Pancreatic Islet Tumors Research Articles

Involvement of interaction of Cav3.2 and nociceptive TRPA1 in pathological pain transmission.

T-type Ca2+ channels and TRPA1 expressed in sensory neurons are involved in pain. We previously demonstrated a functional interaction of these channels under physiological conditions. Here we investigated the possible involvement of these channels in inflammatory pain condition. We also evaluated the relationship of these channels endogenously expressed in RIN-14B, a rat pancreatic islet tumor cell line. In dorsal root ganglion (DRG) neurons innervated inflammatory side, [Ca2+]i increases induced by 15 mM KCl (15K) were enhanced in neurons responded to AITC. This enhancement was not observed in genetically TRPA1-deficient neurons. The T-type and AITC-induced currents were larger in neurons of the inflammatory side than in those of the control one. In DRGs of the inflammatory side, the protein expression of Cav3.2, but not TRPA1, was increased. In RIN-14B, 15K-induced [Ca2+]i increases were decreased by blockers of T-type Ca2+ channel and TRPA1, and by TRPA1-silencing. Immunoprecipitation suggested the coexistent of these channels in sensory neurons and RIN-14B. In mice with inflammation, mechanical hypersensitivity was suppressed by blockers of both channels. These data suggest that the interaction of Cav3.2 with TRPA1 in sensory neurons is enhanced via the augmentation of the activities of both channels under inflammatory conditions, indicating that both channels are therapeutic targets for inflammatory pain.

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.

LBODP065 Rare Cause Of Bone Pain In MEN Syndrome

Abstract Introduction: MEN 1 syndrome is a rare disease in children and young adults, with prevalence reported as 2 per 100,000. MEN 1 caused by mutations in the MEN1 tumor suppressor gene, characterized by 2 or more MEN1 tumors: parathyroid, anterior pituitary, pancreatic islet tumors. To the best of our knowledge, osteomyelitis of the pelvis has not been reported as a complication in the literature in patients who were diagnosed with MEN1Case21 yo male from Honduras with PMHx of remote asthma presented with a common complaint of abdomen pain but found to have a rare diagnosis. He had 2 days of lower abdomen pain associated with non-bilious non bloody vomiting. Initial were significant for calcium 12.9, phosphorus 1.5 magnesium 1.8 and PTH 360. 4 mm renal stone 4 mm was detected on CT abdomen/pelvis. The initial EKG demonstrated J point elevation due to the hypercalcemia. He was admitted to the PICU for continuous telemetry monitoring due to the abnormal EKG related to electrolyte abnormalities. Hypercalcemia treatment was initiated with aggressive hydration, calcitonin administration as well as zoledronic acid administration. The patient's calcium levels improved as well as EKG. The parathyroid scan was positive for a right-sided parathyroid adenoma. He underwent right Inferior Parathyroidectomy, Intraoperative Nerve Monitoring, Cryopreservation of Parathyroid Tissue. As part of his diagnostic work-up for hyperparathyroidism, he was screened for MEN. His pituitary function was abnormal: ACTH and cortisol levels were high, his testosterone and gonadotropins were inappropriately low as well as TSH and T4. A microadenoma was present on brain MRI. No Pancreatic Neuroendocrine Neoplasia was found. The diagnosis of MEN 1 was made. Despite the improvements in his endocrinopathies, he continued to have lower abdomen pain associated with intermittent fevers. He was seen by urology and orthopedics. Initially MRI demonstrated bone erosion thought to be secondary to hypercalcemia but repeat MRI imaging of the pelvis was suspicious for phlegmon vs. developing abscess secondary to osteitis pubis. His blood cultures were positive for MSSA. He was started on antibiotics with continued clinical improvementThis patient presented with one of the most common complaints in the ED, abdomen pain and was found to have two rare and potentially fatal diagnosis which was identified and treated with a good clinical outcome Discussion: This case highlights a rare disease in the setting of osteomyelitis which could easily be missed as a manifestation of bone pain from hypercalcemia. The patient did experience fevers and malaise that was initially attributed to zoledronic acid administration. When his pain did not resolve MRI found osteomyelitis, allowing early antibiotic administration and prevention of sepsis. Presentation: No date and time listed

Mice With RIP-Cre-mediated Deletion of the Long Noncoding RNA Meg3 Show Normal Pancreatic Islets and Enlarged Pituitary.

Maternally expressed gene 3 (MEG3) is a long noncoding RNA (lncRNA) that has been implicated as a tumor suppressor. The expression of MEG3 RNA is downregulated in various human tumors, including pituitary adenoma and pancreatic islet tumors due to MEG3 gene deletion or DNA hypermethylation. Mouse models with conventional germline deletion of Meg3 have shown that Meg3 is essential for perinatal or postnatal development and survival. However, a direct role of Meg3 loss in tumorigenesis has not been shown. To observe a causal relationship between Meg3 loss and tumorigenesis, we have generated a mouse model with conditional deletion of Meg3 mediated by the RIP-Cre transgene that initiated Meg3 deletion in pancreatic islet β cells and anterior pituitary. Meg3 loss did not lead to the development of islet tumors. Interestingly, RIP-Cre-mediated Meg3 loss led to the development of an enlarged pituitary. The genes in the Meg3 region are transcribed together as a 210 kb RNA that is processed into Meg3 and other transcripts. Whether these tandem transcripts play a functional role in the growth of pancreatic endocrine cells and pituitary cells remains to be determined. Our mouse model shows that Meg3 loss leads to hyperplasia in the pituitary and not in pancreatic islets, thus serving as a valuable model to study pathways associated with pituitary cell proliferation and function. Future mouse models with specific inactivation of Meg3 alone or other transcripts in the Meg3 polycistron are warranted to study tissue-specific effects on initiating neoplasia and tumor development.

LUKOIL lets contracts for Perm refinery's proposed FCC complex

Pancreatic neuroendocrine tumors (PNETs) were formerly termed pancreatic islet tumors. These are rare tumors, comprising 2%–3% of all pancreatic tumors. They derive from pluripotent pancreatic stem cells. PNETs can be classified as functional and nonfunctional. Functional tumors secrete hormones that result in a specific clinical presentation. Insulinomas are the most common PNETs. They are usually benign. Glucagonomas and VIPoma are most commonly malignant. Surgical resection, surgical debulking ± systemic treatment may be used. The Zollinger-Ellison syndrome (ZES) is characterized by gastric acid hypersecretion leading to severe acid-related peptic ulcer disease with diarrhea, caused by a gastrinoma, also a neuroendocrine tumor. The gastrinoma triangle (sweep of duodenum/head of pancreas) is the location of 80% of these tumors. The majority of gastrinomas are located in the duodenum. Gastrinomas may be associated with MEN1 (hyperparathyroidism, pituitary tumors, and gastrinoma) in 20% of cases. Surgical resection is the primary treatment of choice for sporadic gastrinoma. Enucleation of small pancreatic tumors, duodenotomy, and enucleation of small duodenal tumors are the most common procedures performed. Liver resection, hepatic artery embolization and/or chemoembolization, microsphere embolization, radiofrequency or cryoablation, and liver transplantation all have been utilized for metastatic disease.

Abstract 71: The immunocytokine PD1-IL2v overcomes immune checkpoint resistance, and combination with an anti-PD-L1 antibody further enhances its anti-tumor activity

Abstract Cancer immunotherapies have shown therapeutic benefits in the clinic, but treatments involving immuno-stimulatory agents such as interleukin 2 (IL-2) are typically accompanied by severe adverse events. One strategy to limit the systemic toxicity of IL-2 is to target it specifically to the tumor microenvironment. Here we evaluated the therapeutic efficacy of the bi-specific immunocytokine PD1-IL2v in the genetically engineered, spontaneous RIP1-Tag5 (RT5) mouse model of pancreatic neuroendocrine tumors (PanNETs). PD1-IL2v is a bi-specific molecule based on a bivalent PD-1 antibody to which a single IL2 variant (IL2v) is fused. PD-1 serves as a targeting moiety to deliver IL2v in cis to PD-1-positive T cells located in the immune tumor microenvironment, and IL2v has been engineered to lack binding to CD25 in order to selectively expand effector T cells but not immuno-suppressive regulatory T cells (Tregs). Although RT5 mice are capable of mounting an adaptive immune response against the tumor-driving oncoprotein, PanNETs developing in RT5 are resistant to immune checkpoint inhibitors. In comparing the untargeted version of IL2v combined with an anti-PD-1 antibody vs. the PD-1 targeted immunocytokine PD1-IL2v, we found that PD1-IL2v, but not the mixture, produced significant anti-tumor activity. PD1-IL2v treatment resulted in a substantial infiltration of CD8+ T cells into the pancreatic islet tumors, which in the context of sustained treatment with PD1-IL2v resulted in tumor regression. Interestingly, following tumor shrinkage, the PanNETs appeared to stabilize and eventually relapsed. Notably, we identified up-regulation of PD-L1 on the tumor vasculature in relapsing tumors as a potential factor in the observed adaptive resistance, which encouraged combining an anti-PD-L1 antibody with PD1-IL2v, leading to enhanced therapeutic benefit. The data obtained in this spontaneous de novo tumor model motivate consideration of evaluating the PD1-IL2v/anti-PD-L1 combination therapy in the clinical setting, in particular in anti-PD-1/anti-PD-L1 resistant tumors infiltrated with PD1+CD8+ T cells. Citation Format: Stephan Wullschleger, Mélanie Tichet, Laura Codarri-Deak, Pablo Umana, Christian Klein, Douglas Hanahan. The immunocytokine PD1-IL2v overcomes immune checkpoint resistance, and combination with an anti-PD-L1 antibody further enhances its anti-tumor activity [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 71.

PCGF3 promotes the proliferation and migration of non-small cell lung cancer cells via the PI3K/AKT signaling pathway

The Polycomb Group Ring Finger 3 (PCGF3) protein has been reported to be significantly upregulated in pancreatic islet tumors and related to signal transduction; however, its detailed mechanisms and biological roles in other tumors, including non-small cell lung cancer (NSCLC), remain unclear. This study investigated the function of PCGF3 in NSCLC and further elucidated its mechanism of action. The immunohistochemical analysis of 86 selected lung cancer tissues revealed that PCGF3 was highly expressed in NSCLC tissues and positively correlated with lymph node metastasis and p-TNM staging. Additionally, PCGF3 promoted cell proliferation in lung cancer by regulating CyclinB1, CyclinD1, and CDK4 expression, and also promoting their migration by regulating RhoA, RhoC, and CDC42. Furthermore, PCGF3 affected both the proliferation and migration of lung cancer cells by regulating the PI3K/AKT pathway, as verified by inhibiting this pathway using LY294002. The findings of this study suggested that PCGF3 is associated with poor prognosis in patients with NSCLC and could therefore be an important biomarker for treating and preventing NSCLC.

MiR-15a/miR-16-1 expression inversely correlates with cyclin D1 levels in Men1 pituitary NETs.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by the combined occurrence of parathyroid, pituitary and pancreatic islet tumours, and is due to mutations of the MEN1 gene, which encodes the tumour suppressor protein menin. Menin has multiple roles in genome stability, transcription, cell division and proliferation, but its mechanistic roles in tumourigenesis remain to be fully elucidated. miRNAs are non-coding single-stranded RNAs that post-transcriptionally regulate gene expression and have been associated with tumour development, although the contribution of miRNAs to MEN1-associated tumourigenesis and their relationship with menin expression are not fully understood. Alterations in miRNA expression, including downregulation of three putative ‘tumour suppressor’ miRNAs, miR-15a, miR-16-1 and let-7a, have been reported in several tumour types including non-MEN1 pituitary adenomas. We have therefore investigated the expression of miR-15a, miR-16-1 and let-7a in pituitary tumours that developed after 12 months of age in female mice with heterozygous knockout of the Men1 gene (Men1+/− mice). The miRNAs miR-15a, miR-16-1 and let-7a were significantly downregulated in pituitary tumours (by 2.3-fold, P < 0.05; 2.1-fold P < 0.01 and 1.6-fold P < 0.05, respectively) of Men1+/− mice, compared to normal WT pituitaries. miR-15a and miR-16-1 expression inversely correlated with expression of cyclin D1, a known pro-tumourigenic target of these miRNAs, and knockdown of menin in a human cancer cell line (HeLa), and AtT20 mouse pituitary cell line resulted in significantly decreased expression of miR-15a (P < 0.05), indicating that the decrease in miR-15a may be a direct result of lost menin expression.

Kmt2a cooperates with menin to suppress tumorigenesis in mouse pancreatic islets

ABSTRACTThe reported incidence of pancreatic neuroendocrine tumors (PanNETs) has increased, due in large part to improvements in detection and awareness. However, therapeutic options are limited and a critical need exists for understanding a more thorough characterization of the molecular pathology underlying this disease. The Men1 knockout mouse model recapitulates the early stage of human PanNET development and can serve as a foundation for the development of advanced mouse models that are necessary for preclinical testing. Menin, the product of the MEN1 gene, has been shown to physically interact with the KMT2A and KMT2B histone methyltransferases. Both the KMT2A and MEN1 genes are located on chromosome 11q, which frequently undergoes loss of heterozygosity (LOH) in PanNETs. We report herein that inactivation of Kmt2a in Men1-deficient mice accelerated pancreatic islet tumorigenesis and shortened the average life span. Increases in cell proliferation were observed in mouse pancreatic islet tumors upon inactivation of both Kmt2a and Men1. The Kmt2a/Men1 double knockout mouse model can be used as a mouse model to study advanced PanNETs.

Imagerie des récepteurs du GLP-1 dans l’exploration des sécrétions inappropriées d’insuline

Endogenous hyperinsulinemic hypoglycemia is a rare disorder (incidence 2–4/Mio/year). In adult patients, the differential diagnosis includes benign and malignant insulinoma, insulinoma in the context of MEN-1 and nesidioblastosis whereby benign insulinoma is the most frequent diagnosis. The preoperative localization of benign insulinomas is challenging using conventional imaging procedures since these lesions are small (0.5–2 cm). GLP-1R imaging (SPECT/CT and PET/CT) using 111 In-DOTA-exendin-4 and a 68 Ga-DOTA-exendin-4, respectively and is an emerging technique targeting the GLP-1R expressed in very high density on insulinomas. All patients included in the prospective studies had a confirmed endogenous hyperinsulinemic hypoglycemia with neurogylcopenic symptoms. GLP-1R imaging using SPECT/CT ( n = 30) showed a sensitivity of 95% in detecting insulinomas (gold standard: histology), whereas conventional imaging exhibited a sensitivity of 47%. The follow-up study that compared GLP-1R SPECT/CT with PET/CT technique (ongoing study) confirmed the high sensitivity of this imaging procedure with an advantage for the PET/CT technique. Patients with NEM-1 develop multicentric pancreatic islet tumors, the most common functional ones being gastrinomas and insulinomas. Surgery is the only treatment option. However, the surgical intervention should be limited as surgery cannot cure patients with MEN-1. It is therefore, mandatory to correctly localize tumor lesions. The experience with 6 patients suggests that adding GLP-1R imaging to conventional imaging is a helpful tool in differentiating insulinomas from other pancreatic islet lesions. Adding GLP-1R imaging to conventional imaging is a very useful technique in preoperatively localizing benign insulinomas and insulinomas in the context of MEN-1.

Carbonyl stress-induced 5-hydroxytriptamine secretion from RIN-14B, rat pancreatic islet tumor cells, via the activation of transient receptor potential ankyrin 1.

Methylglyoxal (MG), a highly reactive dicarbonyl substance, is known as an endogenous carbonyl stress-inducing substance related to various disease states. Irritable bowel syndrome (IBS) is one of the most frequently encountered gastrointestinal disorders and MG is considered to be its causal substance. An increased serum 5-hydroxytryptamine (5-HT) level is related to IBS symptoms and the majority of 5-HT originates from enterochromaffin (EC) cells in the intestine. Here we examine the mechanisms of MG-induced 5-HT secretion using RIN-14B cells derived from a rat pancreatic islet tumor since these cells are used as a model for EC cells. MG increased the intracellular Ca(2+) concentration ([Ca(2+)]i) and 5-HT secretion, both of which were inhibited by the removal of extracellular Ca(2+) and specific transient receptor potential ankyrin 1 (TRPA1) antagonists. MG elicited an inward current under voltage-clamped conditions. Prior application of MG evoked reciprocal suppression of subsequent [Ca(2+)]i responses to allylisothiocyanate, a TRPA1 agonist, and vice versa. Glyoxal, an analog of MG, also evoked [Ca(2+)]i and secretory responses but its potency was much lower than that of MG. The present results suggest that MG promotes 5-HT secretion through the activation of TRPA1 in RIN-14B cells. These results may indicate that TRPA1 is a promising target for the treatment of IBS and that the RIN-14B cell line is a useful model for investigation of IBS.

Tumor-specific promoter-driven adenoviral therapy for insulinoma.

Insulinomas are the most common type of neuroendocrine (NE) pancreatic islet tumors. Patients with insulinomas may develop complications associated with hyperinsulinemia. To increase the treatment options for insulinoma patients, we have tested a conditionally replicating adenovirus that has been engineered in such a way that it can specifically express therapeutic genes in NE tumors. We used a promoter-specific adenoviral vector delivery system that is regulated by an INSM1 (insulinoma-associated-1) promoter, which is silent in normal adult tissues but active in developing NE cells and tumors. Through a series of modifications, using an insulator (HS4) and neuron-restrictive silencer elements (NRSEs), an oncolytic adenoviral vector was generated that retains tumor specificity and drives the expression of a mutated adenovirus E1A gene (Δ24E1A) and the herpes simplex virus thymidine kinase (HSV-tk) gene. The efficacy of this vector was tested in insulinoma-derived MIN, RIN, βTC-1 and pancreatic (Panc-1) cells using in vitro cell survival and in vivo tumor growth assays. Using in vitro insulinoma-derived cell lines and an invivo subcutaneous mouse tumor model we found that the INSM1 promoter-driven viruses were able to replicate specifically in INSM1-positive cells. INSM1-specific HSV-tk expression in combination with ganciclovir treatment resulted in dose-dependent tumor cell killing, leaving INSM1-negative cells unharmed. When we combined the INSM1-promoter driven HSV-tk with Δ24E1A and INSM1p-HSV-tk (K5) viruses, we found that the co-infected insulinoma-derived cells expressed higher levels of HSV-tk and exhibited more efficient tumor suppression than cells infected with INSM1p-HSV-tk virus alone. INSM1 promoter-driven conditionally replicating adenoviruses may serve as a new tool for the treatment of insulinoma and may provide clinicians with additional options to combat this disease.

Multiple Endocrine Neoplasia Type 1 Presenting as Hypoglycemia due to Insulinoma

Multiple endocrine neoplasia (MEN) mutation is an autosomal dominant disorder characterized by the occurrence of parathyroid, pancreatic islet, and anterior pituitary tumors. The incidence of insulinoma in MEN is relatively uncommon, and there have been a few cases of MEN manifested with insulinoma as the first symptom in children. We experienced a 9-year-old girl having a familial MEN1 mutation. She complained of dizziness, occasional palpitation, weakness, hunger, sweating, and generalized tonic-clonic seizure that lasted for 5 minutes early in the morning. At first, she was only diagnosed with insulinoma by abdominal magnetic resonance images of a 1.3 x 1.5 cm mass in the pancreas and high insulin levels in blood of the hepatic vein, but after her father was diagnosed with MEN1. We found she had familial MEN1 mutation, and she recovered hyperinsulinemic hypoglycemia after enucleation of the mass. Therefore, the early genetic identification of MEN1 mutation is considerable for children with at least one manifestation.

Abstract B09: Myc is required for maintenance of KRasG12D-driven pancreatic cancer and its associated microenvironment

Abstract Deregulation of Myc is observed in most, if not all, human cancers making it an attractive therapeutic target. Indeed, recent studies demonstrate that Myc inhibition (via expression of a dominant negative Myc mutant) triggers regression of SV40-driven pancreatic islet tumors and KRasG12D-driven lung tumors in mice. Myc inhibition in insulinomas elicits regression presaged by collapse of the tumor microenvironment and involution of the tumor vasculature. A characteristic feature of human pancreatic ductal adenocarcinoma (PDAC) is an extensive stromal component (desmoplasia) that is thought to contribute to the resistance of PDAC to current therapies. We show that systemic Myc inhibition in KRasG12D-driven mouse PDAC triggers regression of both tumor and its associated desmoplastic stroma, indicating that endogenous Myc function in the tumor cells is required for maintenance of the desmoplastic reaction. Consistent with this, deregulated expression of physiological levels of Myc enhances pancreatic tumor progression and promotes local immune evasion and stromal desmoplasia. Reversal of deregulated Myc activity results in reversion of these features indicating that they are dependent on Myc activity. The ubiquity of Myc in tumor development and maintenance indicates that it serves an essential and non-redundant role, coupling diverse intracellular oncogenic pathways to the tumor microenvironment, and underscoring its credentials as a pharmacological target in cancers driven by different oncogenes and arising in different tissues. Citation Format: Nicole M. Sodir, Lamorna Brown Swigart, Roderik M. Kortlever, Trevor D. Littlewood, Laura Soucek, Mark J. Arends, Gerard I. Evan. Myc is required for maintenance of KRasG12D-driven pancreatic cancer and its associated microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B09.

An Investigation of the Pancreatic Islet Tumor Microenvironment with Time-of-Flight Secondary Ion Mass Spectrometry

An Investigation of the Pancreatic Islet Tumor Microenvironment with Time-of-Flight Secondary Ion Mass Spectrometry

Abstract A52: Myc is required for maintenance of KRasG12D-driven pancreatic cancer and its associated microenvironment

Abstract The frequent occurrence of Myc deregulation in human cancers makes Myc an intriguing potential therapeutic target. This is supported by recent studies demonstrating that Myc inhibition (via expression of a dominant negative Myc mutant) triggers regression of SV40-driven pancreatic islet tumors and KRasG12D-driven lung tumors in mice. In the case of insulinoma, regression is presaged by collapse of the tumor microenvironment and involution of the tumor vasculature. One feature of human pancreatic ductal adenocarcinoma (PDAC) is its extensive stromal component (desmoplasia) that is thought to contribute to the resistance of PDAC to current therapies. We show that systemic Myc inhibition in the well-established KRasG12D-driven PDAC mouse model triggers regression of both tumor and its associated desmoplastic stroma, indicating that endogenous Myc function in the tumor cells is required for maintenance of the desmoplastic reaction. Consistent with this, ectopic expression of physiological levels of deregulated Myc enhances pancreatic tumor progression and drives stromal desmoplasia, and this reverses upon cessation of deregulated Myc. That systemic Myc inhibition causes regression of both tumor and its associated stroma, while ectopic activation of Myc in tumor cells drives both tumor progression and desmoplasia, confirm that Myc serves an essential and non-redundant role, coupling diverse intracellular oncogenic pathways to the tumor microenvironment, and underscoring its credentials as a pharmacological target in cancers driven by different oncogenes and arising in different tissues. Citation Format: Nicole M. Sodir, Laura Soucek, Trevor D. Littlewood, Mark J. Arends, Gerard I. Evan. Myc is required for maintenance of KRasG12D-driven pancreatic cancer and its associated microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A52.

Dynamic epigenetic regulation by menin during pancreatic islet tumor formation.

The tumor suppressor gene MEN1 is frequently mutated in sporadic pancreatic neuroendocrine tumors (PanNET) and is responsible for the familial multiple endocrine neoplasia type 1 (MEN-1) cancer syndrome. Menin, the protein product of MEN1, associates with the histone methyltransferases (HMT) MLL1 (KMT2A) and MLL4 (KMT2B) to form menin-HMT complexes in both human and mouse model systems. To elucidate the role of methylation of histone H3 at lysine 4 (H3K4) mediated by menin-HMT complexes during PanNET formation, genome-wide histone H3 lysine 4 trimethylation (H3K4me3) signals were mapped in pancreatic islets using unbiased chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq). Integrative analysis of gene expression profiles and histone H3K4me3 levels identified a number of transcripts and target genes dependent on menin. In the absence of Men1, histone H3K27me3 levels are enriched, with a concomitant decrease in H3K4me3 within the promoters of these target genes. In particular, expression of the insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) gene is subject to dynamic epigenetic regulation by Men1-dependent histone modification in a time-dependent manner. Decreased expression of IGF2BP2 in Men1-deficient hyperplastic pancreatic islets is partially reversed by ablation of RBP2 (KDM5A), a histone H3K4-specific demethylase of the jumonji, AT-rich interactive domain 1 (JARID1) family. Taken together, these data demonstrate that loss of Men1 in pancreatic islet cells alters the epigenetic landscape of its target genes. Epigenetic profiling and gene expression analysis in Men1-deficient pancreatic islet cells reveals vital insight into the molecular events that occur during the progression of pancreatic islet tumorigenesis.

Neomorphic effects of recurrent somatic mutations in Yin Yang 1 in insulin-producing adenomas

Insulinomas are pancreatic islet tumors that inappropriately secrete insulin, producing hypoglycemia. Exome and targeted sequencing revealed that 14 of 43 insulinomas harbored the identical somatic mutation in the DNA-binding zinc finger of the transcription factor Yin Yang 1 (YY1). Chromatin immunoprecipitation sequencing (ChIP-Seq) showed that this T372R substitution changes the DNA motif bound by YY1. Global analysis of gene expression demonstrated distinct clustering of tumors with and without YY1(T372R) mutations. Genes showing large increases in expression in YY1(T372R) tumors included ADCY1 (an adenylyl cyclase) and CACNA2D2 (a Ca(2+) channel); both are expressed at very low levels in normal β-cells and show mutation-specific YY1 binding sites. Both gene products are involved in key pathways regulating insulin secretion. Expression of these genes in rat INS-1 cells demonstrated markedly increased insulin secretion. These findings indicate that YY1(T372R) mutations are neomorphic, resulting in constitutive activation of cAMP and Ca(2+) signaling pathways involved in insulin secretion.

Metal-mediated oxidative DNA damage induced by methylene blue

BackgroundMethylene blue (MB) is used for various clinical purposes, including chromoendoscopy and methemoglobinemia treatment. However, MB induces tumors of pancreatic islets and small intestine in experimental animals. This finding raises a possibility that MB induces carcinogenicity in these organs via light-independent mechanisms, although MB is known to cause light-dependent DNA damage. MethodsWe investigated the mechanism of MB-induced DNA damage using 32P-5′-end-labeled DNA fragments of human tumor-relevant genes. We investigated the redox reaction of MB by UV–visible spectrometry. ResultsMB induced DNA damage at the 5′-ACG-3′ sequence, a hot spot of the p53 gene, in the presence of NADH and Cu(II). DNA damage was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator. MB induced DNA damage at every nucleotide in the presence of NADH and Fe(III)-ethylenediaminetetraacetic acid, which was inhibited by OH scavengers and catalase. MB significantly increased the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative DNA lesion, in the presence of NADH and metal ions. UV–visible spectrometry revealed that the absorbance of oxidized form of MB at 668nm was decreased by NADH, and the addition of metal ions attenuated the spectral change. ConclusionsMB undergoes NADH-dependent reduction followed by metal ion-mediated reoxidation. Reduced metal ions [Cu(I) and Fe(II)] interact with H2O2, generated during the redox reaction, to produce Cu(I)OOH and OH that cause DNA damage, respectively. These findings suggest that metal-mediated DNA damage contributes to MB-mediated carcinogenesis. General significanceThis study would provide an insight into the mechanism of MB-induced carcinogenesis and its safety assurance for clinical use.

Multiple endocrine neoplasia (MEN) syndromes

Multiple endocrine neoplasia (MEN) syndromes are characterised by the combined occurrence of two or more endocrine tumours in a patient. These autosomal dominant conditions occur in four types: MEN1 due to inactivating MEN1 mutations; MEN2A and MEN2B (MEN3) due to activating mutations of RET and MEN4 due to inactivating cyclin-dependent kinase inhibitor 1B (CDKN1B) mutations. Each MEN syndrome exhibits different combinations of pancreatic islet, anterior pituitary, parathyroid, medullary thyroid and adrenal tumours. This article provides an overview of the clinical features, treatments and molecular genetics of each endocrine tumour syndrome.