Human PanNET Research Articles

A cross-species transcriptomic analysis reveals a novel 2-dimensional classification system explaining the invasiveness heterogeneity of pancreatic neuroendocrine tumor

Pancreatic neuroendocrine tumors (PanNETs), the second most common type of primary pancreatic tumors, display notable heterogeneity in invasiveness. Current knowledge regarding genomic alterations, including DAXX/ATRX, MEN1 mutations, and copy number variations (CNVs), provides some insights into tumor invasiveness. However, the underlying reasons for the significant variation in invasiveness between insulinoma and other types of PanNETs remain unclear. To construct a comprehensive model for the stratification of prognosis, we employed analysis of both the well-established Rip1-Tag2 (RT2) mouse model of PanNETs and human PanNETs with various functional types. Firstly, by applying single-cell and bulk RNA sequencing in PanNETs from different ages and strains of RT2 mice and human PanNETs, we introduced a 2-dimensional (2D) classification system. Based on the 2-D classification system, human PanNETs were mainly classified as benign insulinomas or non-insulinomas subclusters. Non-insulinomas subtypes mainly included gastrinomas, glucagonomas, VIPomas, and NF-PanNETs, which all exhibited potential invasiveness. In addition, we discovered an enrichment of specific CNV patterns and mutations in corresponding human PanNET subclusters. Then we denoted somatic DAXX/ATRX as the ‘second hit’ and confounding factors for invasiveness. Finally, by combining the 2D system, DAXX/ATRX mutation status, and tumor diameter, a group of indolent PanNETs with minimal recurrence risk was identified. In conclusion, our current work constructed a comprehensive model to elucidate the heterogeneity of invasiveness in PanNETs and improve prognostic stratification.

Angiopoietin-2 blockade suppresses growth of liver metastases from pancreatic neuroendocrine tumors by promoting T cell recruitment.

Improving the management of metastasis in pancreatic neuroendocrine tumors (PanNETs) is critical, as nearly half of patients with PanNETs present with liver metastases, and this accounts for the majority of patient mortality. We identified angiopoietin-2 (ANGPT2) as one of the most upregulated angiogenic factors in RNA-Seq data from human PanNET liver metastases and found that higher ANGPT2 expression correlated with poor survival rates. Immunohistochemical staining revealed that ANGPT2 was localized to the endothelial cells of blood vessels in PanNET liver metastases. We observed an association between the upregulation of endothelial ANGPT2 and liver metastatic progression in both patients and transgenic mouse models of PanNETs. In human and mouse PanNET liver metastases, ANGPT2 upregulation coincided with poor T cell infiltration, indicative of an immunosuppressive tumor microenvironment. Notably, both pharmacologic inhibition and genetic deletion of ANGPT2 in PanNET mouse models slowed the growth of PanNET liver metastases. Furthermore, pharmacologic inhibition of ANGPT2 promoted T cell infiltration and activation in liver metastases, improving the survival of mice with metastatic PanNETs. These changes were accompanied by reduced plasma leakage and improved vascular integrity in metastases. Together, these findings suggest that ANGPT2 blockade may be an effective strategy for promoting T cell infiltration and immunostimulatory reprogramming to reduce the growth of liver metastases in PanNETs.

Abstract B054: Histone lysine methyltransferase NSD3 governs transcriptional programs that drive pancreatic neuroendocrine tumors

Abstract Pancreatic Neuroendocrine Tumors (PanNETs) are the most common and lethal neuroendocrine malignancies. For decades, standard chemotherapeutic regimens and targeted therapeutics have been used in advanced patients with little to no avail. Thus, there is a critical unmet need to develop novel and effective precision therapeutics. Additionally, pre-clinical models that faithfully recapitulate disease are urgently needed for translational studies. Here, we directly address these challenges by characterizing a novel genetically engineered mouse model of PanNETs as well as identifying histone lysine methyltransferase (KMT) NSD3 as a critical oncogenic driver. We identified KMT NSD3 to be upregulated through a meta-analysis of human PanNET RNA-Seq data. Previous literature indicates that NSD3 canonically di-methylates Histone H3 lysine residue 36 (H3K36me2) selecting transcriptional and gene expression programs with potential cancer implications. Thus, we sought to identify the role of NSD3 and H3K36 di-methylation dynamics in PanNET tumorigenesis. Our initial findings indicate that loss of NSD3 decreases proliferation and di-methylation of H3K36 in vitro using PanNET PDX cell lines. Additionally, we generated a novel in vivo model focusing on the pancreatic islet-specific loss of conditional Men1Lox/Lox, AtrxLox/Lox, and PtenLox/Lox alleles, alterations of which are representative of human disease. Proof-of-concept studies were done comparing time-matched wild-type and our PanNET model, revealing that our model has increased islet hyperplasia and proliferation. Additionally, to analyze the role of NSD3 in vivo, we generated a conditional Nsd3Lox/Lox knockout mouse which I have crossed to our PanNET model. Preliminary studies suggest that Nsd3 depletion significantly attenuates tumor growth in vivo. Furthermore, our lab conducted in vitro methylation assays that identified a gain-of-function point mutation NSD3T1232A that increases the catalytic activity of NSD3 at H3K36me2. Thus, to understand the role of the enzymatic activity of NSD3 in PanNET tumorigenesis, we conducted studies in vitro where we reconstituted NSD3 wild-type, NSD3Y1174A enzymatically deficient, and NSD3T1232A in NSD3 depleted cells. Preliminary findings indicate that NSD3T1232A increases H3K36me2 and significantly increases proliferation, while NSD3WT rescues H3K36me2 and proliferation, and NSD3Y1174A does not rescue H3K36me2 and the phenotype observed. Lastly, to leverage NSD3 hyperactivity in vivo, we generated an NSD3T1232A mouse (Rosa26LSL-NSD3T1232A) and crossed with our initial PanNET model. Our results demonstrate that NSD3T1232A significantly decreases survival, accelerates tumor burden, and proliferation when compared to time-matched control and NSD3 deficient mice. Together, these studies provide insights into the role of NSD3 in PanNET tumorigenesis. Future portions of testing will establish the specific cellular and molecular modes of action of NSD3 and establish a potential therapeutic target. Citation Format: Mary E. Fuentes, Simone C. Hausmann, Natasha M. Flores, Xiaoyin Lu, Pawel K. Mazur. Histone lysine methyltransferase NSD3 governs transcriptional programs that drive pancreatic neuroendocrine tumors [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B054.

Three-Dimensional Primary Cell Culture: A Novel Preclinical Model for Pancreatic Neuroendocrine Tumors

Molecular mechanisms underlying the development and progression of pancreatic neuroendocrine tumors (PanNETs) are still insufficiently understood. Efficacy of currently approved PanNET therapies is limited. While novel treatment options are being developed, patient stratification permitting more personalized treatment selection in PanNET is yet not feasible since no predictive markers are established. The lack of representative in vitro and in vivo models as well as the rarity and heterogeneity of PanNET are prevailing reasons for this. In this study, we describe an in vitro 3-dimensional (3-D) human primary PanNET culture system as a novel preclinical model for more personalized therapy selection. We present a screening platform allowing multicenter sample collection and drug screening in 3-D cultures of human primary PanNET cells. We demonstrate that primary cells isolated from PanNET patients and cultured in vitro form islet-like tumoroids. Islet-like tumoroids retain a neuroendocrine phenotype and are viable for at least 2 weeks in culture with a high success rate (86%). Viability can be monitored continuously allowing for a per-well normalization. In a proof-of-concept study, islet-like tumoroids were screened with three clinically approved therapies for PanNET: sunitinib, everolimus and temozolomide. Islet-like tumoroids display varying in vitro response profiles to distinct therapeutic regimes. Treatment response of islet-like tumoroids differs also between patient samples. We believe that the presented human PanNET screening platform is suitable for personalized drug testing in a larger patient cohort, and a broader application will help in identifying novel markers predicting treatment response and in refining PanNET therapy.

A set of microRNAs coordinately controls tumorigenesis, invasion, and metastasis

MicroRNA-mediated gene regulation has been implicated in various diseases, including cancer. This study examined the role of microRNAs (miRNAs) during tumorigenesis and malignant progression of pancreatic neuroendocrine tumors (PanNETs) in a genetically engineered mouse model. Previously, a set of miRNAs was observed to be specifically up-regulated in a highly invasive and metastatic subtype of mouse and human PanNET. Using functional assays, we now implicate different miRNAs in distinct phenotypes: miR-137 stimulates tumor growth and local invasion, whereas the miR-23b cluster enables metastasis. An algorithm, Bio-miRTa, has been developed to facilitate the identification of biologically relevant miRNA target genes and applied to these miRNAs. We show that a top-ranked miR-137 candidate gene, Sorl1, has a tumor suppressor function in primary PanNETs. Among the top targets for the miR-23b cluster, Acvr1c/ALK7 has recently been described to be a metastasis suppressor, and we establish herein that it is down-regulated by the miR-23b cluster, which is crucial for its prometastatic activity. Two other miR-23b targets, Robo2 and P2ry1, also have demonstrable antimetastatic effects. Finally, we have used the Bio-miRTa algorithm in reverse to identify candidate miRNAs that might regulate activin B, the principal ligand for ALK7, identifying thereby a third family of miRNAs-miRNA-130/301-that is congruently up-regulated concomitant with down-regulation of activin B during tumorigenesis, suggestive of functional involvement in evasion of the proapoptotic barrier. Thus, dynamic up-regulation of miRNAs during multistep tumorigenesis and malignant progression serves to down-regulate distinctive suppressor mechanisms of tumor growth, invasion, and metastasis.

Tumour cells mirror embryonic developmental programs to acquire invasive and metastatic capabilities

Abstract Background Pancreatic Neuroendocrine Tumours (PanNETs) are the second most common form of pancreatic cancer. We previously identified two principal subtypes of PanNET: insulinomas (islet tumors; IT) and metastasis-like primaries (MLP), corresponding to the low- and high-grade classification of human PanNETs. This study describes the mechanisms by which PanNETs progress from IT to more aggressive MLP tumors and eventually metastasize. Methods We profiled single cell transcriptomes, bulk mRNA and miRNA transcriptomes and the proteomes of primary and metastasis specimens from the genetically engineered mouse model of PanNETs (Rip1 Tag2). Results We found that the IT tumours maintained the expression of mature s cell markers. In contrast, the MLP tumours expressed pancreatic progenitor markers. By integrating the data on s-cell differentiation from pancreatic progenitors to mature s cells, we demonstrated that the tumour progression from IT to MLP follows the reverse embryonic and postnatal developmental path. Furthermore, we identified Hmgb3 and miR-181cd cluster as the MLP master regulators. Over-expression of the miR-181cd cluster in IT-like cancer cell-lines resulted in the acquisition of the MLP gene signature and MLP morphologic phenotypes, in addition to activation Hmgb3 expression. This suggested a central role for Hmgb3 in initiating the MLP program. Furthermore, inhibiting the expression of Hmgb3 in MLP-like cancer cell-lines resulted in a significant growth decrease, demonstrating the importance of Hmgb3 in the maintenance of MLP-like cell state in vitro. Using transcriptomic data from human patients, we evaluated the relevance of the MLP program in human tumours. We established that aggressive human PanNET tumours also follow the same reverse developmental trajectory of dedifferentiation. Notably, patients with high MLP genes expression had a worse prognosis. Conclusions These data demonstrate dedifferentiation as a mechanism by which malignant neuroendocrine cancer cells acquire progenitor-like features, enabling them to become more aggressive and metastatic. In addition, miR-181cd cluster and Hmgb3 act as the core regulators in the initiation of dedifferentiation and maintenance of progenitor-like features in tumour cells. Legal entity responsible for the study CMSO Group at ISREC, EPFL. Funding Swiss National Science Foundation. Disclosure All authors have declared no conflicts of interest.

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.

Hypo-methylation mediates chromosomal instability in pancreatic NET.

DAXX and or ATRX loss occur in 40% of pancreatic neuroendocrine tumors (PanNETs). PanNETs negative for DAXX or ATRX show an increased risk of relapse. The tumor-associated pathways activated upon DAXX or ATRX loss and how this event may induce chromosomal instability (CIN) and alternative lengthening telomeres (ALT) are still unknown. Both DAXX and ATRX are involved in DNA methylation regulation. DNA methylation of heterochromatin and of non-coding sequences is extremely important for the maintenance of genomic stability. We analyzed the association of DAXX and/or ATRX loss and CIN with global DNA methylation in human PanNET samples and the effect of DAXX knock-down on methylation and cell proliferation. We assessed LINE1 as well as global DNA methylation in 167 PanNETs, and we found that DAXX and or ATRX-negative tumors and tumors with CIN were hypomethylated. DAXX knock-down in PanNET cell lines blocked cells in G1/G0 phase and seemed to increase CIN in QGP-1 cells. However, no direct changes in DNA methylation were observed after DAXX knock-down in vitro In conclusion, our data indicate that epigenetic changes are crucial steps in the progression of PanNETs loss and suggest that DNA methylation is the mechanism via which CIN is induced, allowing clonal expansion and selection.

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.

Abstract 2862: The balance of divergent IRE1α signaling regulates pancreatic neuroendocrine tumor growth

Abstract The Unfolded Protein Response (UPR) is an intracellular signaling pathway that communicates the protein folding status of the endoplasmic reticulum (ER) to the nucleus to maintain ER homeostasis. Hypoxia, nutrient deprivation, proteasome dysfunction, or sustained demands on the secretory pathway–conditions often encountered by solid tumor cells–lead to the accumulation of misfolded proteins in the ER and cause “ER stress.” However, the precise role of UPR signaling in the development and maintenance of cancer remains controversial. We reasoned that pancreatic neuroendocrine tumors (PanNETs) might be one class of solid tumor that may be particularly sensitive to protein folding stress due to their high secretory activity. The Master UPR regulator IRE1α is an ER transmembrane kinase/endoribonuclease (RNase) that acts as a critical life-death switch depending on the level of ER stress. We analyzed primary human PanNET samples for activation of UPR components and found strong evidence of ER stress and IRE1α hyperactivation in this tumor type. We used a variety of chemical-genetic tools to selectively manipulate IRE1α's homeostatic and apoptotic outputs in a PanNET xenograft model and found that the precise balance of IRE1α signaling is critical for tumor growth in vivo. Disrupting this balance by pushing the pathway in either direction is detrimental to PanNET survival. Moreover, targeting IRE1α with highly selective small molecules in a preclinical mouse model phenocopied the results of genetic manipulation. Our results indicate that IRE1α is an important regulator of PanNET cell survival and growth, and provide a strong rationale for therapeutically targeting this kinase in PanNETs and other cancers that experience high levels of ER stress. Citation Format: Jenny Y. Qi, Paul C. Moore, Maike Thamsen, Rajarshi Ghosh, Micah J. Gliedt, Dustin J. Maly, Bradley J. Backes, Feroz R. Papa, Scott A. Oakes. The balance of divergent IRE1α signaling regulates pancreatic neuroendocrine tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2862.

Abstract B33: Beyond apoptosis, Bcl-xL in metastasis

Abstract Bcl-xL, a negative regulator of mitochondria-mediated apoptosis, is frequently overexpressed in cancer to promote survival. Bcl-xL can also promote migration and/or metastasis. However, it is unclear whether this metastatic function is mediated via – or independent of – its anti-apoptotic activity and its residence at mitochondria. Here, we show that Bcl-xL promotes migration of cells defective in mitochondria-mediated apoptosis. Importantly, Bcl-xL mutants lacking anti-apoptotic activity still induce epithelial-mesenchymal transition (EMT), upregulate TGFβ, and increase metastasis of pancreatic neuroendocrine tumor (panNET) cells. In the RIP-Tag; RIP-tva mouse model, infection of pancreatic lesions with avian retroviruses carrying such Bcl-xL mutants promotes metastasis as potently as wild-type Bcl-xL does. Furthermore, engineered Bcl-xL targeted to the nucleus, but not mitochondria or outside of nucleus, promotes EMT and alters EMT-associated factors. In addition, nuclear Bcl-xL can be found in human metastatic panNET samples. Taken together, these data demonstrate that the metastatic function of Bcl-xL is independent of its anti-apoptotic activity and its residence at mitochondria. Citation Format: Soyoung Choi, Zhengming Chen, Laura Tang, Yuanzhang Fang, Yi Li, Xuejun Jiang, Yi-Chieh Nancy Du. Beyond apoptosis, Bcl-xL in metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B33.

Targeting Focal Adhesion Kinase and Resistance to mTOR Inhibition in Pancreatic Neuroendocrine Tumors.

Focal adhesion kinase (FAK) mediates survival of normal pancreatic islets through activation of AKT. Upon malignant transformation of islet cells into pancreatic neuroendocrine tumors (PanNETs), AKT is frequently overexpressed and mutations in the AKT/mTOR pathway are detected. Because mTOR inhibitors rarely induce PanNET tumor regression, partly because of feedback activation of AKT, novel combination strategies are needed to target FAK/AKT/mTOR signaling. We characterized the activation of FAK in PanNETs using immunohistochemistry and Western blot analysis and tested the FAK inhibitor PF-04554878 in human PanNET cells in vitro and in vivo (at least three mice per group). In addition, we evaluated the effect of combined FAK and mTOR inhibition on PanNET viability and apoptosis. All statistical tests were two-sided. We found that FAK is overexpressed and hyperphosphorylated in human PanNETs and that PF-04554878 strongly inhibited FAK (Tyr397) autophosphorylation in a dose-dependent manner. We found that PF-04554878 inhibited cell proliferation and clonogenicity and induced apoptosis in PanNET cells. Moreover, oral administration of PF-04554878 statistically significantly reduced tumor growth in a patient-derived xenograft model of PanNET (P = .02) and in a human PanNET xenograft model of peritoneal carcinomatosis (P = .03). Importantly, PF-04554878 synergized with the mTOR inhibitor everolimus by preventing feedback AKT activation. We demonstrate for the first time that FAK is overexpressed in PanNETs and that inhibition of FAK activity induces apoptosis and inhibits PanNET proliferation. We found that the novel FAK inhibitor PF-04554878 synergizes with everolimus, a US Food and Drug Administration-approved agent for PanNETs. Our findings warrant the clinical investigation of combined FAK and mTOR inhibition in PanNETs.

The bromodomain and extra-terminal inhibitor CPI203 enhances the antiproliferative effects of rapamycin on human neuroendocrine tumors.

Endogenous c-MYC (MYC) has been reported to be a potential pharmacological target to trigger ubiquitous tumor regression of pancreatic neuroendocrine tumors (PanNETs) and lung tumors. Recently inhibitors of bromodomain and extra-terminal (BET) family proteins have shown antitumor effects through the suppression of MYC in leukemia and lymphoma. In this paper, we investigated the antitumor activity of a BET protein bromodomain inhibitor (BETi) CPI203 as a single agent and in combination with rapamycin in human PanNETs. We found that exposure of human PanNET cell lines to CPI203 led to downregulation of MYC expression, G1 cell cycle arrest and nearly complete inhibition of cell proliferation. In addition, overexpression of MYC suppressed the growth inhibition caused by CPI203 and knockdown of MYC phenocopied the effects of CPI203 treatment. These findings indicate that suppression of MYC contributed to the antiproliferative effects of BETi inhibition in human PanNET cells. Importantly, CPI203 treatment enhanced the antitumor effects of rapamycin in PanNET cells grown in monolayer and in three-dimensional cell cultures, as well as in a human PanNET xenograft model in vivo. Furthermore, the combination treatment attenuated rapamycin-induced AKT activation, a major limitation of rapamycin therapy. Collectively, our data suggest that targeting MYC with a BETi may increase the therapeutic benefits of rapalogs in human PanNET patients. This provides a novel clinical strategy for PanNETs, and possibly for other tumors as well.

Attenuation of the Retinoblastoma Pathway in Pancreatic Neuroendocrine Tumors Due to Increased Cdk4/Cdk6

In mice, genetic changes that inactivate the retinoblastoma tumor suppressor pathway often result in pancreatic neuroendocrine tumors (Pan-NETs). Conversely, in humans with this disease, mutations in genes of the retinoblastoma pathway have rarely been detected, even in genome-wide sequencing studies. In this study, we took a closer look at the role of the retinoblastoma pathway in human Pan-NETs. Pan-NET tumors from 92 patients were subjected to immunohistochemical staining for markers of the retinoblastoma pathway. To search for amplifications of retinoblastoma pathway genes, genomic DNAs from 26 tumors were subjected to copy number analysis. Finally, a small-molecule activator of the retinoblastoma pathway was tested for effects on the growth of two Pan-NET cell lines. A majority of tumors expressed high amounts of Cdk4 or its partner protein cyclin D1. High amounts of phosphorylated Rb1 were present in tumors that expressed high levels of Cdk4 or cyclin D1. The copy numbers of Cdk4 or the analogous kinase gene Cdk6 were increased in 19% of the tumors. Growth of the human Pan-NET cell line QGP1 was inhibited in a xenograft mouse model by the Cdk4/6 inhibitor, PD 0332991, which reactivates the retinoblastoma pathway. Inactivation of the retinoblastoma pathway was indicated for most Pan-NETs. Gene amplification and overexpression of Cdk4 and Cdk6 suggests that patients with Pan-NETs may respond strongly to Cdk4/6 inhibitors that are entering clinical trials.