), mitotic rate (0.0% vs 31.3%), and grade (20.0% vs 80.4%, P < .01). Treatment with 5-azacytidine and chemotherapy resulted in a reduction of cell proliferation compared to chemotherapy alone in BON1 (77.3% vs 53.1%, P < .001) and STC (73.4% vs 34.2%, P < .001). Treatment with 5-azacytidine and chemotherapy resulted in upregulation of tumor suppressors CDKN1A (7.6 rel. fold, P < .001), BRCA2 (4.3 rel. fold, P < .001), and CDH1 (6.0 rel. fold, P= .026) in BON1 and CDKN1a (14.5 rel. fold, P < .001) and CDH (17.5 rel. fold, P < .001) in STC. In microarray, 5-azacytidine drove global genetic changes in combination treatment. Invivo tumors treated with chemotherapy measured 88.6 ± 19.54 mm3 vs 52.89 ± 10.51 mm3 in those treated with combination therapy (P= .009). Epigenetic dysregulation with DNA methyltransferase 1 is associated with pancreatic neuroendocrine tumors and is a potential targetable strategy. 5-azacytidine and chemotherapy in combination can reduce cell proliferation, upregulate silenced tumor suppressor genes, and decrease invivo tumors in pancreatic neuroendocrine tumors.

Molecular alterations and therapeutic targets in pancreatic neuroendocrine tumors

Human pancreatic neuroendocrine tumors (PanNETs) are a rare, deadly tumor type that is sporadic or arises in the background of a hereditary syndrome. A critical genetic event in sporadic tumors is inactivation of the gene menin 1 (MEN1) on chromosome 11, and indeed, PanNETs occur in patients with the hereditary syndrome multiple endocrine neoplasia type 1 (MEN1) due to germline mutations in the gene. Here, we review the recent progress in the field of molecular genetics and therapeutic targets of PanNETs. The key genomic alterations, including MEN1, ATRX/DAXX, mammalian target of rapamycin (mTOR), DNA damage and repair associated genes, vascular endothelial growth factor receptor (VEGFR) and SSTRs, and epigenetic aberrations in PanNETs are discussed. In addition, the commonly used preclinical models for PanNETs are enumerated.

FAM83A promotes the progression and metastasis of human pancreatic neuroendocrine tumors by inducing the epithelial-mesenchymal transition via the PI3K/AKT and ERK pathways.

Family with sequence similarity 83, member A (FAM83A) has been reported to play an important role in cancer progression and metastasis. The purpose of this study was to clarify the role and mechanism of FAM83A in pancreatic neuroendocrine tumors (PanNETs). PanNET specimens and adjacent nontumor pancreatic tissues obtained from 68 patients who underwent curative surgery for PanNETs were assessed for FAM83A expression using immunochemical staining. The relationships between FAM83A expression, clinicopathological parameters and prognosis were statistically analyzed. PanNET cell lines were used to study the role of FAM83A in the progression and metastasis of PanNETs in vitro and in vivo. FAM83A was overexpressed in PanNET specimens compared with adjacent nontumor tissues. Furthermore, FAM83A expression was closely associated with lymph node metastasis (P = 0.02), perineural invasion (P = 0.001), WHO classification (P = 0.039), AJCC stage (P = 0.01) and shorter disease-free survival in patients with PanNETs (P < 0.001). FAM83A overexpression effectively promoted PanNET cell proliferation, migration, invasion and growth both in vitro and in vivo, whereas FAM83A inhibition exerted the opposite effects. Subsequent mechanistic investigations revealed that FAM83A promotes the progression and metastasis of PanNETs by inducing epithelial-mesenchymal transition (EMT) via the PI3K/AKT and ERK pathways. FAM83A plays an important role in the progression and metastasis of PanNET by inducing the EMT via the activation of the ERK and PI3K/AKT pathways and may serve as a valuable molecular target in PanNET treatment.

FAM83A promotes the progression and metastasis of human pancreatic neuroendocrine tumors by inducing the epithelial-mesenchymal transition via the PI3K/AKT and ERK pathways

Background: Family with sequence similarity 83, member A (FAM83A) has been reported to play an important role in cancer progression and metastasis. The purpose of this study was to clarify the role and mechanism of FAM83A in pancreatic neuroendocrine tumors (PanNETs). Methods: PanNET specimens and adjacent nontumor pancreatic tissues obtained from 68 patients who underwent curative surgery for PanNETs were assessed using immunochemical staining to identify FAM83A expression. The relationships between FAM83A expression, clinicopathological parameters and prognosis were statistically analyzed. PanNET cell lines were used to study the role of FAM83A in the progression and metastasis of PanNETs in vitro and in vivo. Results: FAM83A was overexpressed in PanNET specimens compared with adjacent nontumor tissues. Furthermore, FAM83A expression was closely associated with lymph node metastasis (P = 0.02), perineural invasion (P = 0.001), the WHO classification (P = 0.039), AJCC stage (P = 0.01) and shorter disease-free survival in patients with PanNETs (P &lt; 0.001). Enforced expression of FAM83A effectively promoted PanNET cell proliferation, migration, invasion and growth both in vitro and in vivo, whereas FAM83A inhibition exerted the opposite effects. Subsequent mechanistic investigations revealed that FAM83A promoted the progression and metastasis of PanNETs by inducing epithelial-mesenchymal transition (EMT) via the PI3K/AKT and ERK pathways. More importantly, specific inhibition of either the AKT or ERK pathways partially rescued the effect of FAM83A overexpression on promoting PanNET progression and metastasis. Conclusions: FAM83A plays an important role in the progression and metastasis of PanNET by inducing the EMT via the activation of the ERK and PI3K/AKT pathways and may serve as a valuable molecular target in PanNET treatment.

Can laboratory x-ray virtual histology provide intraoperative 3D tumor resection margin assessment?

Purpose: Surgery is an essential part of the curative plan for most patients affected with solid tumors. The outcome of such surgery, e.g., recurrence rates and ultimately patient survival, depends on several factors where the resection margin is of key importance. Presently, the resection margin is assessed by classical histology, which is time-consuming (several days), destructive, and basically only gives two-dimensional information. Clearly, it would be advantageous if immediate feedback on tumor extension in all three dimensions were available to the surgeon intraoperatively. Approach: We investigate a laboratory propagation-based phase-contrast x-ray computed tomography system that provides the resolution, the contrast, and, potentially, the speed for this purpose. The system relies on a liquid-metal jet microfocus source and a scintillator-coated CMOS detector. Our study is performed on paraffin-embedded non-stained samples of human pancreatic neuroendocrine tumors, liver intrahepatic cholangiocarcinoma, and pancreatic serous cystic neoplasm (benign). Results: We observe tumors with distinct and sharp edges having cellular resolution ( ) as well as many assisting histological landmarks, allowing for resection margin assessment. All x-ray data are compared with classical histology. The agreement is excellent. Conclusion: We conclude that the method has potential for intraoperative three-dimensional virtual histology.

Targeting HDACs in Pancreatic Neuroendocrine Tumor Models.

Compared to pancreatic adenocarcinoma (PDAC), pancreatic neuroendocrine tumors (PanNET) represent a rare and heterogeneous tumor entity. In addition to surgical resection, several therapeutic approaches, including biotherapy, targeted therapy or chemotherapy are applicable. However, primary or secondary resistance to current therapies is still challenging. Recent genome-wide sequencing efforts in PanNET identified a large number of mutations in pathways involved in epigenetic modulation, including acetylation. Therefore, targeting epigenetic modulators in neuroendocrine cells could represent a new therapeutic avenue. Detailed information on functional effects and affected signaling pathways upon epigenetic targeting in PanNETs, however, is missing. The primary human PanNET cells NT-3 and NT-18 as well as the murine insulinoma cell lines beta-TC-6 (mouse) and RIN-T3 (rat) were treated with the non-selective histone-deacetylase (HDAC) inhibitor panobinostat (PB) and analyzed for functional effects and affected signaling pathways by performing Western blot, FACS and qPCR analyses. Additionally, NanoString analysis of more than 500 potentially affected targets was performed. In vivo immunohistochemistry (IHC) analyses on tumor samples from xenografts and the transgenic neuroendocrine Rip1Tag2-mouse model were investigated. PB dose dependently induced cell cycle arrest and apoptosis in neuroendocrine cells in human and murine species. HDAC inhibition stimulated redifferentiation of human primary PanNET cells by increasing mRNA-expression of somatostatin receptors (SSTRs) and insulin production. In addition to hyperacetylation of known targets, PB mediated pleitropic effects via targeting genes involved in the cell cycle and modulation of the JAK2/STAT3 axis. The HDAC subtypes are expressed ubiquitously in the existing cell models and in human samples of metastatic PanNET. Our results uncover epigenetic HDAC modulation using PB as a promising new therapeutic avenue in PanNET, linking cell-cycle modulation and pathways such as JAK2/STAT3 to epigenetic targeting. Based on our data demonstrating a significant impact of HDAC inhibition in clinical relevant in vitro models, further validation in vivo is warranted.

CDK5 Activation Drives Pancreatic Neuroendocrine Tumorigenesis and is a Therapeutic Target

Introduction: Pancreatic neuroendocrine tumors(PNET) are frequently seen with significant morbidity/mortality. Most PNET are non-functional, yet existing animal models are functional. Therefore, they do not accurately recapitulate human disease. We hypothesize that Cdk5 activity (previously shown to be important in other neuroendocrine diseases) plays a role in PNET pathogenesis and maybe a therapeutic target. Methods: Cdk5 expression was quantified in human PNET(hPNET) tissues. hPNET cell lines were treated with Cdk5 inhibitors. Bi-transgenic mice were created with inducible Cdk5 activity under control of the tet-operon. Growing/arrested tumors were studied for phosphorylation sites that were upregulated in growing tumors. Short interfering peptides (SIPs) were generated to these targets to identify potential downstream signaling pathways. Results: All hPNET samples tested had increased Cdk5 expression(Figure A). A Cdk4/5 inhibitor(IndolamineA) affected growth of hPNET cells in a dose-dependent manner without effecting fibroblasts(Figure B). Cdk4 inhibition(IndolamineB) did not effect these cells. All transgenic mice developed PNET with most nonfunctional. Phosphoproteomic analysis identified 50 potential Cdk5 targets upregulated in growing tumors. SIPs to 15 sites inhibited growth of BON cells but not fibroblasts(Figure D, top). Six of these sites included known proteins implicated in human tumorigenesis (but unknown in hPNET). Phosphorylation of each was reduced in BON cells with Cdk5 inhibition, implicating each in PNET formation and growth. Conclusion: Aberrant Cdk5 activity is seen in all hPNET and is a potential therapeutic target. We have created the first transgenic model of PNET that behaves like human disease and have identified molecular pathways that are being activated in PNET.

Generation and characterization of CRISPR/Cas9-mediated MEN1 knockout BON1 cells: a human pancreatic neuroendocrine cell line

Among patients with the rare diagnosis of pancreatic neuroendocrine tumor (P-NET), a substantial proportion suffer from the inherited cancer syndrome multiple endocrine neoplasia type 1 (MEN1), which is caused by germline mutations of the MEN1 suppressor gene. Somatic mutations and loss of the MEN1 protein (menin) are frequently also found in sporadic P-NETs. Thus, a human neuroendocrine pancreatic cell line with biallelic inactivation of MEN1 might be of value for studying tumorigenesis. We used the polyclonal human P-NET cell line BON1, which expresses menin, serotonin, chromogranin A and neurotensin, to generate a monoclonal stable MEN1 knockout BON1 cell line (MEN1-KO-BON1) by CRISPR/Cas9 editing. Changes in morphology, hormone secretion, and proliferation were analyzed, and proteomics were assessed using nanoLC-MS/MS and Ingenuity Pathway Analysis (IPA). The menin-lacking MEN1-KO-BON1 cells had increased chromogranin A production and were smaller, more homogenous, rounder and grew faster than their control counterparts. Proteomic analysis revealed 457 significantly altered proteins, and IPA identified biological functions related to cancer, e.g., posttranslational modification and cell death/survival. Among 39 proteins with at least a two-fold difference in expression, twelve are relevant in glucose homeostasis and insulin resistance. The stable monoclonal MEN1-KO-BON1 cell line was found to have preserved neuroendocrine differentiation, increased proliferation, and an altered protein profile.

Multiple Endocrine Neoplasia Type 1 (MEN1) 5'UTR Deletion, in MEN1 Family, Decreases Menin Expression.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic and pituitary tumors, and is due to mutations in the coding region of the MEN1 gene, which encodes menin. We investigated a family with identical twins that had MEN1, with different MEN1 tumors. DNA sequence analysis of the MEN1 coding region had not identified any abnormalities and we hypothesized that deletions and mutations involving the untranslated regions may be involved. Informed consent and venous blood samples were obtained from five family members. Sanger DNA sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses were performed using leukocyte DNA. This revealed a heterozygous 596bp deletion (Δ596bp) between nucleotides -1087 and -492 upstream of the translation start site, located within the MEN1 5' untranslated region (UTR), and includes the core promoter and multiple cis-regulatory regions. To investigate the effects of this 5'UTR deletion on MEN1 promoter activity, we generated luciferase reporter constructs, containing either wild-type 842bp or mutant 246bp MEN1 promoter, and transfected them into human embryonic kidney HEK293 and pancreatic neuroendocrine tumor BON-1 cells. This revealed the Δ596bp mutation to result in significant reductions by 37-fold (p < 0.0001) and 16-fold (p < 0.0001) in luciferase expression in HEK293 and BON-1 cells, respectively, compared to wild-type. The effects of this 5'UTR deletion on MEN1 transcription and translation were assessed using qRT-PCR and Western blot analyses, respectively, of mRNA and protein lysates obtained from Epstein-Barr-virus transformed lymphoblastoid cells derived from affected and unaffected individuals. This demonstrated the Δ596bp mutation to result in significant reductions of 84% (p < 0.05) and 88% (p < 0.05) in MEN1 mRNA and menin protein, respectively, compared to unaffected individuals. Thus, our results report the first germline MEN1 5'UTR mutation and highlight the importance of investigating UTRs in MEN1 patients who do not have coding region mutations. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

GCGR Loss-of-Function Mutations Promote Amino Acid-Dependent Pancreatic Neuroendocrine Tumor Initiation from a Subpopulation of Alpha Cells

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Dysregulated hormone production and the absence of obvious driver mutations mask tumor origins although they presumably arise from endocrine cell lineages such as glucagon-producing alpha cells or insulin-expressing beta cells. A reciprocal feedback circuit involving pancreatic glucagon and hepatic amino acids regulates alpha cell mass such that loss of hepatic glucagon receptor (GCGR) signaling drives alpha cell hyperproliferation and tumor formation. This genetically-defined system is an ideal model for parsing the mechanisms of PNET origin and progression. Utilizing a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we found that elevated plasma amino acids drive reversion to a proliferative SLC38A5+ embryonic progenitor-like status. Chronic activation of this mTOR-dependent state drives tumor initiation and single cell profiling reveals tumors originate from a subpopulation of alpha cells. Profiling RNA, tumor signaling, and immune content of biopsies from rare human PNETs found in individuals with germ-line GCGR loss-of-function variants showed active mTOR and prominent tumor cell-associated expression of amino acid transporter SLC7A8. This work, for the first time, identifies metabolic control of an adult-to-progenitor alpha cell identity switch and shows that chronic maintenance of progenitor identity in the adult organ drives proliferation and tumor initiation.

MiR-431 Promotes Metastasis of Pancreatic Neuroendocrine Tumors by Targeting DAB2 Interacting Protein, a Ras GTPase Activating Protein Tumor Suppressor

The incidence of pancreatic neuroendocrine tumor (PNET) is increasing, and it presents with various clinical manifestations and an unfavorable survival rate. A better understanding of the drivers of PNET tumorigenesis is urgently needed. Distinct miRNA signatures have been identified for different stages of tumorigenesis in both human and mouse PNETs. The functions of these miRNAs are poorly understood. miR-431 is the most up-regulated miRNA in the metastatic signature. However, it is unknown whether miR-431 contributes to metastasis of PNETs. Herein, we show that miR-431 overexpression activates Ras/extracellular signal-regulated kinase (Erk) signaling and promotes epithelial-mesenchymal transition, migration/invasion invitro, and metastasis in both xenograft and spontaneous mouse models of PNET. Treatment of PNET cells with Erk inhibitor or locked nucleic acids sequestering miR-431 inhibits invasion. Four target prediction modules and dual-luciferase reporter assays were used to identify potential mRNA targets of miR-431. A Ras GTPase activating protein tumor suppressor (RasGAP), DAB2 interacting protein (DAB2IP), was discovered as an miR-431 target. Overexpression of DAB2IP's rat homolog, but not its mutant defective in Ras GTPase activating protein activity, reverses miR-431's effect on promoting invasion, Erk phosphorylation, and epithelial-mesenchymal transition of PNETs. Taken together, miR-431 silences DAB2IP to active Ras/Erk and promote metastasis of PNETs. miR-431 may be targeted to manage metastatic PNETs.

Parallel Signaling through IRE1α and PERK Regulates Pancreatic Neuroendocrine Tumor Growth and Survival.

Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers.

A growth model of neuroendocrine tumor surrogates and the efficacy of a novel somatostatin-receptor–guided antibody-drug conjugate: Perspectives on clinical response?

BackgroundAs patient-derived xenografts and other preclinical models of neuroendocrine tumors for testing personalized therapeutics are lacking, we have developed a perfused, 3D bioreactor model to culture tumor surrogates from patient-derived neuroendocrine tumors. This work evaluates the duration of surrogate culture and surrogate response to a novel antibody-drug conjugate. MethodsTwenty-seven patient-derived neuroendocrine tumors were cultured. Histologic sections of a pancreatic neuroendocrine tumor xenograft (BON-1) tumor were assessed for SSTR2 expression before tumor implantation into 2 bioreactors. One surrogate was treated with an antibody-drug conjugate composed of an anti-mitotic Monomethyl auristatin-E linked to a somatostatin receptor 2 antibody. Viability and therapeutic response were assessed by pre-imaging incubation with IR-783 and the RealTime-Glo AnnexinV Apoptosis and Necrosis Assay (Promega Corporation, Madison, WI) over 6 days. A primary human pancreatic neuroendocrine tumor was evaluated similarly. ResultsMean surrogate growth duration was 34.8 days. Treated BON-1 surrogates exhibited less proliferation (1.2 vs 1.9-fold) and greater apoptosis (1.5 vs 1.1-fold) than controls, whereas treated patient-derived neuroendocrine tumor bioreactors exhibited greater degrees of apoptosis (13- vs 9-fold) and necrosis (2.5- vs 1.6-fold). ConclusionPatient-derived neuroendocrine tumor surrogates can be cultured reliably within the bioreactor. This model can be used to evaluate the efficacy of antibody-guided chemotherapy ex vivo and may be useful for predicting clinical responses.

Abstract 3196: Combination immunotherapy with PD-1 and CXCR4 blockade activates antitumor immunity against pancreatic neuroendocrine tumors

Abstract Introduction: Although pancreatic neuroendocrine tumors (PNET) are less common and less deadly than pancreatic adenocarcinoma, they remain a considerable source of morbidity and mortality. The cases increasingly discovered due to advanced cross sectional imaging and the majority of PNETs are malignant when diagnosed. Immunotherapy for PNET has not yet been successfully demonstrated, although we and others have previously shown that they often contain robust T cell infiltrates. We hypothesized that combined immunotherapy could reactivate endogenous antitumor activity in organotypic tumor slice cultures of human PNET. Methods: Human PNET tumors were collected from the operating room and 250μm slices were cut using a vibratome and place on 0.4μm pore size membrane inserts (6 tumors). Slice culture survival testing was conducted using immunohistochemistry (IHC), the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay, or live immunofluorescence imaging. To test the combining immunotherapeutic effects, slices were treated with a PD-1 blocking monoclonal antibody or isotype control, with or without the CXCR4 inhibitor, AMD3100. For time-lapse live imaging, slices were stained with fluorescently conjugated antibodies for CD8 and EpCAM, as well as a reagent that binds to activated caspase 3/7 enzymes to indicate induction of apoptosis. The live slice was first imaged alone to serve as a non-treated control, then anti-PD1 antibody and AMD3100 were applied, and the slice imaged immediately afterward to monitor response after treatment. Results: We confirmed that cultured slices maintain their baseline morphology and architecture over 9 days in culture. The MTT assay showed stable metabolic activity over the same period. The demonstration of the live and dynamic microenvironment was performed first via live multicolor IF including PNET cells (EpCAM+) and immune cells (both CD45+ and CD4+ cells) within the microenvironment. IHC demonstrated 10% or 12% increased of cleaved-Caspase-3+ cells with combined PD-1 and CXCR4 blockade, as compared to control, after 2 days or 7 days in culture. To confirm the role of cytotoxic T cells, live stained slices were imaged before and after addition of PD-1 blockade and AMD3100. We demonstrated that caspase activation was increased in EpCAM+ cells proximal to CD8+ T cells immediately following combination drug treatment. Furthermore, CD8+ cells proximity to EPCAM+ cells (&amp;lt;20 µm) was significantly increased after addition of PD-1 blockade and AMD3100. There was no difference in the proportion of apoptotic EpCAM+ cells that did not have a CD8+ cell nearby, suggesting that apoptosis induction is specifically due to cytotoxic T cell function. Conclusion: Our study demonstrates that combination PD-1 and CXCR4 blockade enhances CD8+ T cell migration and antitumor activity in human PNET tumor slice cultures. Citation Format: Xiuyun Jiang, Kevin Sullivan, Kevin Labadie, Sara K. Daniel, David Seo, Arezou Abbasi, Teresa Kim, Raymond Yeung, Venu Pillarisetty. Combination immunotherapy with PD-1 and CXCR4 blockade activates antitumor immunity against pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3196.

Placenta-Specific 8 Is Overexpressed and Regulates Cell Proliferation in Low-Grade Human Pancreatic Neuroendocrine Tumors

Background/Aims: Many aspects of the biology of pancreatic neuroendocrine tumors (PanNETs), including determinants of proliferative, invasive, and metastatic potential, remain poorly understood. Placenta-specific 8 (PLAC8), a gene with unknown molecular function, has been reported to have tumor-promoting roles in different human malignancies, including exocrine pancreatic cancer. Since preliminary data suggested deregulation of PLAC8 expression in PanNET, we have performed detailed analyses of PLAC8 expression and function in human PanNET. Methods: Primary tissue from PanNET patients was immunohistochemically stained for PLAC8, and expression was correlated with clinicopathological data. In vitro, PLAC8 expression was inhibited by siRNA transfection in PanNET cell lines and effects were analyzed by qRT-PCR, Western blot, and proliferation assays. Results: We report that PLAC8 is expressed in the majority of well-differentiated human PanNETs, predominantly in early-stage and low-grade tumors. SiRNA-mediated knockdown of PLAC8 in PanNET cells resulted in decreased proliferation and viability, while apoptosis was not induced. Mechanistically, these effects were mediated by attenuation of cell cycle progression, as Western blot analyses demonstrated upregulation of the tumor suppressor p21/CDKN2A and downregulation of the cell cycle regulator Cyclin D1 as well as reduced levels of phosphorylated ribosomal protein s6 and retinoblastoma protein. Conclusion: Our findings establish PLAC8 as a central mediator of cell growth in a subset of human PanNET, providing evidence for the existence of distinct molecular subtypes within this class of tumors.

Daxx Functions Are p53-Independent In Vivo.

Mutations in the death domain-associated protein (DAXX) have been recently identified in a substantial proportion of human pancreatic neuroendocrine tumors (PanNETs). Remarkably, however, little is known about the physiologic role(s) of DAXX despite in vitro studies suggesting potential functions. Most prominently, and supported by tumor sequencing data, DAXX functions in concert with alpha thalassemia/mental retardation X-linked (ATRX) as a histone chaperone complex for the H3.3 variant. Studies have also identified potential roles in apoptosis, transcription, and negative regulation of the p53 tumor suppressor pathway. Herein, a mouse modeling approach was used to specifically address the latter and no significant genetic interaction between Daxx and the p53 pathway was determined. The embryonic lethal phenotype of Daxx loss is not p53-dependent. In addition, Daxx heterozygosity does not sensitize mice to a sublethal dose of ionizing radiation or alter the survival or tumor phenotype of Mdm2 transgenic mice. However, the data support a tumor suppressor role for DAXX as low-dose ionizing radiation produced a higher proportion of carcinomas in Daxx heterozygous mice than wild-type controls.Implications: While DAXX has important in vivo functions, they are independent of an inhibitory role on the p53 tumor suppressor pathway. Mol Cancer Res; 16(10); 1523-9. ©2018 AACR.

Reduced menin expression impairs rapamycin effects as evidenced by an increase in mTORC2 signaling and cell migration

BackgroundMammalian target of rapamycin (mTOR) is a master regulator of various cellular responses by forming two functional complexes, mTORC1 and mTORC2. mTOR signaling is frequently dysregulated in pancreatic neuroendocrine tumors (PNETs). mTOR inhibitors have been used in attempts to treat these lesions, and prolonged progression free survival has been recorded. If this holds true also for the multiple endocrine neoplasia type 1 (MEN1) associated PNETs is yet unclear. We investigated the relationship between expression of the MEN1 protein menin and mTOR signaling in the presence or absence of the mTOR inhibitor rapamycin.MethodsIn addition to use of menin wild type and menin-null mouse embryonic fibroblasts (MEFs), menin was silenced by siRNA in pancreatic neuroendocrine tumor cell line BON-1. Panels of protein phosphorylation, as activation markers downstream of PI3k-mTOR-Akt pathways, as well as menin expression were evaluated by immunoblotting. The impact of menin expression in the presence and absence of rapamycin was determinate upon Wound healing, migration and proliferation in MEFs and BON1 cells.ResultsPDGF-BB markedly increased phosphorylation of mTORC2 substrate Akt, at serine 473 (S473) and threonine 450 (T450) in menin−/− MEFs but did not alter phosphorylation of mTORC1 substrates ribosomal protein S6 or eIF4B. Acute rapamycin treatment by mTORC1-S6 inhibition caused a greater enhancement of Akt phosphorylation on S473 in menin−/− cells as compared to menin+/+ MEFs (116% vs 38%). Chronic rapamycin treatment, which inhibits both mTORC1and 2, reduced Akt phosphorylation of S473 to a lesser extent in menin−/− MEFs than menin+/+ MEFs (25% vs 75%). Silencing of menin expression in human PNET cell line (BON1) also enhanced Akt phosphorylation at S473, but not activation of mTORC1. Interestingly, silencing menin in BON1 cells elevated S473 phosphorylation of Akt in both acute and chronic treatments with rapamycin. Finally, we show that the inhibitory effect of rapamycin on serum mediated wound healing and cell migration is impaired in menin−/− MEFs, as well as in menin-silenced BON1 cells.ConclusionsMenin is involved in regulatory mechanism between the two mTOR complexes, and its reduced expression is accompanied with increased mTORC2-Akt signaling, which consequently impairs anti-migratory effect of rapamycin.