An update on genetically engineered mouse models of pancreatic neuroendocrine neoplasms

in Endocrine-Related Cancer
Authors:
Tiago Bordeira Gaspari3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal
Ipatimup – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal
ICBAS – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto, Porto, Portugal
FMUP – Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Search for other papers by Tiago Bordeira Gaspar in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0002-7410-7869
,
José Manuel Lopesi3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal
Ipatimup – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal
FMUP – Faculdade de Medicina da Universidade do Porto, Porto, Portugal
Department of Pathology, Centro Hospitalar e Universitário de São João, Porto, Portugal

Search for other papers by José Manuel Lopes in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0001-8597-3474
,
Paula Soaresi3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal
Ipatimup – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal
FMUP – Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Search for other papers by Paula Soares in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0001-9607-6998
, and
João Vinagrei3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal
Ipatimup – Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal
FMUP – Faculdade de Medicina da Universidade do Porto, Porto, Portugal

Search for other papers by João Vinagre in
Current site
Google Scholar
PubMed
Close
https://orcid.org/0000-0002-0871-1427
View More View Less

Correspondence should be addressed to P Soares or J Vinagre: psoares@ipatimup.pt or jvinagre@ipatimup.pt
Free access

Pancreatic neuroendocrine neoplasms (PanNENs) are rare and clinically challenging entities. At the molecular level, PanNENs’ genetic profile is well characterized, but there is limited knowledge regarding the contribution of the newly identified genes to tumor initiation and progression. Genetically engineered mouse models (GEMMs) are the most versatile tool for studying the plethora of genetic variations influencing PanNENs’ etiopathogenesis and behavior over time. In this review, we present the state of the art of the most relevant PanNEN GEMMs available and correlate their findings with the human neoplasms’ counterparts. We discuss the historic GEMMs as the most used and with higher translational utility models. GEMMs with Men1 and glucagon receptor gene germline alterations stand out as the most faithful models in recapitulating human disease; RIP-Tag models are unique models of early-onset, highly vascularized, invasive carcinomas. We also include a section of the most recent GEMMs that evaluate pathways related to cell cycle and apoptosis, Pi3k/Akt/mTOR, and Atrx/Daxx. For the latter, their tumorigenic effect is heterogeneous. In particular, for Atrx/Daxx, we will require more in-depth studies to evaluate their contribution; even though they are prevalent genetic events in PanNENs, they have low/inexistent tumorigenic capacity per se in GEMMs. Researchers planning to use GEMMs can find a road map of the main clinical features in this review, presented as a guide that summarizes the chief milestones achieved. We identify pitfalls to overcome, concerning the novel designs and standardization of results, so that future models can replicate human disease more closely.

Abstract

Pancreatic neuroendocrine neoplasms (PanNENs) are rare and clinically challenging entities. At the molecular level, PanNENs’ genetic profile is well characterized, but there is limited knowledge regarding the contribution of the newly identified genes to tumor initiation and progression. Genetically engineered mouse models (GEMMs) are the most versatile tool for studying the plethora of genetic variations influencing PanNENs’ etiopathogenesis and behavior over time. In this review, we present the state of the art of the most relevant PanNEN GEMMs available and correlate their findings with the human neoplasms’ counterparts. We discuss the historic GEMMs as the most used and with higher translational utility models. GEMMs with Men1 and glucagon receptor gene germline alterations stand out as the most faithful models in recapitulating human disease; RIP-Tag models are unique models of early-onset, highly vascularized, invasive carcinomas. We also include a section of the most recent GEMMs that evaluate pathways related to cell cycle and apoptosis, Pi3k/Akt/mTOR, and Atrx/Daxx. For the latter, their tumorigenic effect is heterogeneous. In particular, for Atrx/Daxx, we will require more in-depth studies to evaluate their contribution; even though they are prevalent genetic events in PanNENs, they have low/inexistent tumorigenic capacity per se in GEMMs. Researchers planning to use GEMMs can find a road map of the main clinical features in this review, presented as a guide that summarizes the chief milestones achieved. We identify pitfalls to overcome, concerning the novel designs and standardization of results, so that future models can replicate human disease more closely.

Introduction

Pancreatic neuroendocrine neoplasms (PanNENs) are rare entities that constitute the second most common pancreatic malignancy (1–2% of all pancreatic neoplasms) following pancreatic ductal adenocarcinoma (PDAC) (Lawrence et al. 2011, Dasari et al. 2017). PanNENs originate from a network of endocrine cells that includes islet cells and pluripotent precursors in the pancreatic ductal epithelium (Asa 2011).

Clinically, PanNENs can be divided into functioning PanNENs (F-PanNENs), such as insulinoma, glucagonoma, and gastrinoma (15–30% of all PanNENs), or non-functioning PanNENs (NF-PanNENs) that comprise the most part (Metz & Jensen 2008, Batukbhai & De Jesus-Acosta 2019, Klöppel et al. 2021). While F-PanNENs actively secrete hormones, thus triggering well-described clinical syndromes, NF-PanNENs are usually detected at a late disease stage, only when patients experience symptoms derived from tumor mass effects or metastatic lesions, being frequently associated with a poorer prognosis (Metz & Jensen 2008, Scott & Howe 2019, Wang et al. 2019).

PanNENs usually occur sporadically, but in 10% of the cases, they can be present as a component of familial syndromes (Jensen et al. 2008, Chen et al. 2012, McKenna & Edil 2014). The most representative is the multiple endocrine neoplasia type 1 (MEN1) syndrome, where over 95% of patients develop multiple pancreatic neuroendocrine tumors (PanNETs) (Sadowski & Triponez 2015, Jensen & Norton 2017). PanNETs can also be part of the spectrum of other hereditary cancer syndromes, namely von Hippel–Lindau (VHL), neurofibromatosis type 1 (NF1), tuberous sclerosis complex (TSC1/TSC2), and Mahvash disease from glucagon receptor (GCGR) mutations (Jensen et al. 2008, Chen et al. 2012, Yu 2018, Geurts 2020).

Histologically, WHO criteria direct pathologists on human PanNEN classification and grading based on the proliferative activity of the neoplastic cells (Perren et al. 2017): low-grade (G1) (mitoses <2/10 high-power field (HPF) or Ki67 index <3%) and intermediate-grade (G2) (mitoses of 2–20 HPF or Ki67 index of 3–20%) well-differentiated (WD) tumors (PanNETs) and high-grade (G3) neoplasms (mitoses >20/10 HPF or Ki67 index > 20%) that could either be WD (PanNETs G3) or poorly differentiated (PD) pancreatic neuroendocrine carcinomas (PanNECs).

Long-term survival is achieved in some PanNEN patients, although the overall 5-year and 10-year survival rates are 46 and 31%, respectively (Brooks et al. 2019, Wang et al. 2019, Chen et al. 2021). PanNETs G3 and PanNECs are uncommon (<10%) and present the worst prognosis of the pancreatic NEN spectrum (Yao et al. 2008, Chen et al. 2021). Despite the substantial progress made in the clinicians’ awareness and diagnostic techniques during the last decade that contributed to the rapidly rising annual incidence worldwide (Dasari et al. 2017, Brooks et al. 2019, Das & Dasari 2021), PanNENs are clinically challenging because of the limited understanding of their molecular basis (Raj & Reidy-Lagunes 2016, Akirov et al. 2019, Batukbhai & De Jesus-Acosta 2019).

Using whole exome sequencing, Jiao and colleagues identified, a decade ago, novel and frequently mutated genes in PanNET (Jiao et al. 2011). Since then, multiple works have assessed the mutation frequencies of the genes involved in PanNET and PanNEC tumorigenesis (Jiao et al. 2011, Cao et al. 2013, Scarpa et al. 2017, Cives et al. 2019, Hong et al. 2020, Song et al. 2021). Besides the menin 1 gene (MEN1) (mutated in 21–44%), novel genes started to be regarded as essential players in PanNEN tumorigenesis, such as the ATRX chromatin remodeler (ATRX) (11–17%), the death domain-associated protein (DAXX) (14–25%), and members of the mechanistic target of rapamycin kinase (mTOR) (9–15%) (Jiao et al. 2011, Scarpa et al. 2017, Cives et al. 2019, Hong et al. 2020). Currently, it is recognized that mutations in ATRX, DAXX, or MEN1 are characteristic of PanNETs but generally absent in PanNECs, in which TP53 and RB1 mutations are more represented. This points out that human PanNECs share a more similar genetic signature to PDACs (Jiao et al. 2011, Yachida et al. 2012, Kim et al. 2020). TP53 and RB1 mutations have been detected in 3–4% and 2% of human PanNEN studied samples, respectively (Jiao et al. 2011, Scarpa et al. 2017, Cives et al. 2019). PDAC’s specific KRAS mutations can occur in PanNECs up to 49%, and a correlation with poor prognosis is also reported (Yuan et al. 2014, Hijioka et al. 2017). YY1 mutations, although residual in the overall spectrum of PanNEN mutations, are rare in NF-PanNENs and particularly prevalent in insulinomas (18–30%) (Cao et al. 2013, Hong et al. 2020, Song et al. 2021).

Generation of PanNEN mouse models

Genetically engineered mouse models (GEMMs) are excellent tools for investigating PanNENs, from prognostic factors to targeted drug therapies, by allowing specific genetic manipulation, relatively short-term outcomes, and easy access to pancreatic tissue virtually at any stage of tumorigenesis (Babu et al. 2013, Yu 2016, Kawasaki et al. 2018). They are not the only option, and in recent years, substantial advances in other animal and non-animal PanNEN models have been presented (e.g. other rodents, zebrafish, Xenopus frogs, and organoids). For instance, zebrafish lines evolved considerably during the last decade and are now being used in many preclinical settings in PanNETs and PanNECs (Vitale et al. 2014, Gaudenzi et al. 2020); Xenopus tropicalis frogs were also recently endorsed as PanNEC models (Naert et al. 2020); both are useful models to study the NE system (Salanga & Horb 2015, McNamara et al. 2018, Gaudenzi et al. 2020). Patient-derived xenografts (PDXs) provide a relevant, specific drug screening application capacity in a more personalized approach. However, PDXs have low engraftment rates in rodents (Yang et al. 2016), and they are not perfect for studying PanNEN initiation and progression since these animals lack a competent immune system. PanNEN organoids are rapidly emerging in the literature as an alternative to animal models, with impactful utility in the personalization of anticancer therapy (Kawasaki et al. 2020). When selecting the perfect model, the main advantages and limitations of the PanNEN models must be pondered to determine the best model applicability (a summary is presented in Fig. 1).

Figure 1
Figure 1

Advantages and limitations of the main PanNEN models. GEP, gastroenteropancreatic; NE, neuroendocrine; PanNEN, pancreatic neuroendocrine neoplasm; PDX, patient-derived xenograft; TFs, transcription factors; ZF, zebrafish. Created with BioRender.com. A full color version of this figure is available at https://doi.org/10.1530/ERC-22-0166.

Citation: Endocrine-Related Cancer 29, 12; 10.1530/ERC-22-0166

Given their unique advantages, GEMMs dominate the PanNEN animal models, and this review will only focus on them. In PanNENs, the most validated mouse models have been genetically programmed: (1) to conditionally activate oncogenes under relevant promoters (e.g. insulin and preproglucagon) or (2) to mimic PanNET syndromes, with MEN1 being the most studied model. More recently, some GEMMs whose genetic manipulation aimed at inducing PanNEN tumorigenesis, but resulted in other outcomes, have also been studied and are worth discussing.

Embryonic stem cells have been used for decades to alter virtually any gene of interest in the genome of mammals. This technology, known as gene targeting or knockout (KO), occurs through homologous recombination (HR) between the newly introduced DNA and the receptor mice genome, and this is the base of all the GEMMs we will discuss (Capecchi 1989). Global (or constitutive) KO GEMMs have been used to reproduce human hereditary syndromes. Since many genes play essential roles in embryonic and/or early postnatal development, a Cre-loxP technology was developed to circumvent premature lethality by allowing spatial and/or temporal HR control (Deng 2012). This tool has been extensively used to generate conditional mouse cancer models. It enables the deletion of one or more specific gene exons, leading to complete or partial protein loss when targeting tumor suppressors or the introduction or activation of oncogenes. This way, it is possible to study its effects in particular tissues and more efficiently reproduce spontaneous carcinogenesis. In the context of PanNENs, conditional GEMMs are usually the offspring of a mouse strain carrying conditional KO alleles crossed with another mouse strain that overexpresses Cre recombinase (Cre) under a specific gene promoter. Variations using the rat insulin promoter (RIP), exclusively expressed in beta (β) cells, and the preproglucagon promoter (Glu), expressed solely in alpha (α) cells, are some representative examples (Deng 2012).

We reviewed the main findings among all the revisited 60 studies on PanNEN GEMMs; the information was divided into two major groups: (1) historic PanNEN mouse models (RIP-Tag GEMMs of insulinoma, Glu-Tag GEMMs of glucagonoma, Mahvash disease, and Men1 GEMMs), whose details are depicted in Supplementary Tables 1, 2, and 3 (see section on supplementary materials given at the end of this article) and (2) novel PanNEN mouse models, which include GEMMs that disrupted cell cycle/apoptosis players, Pi3k/Akt/mTOR, and Atrx/Daxx pathways, and whose details are available in Supplementary Tables 4, 5, and 6. In the supplementary tables, a detailed data summary of the studied GEMMs can be found, including (1) GEMM name and background strain(s); (2) mechanism of genetic modification; (3) type and the onset of PanNEN; (4) presence and the onset of metastases and other lesions; (5) PanNEN behavior and presence of a multistep tumorigenic pathway; (6) presence and the onset of islet hyperplasia; (7) information regarding glycemia and insulinemia; and (8) some relevant notes.

Historic PanNEN mouse models

RIP-Tag GEMMs of insulinoma

The first animal model of PanNEN was described in 1985 by Hanahan et al. the RIP-Tag GEMM – also known as RIP1-Tag2 or simply RT2 (Hanahan 1985). It was developed to express a viral oncogene conditionally, the simian virus 40 (SV40) large T antigen, under the rat insulin II (Ins2) promoter (RIP1), resulting in p53 and retinoblastoma-associated protein (Rb) tumor-suppressor pathway inactivation, specifically in islet β cells. Despite being a rare event in human PanNENs and not a mechanism of tumor initiation, p53 total or partial silencing through aberrant activation of negative regulators of the p53 pathway was present in 70% of PanNETs patients in a large case series (Hu et al. 2010, Jiao et al. 2011). RT2 is the most used GEMM in research, described as an aggressive insulinoma model with a well-characterized multistage tumor progression pathway (Hanahan 1985, Rindi et al. 1990, Grant et al. 1991, Inoue et al. 2002, Lopez & Hanahan 2002, Hunter et al. 2013). At the embryonic stage, the T antigen begins to be expressed. After 2 months, β-cell hyperplasia and/or dysplasia and angiogenic phenomena occur; multiple tumors develop between 2.5 and 5 months (Hanahan 1985). The tumor spectrum of RT2 mice encompasses highly vascularized PanNENs with well-defined margins to invasive carcinomas (ICs) (also called malignant insulinomas) of two types (IC-1 and IC-2), according to the extension of the adjacent exocrine tissue invasion (Lopez & Hanahan 2002). In RT2 models, such malignant insulinomas exhibit aggressive behavior, with marked nuclear pleomorphism and frequent mitoses but infrequent distant metastases (Grant et al. 1991, Perl et al. 1998, Mandriota et al. 2001, Lopez & Hanahan 2002, Casanovas et al. 2005a , Du et al. 2007, Hager et al. 2009, Hunter et al. 2013, Kobayashi et al. 2019). Hunter et al. reported the coexistence of Id-1-positive poorly differentiated invasive carcinomas (PDICs) that lacked expression of insulin, chromogranin A, and synaptophysin (Syn) in a proportion of 1:9 with insulinomas (Hunter et al. 2013). In general, RT2 mice benefit from a high-sugar diet to prevent sudden premature death that can occur around 23 months ascribed to hypoglycemia due to insulinomas (Perl et al. 1998, Herzig et al. 1999, Mandriota et al. 2001, Porcellini et al. 2015).

It is worth noting that a substantial amount of GEMMs, like some RT2 models, used the term ‘insulinomas’ to classify insulin-expressing PanNENs, regardless of the insulin secretory profile. Also, considering the different nomenclature between human and murine PanNENs, in all revisited GEMMs, the retrieved information regarding tumor histopathological classification was categorized as follows: (1) islet adenoma (IA) or (2) islet IC, which should not be directly compared with human PanNECs.

Glu-Tag GEMMs of glucagonoma

The preproglucagon (Glu) gene is expressed in the pancreatic α cells, intestinal epithelium, and central nervous system. It encodes the preproglucagon polypeptide, cleaved to generate glucagon in the pancreatic α cells and glucagon-like peptides (GLP) 1 and 2 in the gut (Efrat et al. 1988). Similar to RT2, Glu-driven expression of Tag causes moderate to aggressive glucagonomas that arise in a multistep pathway (Efrat et al. 1988, Rindi et al. 1991, Lee et al. 1992, Asa et al. 1996). In the Glu2-Tag mice, the transgene is expressed in the pancreas and the brain under an 850-base pair (bp) rat Glu promoter. Hyperplasia of α cells occurs at 5 months, rare dysplastic islets are visible at 6 months, and glucagonomas develop around 9–12 months (Efrat et al. 1988, Rindi et al. 1991). Mice euthanized at 18 months have a tumor spectrum resembling sporadic human glucagonomas, varying from benign adenomas (IA) to locally ICs and lymph node metastases. Tumor cells express various neuroendocrine markers and glucagon, suggesting that they arise from differentiated and mature endocrine cells.

In 1992 Lee et al. reported a different glucagon-expressing tumor model by expressing the same viral oncogene under a more extended portion (2500 bp) of the same promoter (Glu-Tag) (Lee et al. 1992). These mice developed irregular islets, with cells containing pleomorphic nuclei during the first month of life (1–3 weeks), and exhibited islet hyperplasia with architectural disruption and peripherally located pleomorphic cells with hyperchromatic nuclei by week 4. Mice that lived through 3 months exhibited large PanNENs composed of pleomorphic cells alongside the development of infiltrative and metastasizing intestinal neuroendocrine carcinomas. These authors did not claim to have developed a glucagonoma GEMM, as they did not assess glucagonemia levels; they did not report the development of IC. Despite the early PanNEN onset, a drawback of this model was that a certain percentage of mice became progressively wasted and died of cachexia within 1–3 months (Lee et al. 1992, Asa et al. 1996).

Mahvash disease: other glucagonoma GEMMs

Glucagon receptor deletion

Mice with global homozygous deletion of the glucagon receptor (Gcgr–/–) were initially developed to explore the role of glucagon signaling in diabetes pathogenesis (Parker et al. 2002). These mice present hyperglucagonemia and pancreatic α-cell hyperplasia and are resistant to diabetes, thus closely mimicking the human Mahvash disease (Zhou et al. 2009, Yu et al. 2011, Sipos et al. 2015). At 2–3 months, Gcgr–/– mice exhibit pancreatomegaly and α-cell hyperplasia; at 5–7 months, the islet cell mass is 10-fold larger with nesidioblastosis, hyperplasia, and mild dysplasia; at 10–12 months, all mice bear micro-PanNETs (< 1 mm), as more than 80% present highly vascular PanNETs (>1 mm); at 18 months, all Gcgr–/– mice have grossly visible PanNETs. Surprisingly, rather than increased proliferation of existing α cells, hyperplasia was due to the neogenesis of α cells from the ductal epithelium. Therefore, at 10–12 months, each stage of tumorigenesis, from hyperplasia to gross PanNET, coexisted with all others, suggesting that new neoplastic α cells continuously emerge in this model. Besides the tumor frequency, Gcgr+/– mice do not have major phenotypic alterations compared to wild-type (WT). Distant metastasis to the liver and skin occurred in 20% of mice that die prematurely. Interestingly, abnormal cytoplasmic expression of menin is observed in α cells of neoplastic and dysplastic islets, suggesting that menin dysregulation may play a role in transforming hyperplastic α cells (Parker et al. 2002, Yu et al. 2011).

Other strategies

Like Gcgr / model, two other GEMMs that disrupt the glucagon signaling pathway were developed: the prohormone convertase-2 (Pc2) model (Jones et al. 2014) and the glucagon gene (Gcg) model (Hayashi et al. 2009, Takano et al. 2015). Pc2 cleaves proglucagon to produce mature glucagon. Jones et al. studied a Pc2–/– GEMM that developed glucagonomas within 6–12 months. The authors indicated that tumors were glucagonomas, either IA or IC, associated with local invasion and rarely distant metastases. The tumorigenesis process is apparently very similar to that in the Gcgr–/– mice, although no metastases were reported (Jones et al. 2014). These mice resist to diabetes and exhibit pancreatic α-cell hyperplasia at 3 months. By disrupting Gcg with a GFP knock-in, Hayashi et al. generated another PanNEN GEMM with early postnatal islet hyperplasia, glucagonoma onset at 8 months, and distant metastases to the liver and lungs. Contrarily to Gcgr–/– and Pc2–/– mice that present low blood glucose levels and increased serum GLP-1 levels, adult Gcggfp/gfp show hyperplasia of GFP-positive α-like cells but are normoglycemic and lack GLP-1. Therefore, neither GLP-1 nor sustained hypoglycemia is required in these mice to develop hyperplasia of α-like cells (Hayashi et al. 2009).

These three models (Gcgr–/–, Gcggfp/gfp, and Pc2–/–) share essential features. The pancreatic α cells likely do not have intrinsic abnormalities to drive hyperplasia. In the Pc2–/– GEMM, α-cell hyperplasia can even be reversible upon glucose delivery (Webb et al. 2002). The α-cell hyperplasia, dysplasia, and PanNENs most likely result from the loss of negative feedback on the normal α cells or their precursors, derived from glucagon signaling disruption, providing a permissive environment where additional genetic changes can affect α cell architecture (Ouyang et al. 2011, Yu et al. 2012).

Multiple endocrine neoplasia type 1

MEN1 is an autosomal dominant syndrome, mainly characterized by developing tumors in the pituitary, and/or parathyroid glands, and/or the pancreas due to inactivating mutations in the MEN1 tumor-suppressor gene (Thakker 2010). MEN1 encodes a 610-amino acid tumor suppressor, menin, widely expressed in neuroendocrine (NE) and non-NE tissues. MEN1 mutations on chromosome 11q13 are identified in inherited and de novo cases (Agarwal et al. 2005, Lemos & Thakker 2008). PanNETs arise in 30–80% of MEN1 patients, including insulinoma, glucagonoma, gastrinoma, other F-PanNETs, and NF-PanNETs (Thakker 2010, Marini et al. 2021). Usually, PanNETs in MEN1 patients are multiple and have an early onset (Klein Haneveld et al. 2021). As a hub gene, mutations in the MEN1 gene will affect all the core pathways involved in PanNET tumorigenesis (e.g. PI3K/AKT/mTOR, chromatin remodeling, and DNA damage) (Mafficini & Scarpa 2018).

To mimic human MEN1 syndrome more closely, a GEMM is designed to harbor a germline mutation. Germline homozygous deletion of Men1 is embryonically lethal in mice. Heterozygous menin deletion results in PanNETs (predominantly insulinoma) and other abnormalities (Crabtree et al. 2001, Bertolino et al. 2003a,b). GEMMs that conditionally disrupt Men1 (in β- or α-cell populations) reproduce the most prevalent somatic genetic event in PanNETs (Jiao et al. 2011, Cao et al. 2013, Scarpa et al. 2017).

Germline Men1 deletion

The first two Men1 GEMMs were described 20 years ago. Both generated nonselective Men1 KO mice by targeting by HR exons 1 and 2 or exons 3–8 (Crabtree et al. 2001) and exon 3 of the Men1 gene (Bertolino et al. 2003b). Both authors reported heterozygous individuals to develop pancreatic islet cell hyperplasia at 8–9 months. In Crabtree’s models (Men1TSM/+ and Men1ΔN3-8/+), the first PanNENs appear by 9 months and consist solely of insulinomas (Crabtree et al. 2001), while in Bertolino’s model (Men1+/T) a broader spectrum of tumors appears between 8 and 12 months, consisting in a set of IA and frank PanNENs (insulinomas, glucagonomas, gastrinomas, and mixed tumors (classification based on immunohistochemistry (IHC))), associated with lymph node metastases (Bertolino et al. 2003b). Both models gave rise to frequent parathyroid, adrenocortical, and pituitary NE tumors (starting from 8 to 9 months) and rare thyroid follicular adenomas and carcinomas (later onset, after 16–19 months). Mice were able to reach long lifespans (Crabtree et al. 2001, Bertolino et al. 2003b). In Crabtree’s model, larger multicentric tumors were reported by 16 months; by 22 months, one-third of mice had insulinomas. The tumor development exhibited a pattern starting with hyperplastic islets, then focal atypia, followed by frank tumors. Hypoglycemia was not reported as a cause of death, despite the high insulin levels produced by the insulinomas (Crabtree et al. 2001). In Bertolino’s model, 61% of heterozygotes develop carcinomas by 19-26 months; the authors did not mention its invasive potential. Insulin and glucagon production was evident in tumors from younger animals. Still, its expression was lost at advanced stages, probably due to dedifferentiation (Bertolino et al. 2003b). In both models, tumor cells of all organs revealed loss of the WT Men1 allele.

Loffler et al. and Harding et al. used two different Men1 constitutive KO GEMMs. Nonetheless, they obtained similar results to those obtained by Crabtree et al. and Bertolino et al. with the development of IA, either insulinomas (mostly) or glucagonomas (classification based on IHC), other NE tumors, and non-NE tumors including ovary and testicular between 9 and 12 months (Loffler et al. 2007, Harding et al. 2009).

Bai et al., using Crabtree’s model, reproduced the same phenotype in a C57BL/6 (B6) background but were also able to anticipate tumor formation using two double-mutated mice lines using p27 and p18 protein-coding cyclin-dependent kinase inhibitors 1b and 2c (Cdkn1b and Cdkn2c), respectively (Crabtree et al. 2001, Bai et al. 2007). Men1+/–;Cdkn1b–/– and Men1+/–;Cdkn2c–/– gave rise to insulin-expressing tumors, but the latter had a more aggressive behavior (earlier onset and IC formation by 12 months). Thus, the authors found that menin may interact differently with p27 and p18, as a functional synergistic interplay seems to exist between Cdkn2c and Men1 and not with Cdkn1b (Bai et al. 2007).

In 2014, Gillam et al. described two double transgenic models using two other cell cycle regulators, cyclin-dependent kinases (Cdk) 2 and 4: Men1+/–;Cdk2+/– and Men1+/–;Cdk4+/–. While the first GEMM resulted in insulin-expressing tumor formation at 15 months, together with pituitary NE tumors, the second did not develop any tumor and exhibited marked islet hypoplasia. The authors suggested that Cdk4-Rb pathway may be required for the tumorigenic pathway (Gillam et al. 2015).

β cell-specific Men1 deletion

GEMMs of β cell-specific Men1 deletion develop insulinomas with earlier onset than that of mice with germline Men1 deletion. Differences in specific ways of deleting Men1 and the genetic backgrounds contributed to a slight variation in lesion onset: β-cell hyperplasia by 1–5 months, which can exhibit features of atypia or dysplasia, followed by multiple insulinomas by 5–12 months (Bertolino et al. 2003c , Crabtree et al. 2003, Jiang et al. 2014, Lines et al. 2017).

Based on their previous models, Crabtree et al. and Bertolino et al. developed novel GEMMs using conditional gene targeting. In 2003, Crabtree et al. described a β cell-specific homozygous deletion of exons 3–8 of Men1 (Crabtree et al. 2003). When using RIP-Cre transgene (668 bp), islet hyperplasia was already pronounced with atypia at 1 month and insulinomas (IA) were diagnosed between 5 and 7 months and grew 20- to 26-fold more than controls at 15 months, without progressing to carcinoma. For the first time, a model of the multistage pathway was proposed. The severity of the lesions correlated with the Cre recombinase expression, supporting the fact that Men1 loss may be in its origin. Two other RIP-Cre transgenes were studied, resulting in later lesion onset. In the same year, Bertolino et al. reported a model that induced islet hyperplasia at 2 months that progressed with aging (Bertolino et al. 2003c). More than 40% of mice developed insulinomas by 6 months and 100% by 10 months. After 10 months, multifocal tumors and ‘advanced’ (non-invasive) carcinomas were seen, unlike in the model described by Crabtree et al. (2003). Insulin immunoreactivity also decreased with the progression of tumors, suggesting dedifferentiation, similar to Men1T/+ mice. Based on the normal development of endocrine tissues in this model, the authors argued that menin is not essential for islet-cell development (Bertolino et al. 2003b,c). Since it takes several months for insulinoma to develop even in the complete absence of Men1, additional genetic factors are likely required for neoplastic transformation.

Biondi et al. reported a similar β cell-specific Men1 KO model (Biondi et al. 2004). At necropsy at 12 months, all mice harbored multifocal islet cell hyperplasia, and one-third developed insulin-positive adenomas; interestingly, prolactinoma formation was diagnosed in these mice at 9 months revealing promoter leakage. More recently, using a temporal inducible RIP-Cre model, Lines et al. reported similar results as the previous models, with islet hyperplasia and insulinoma present at the age of 5 months (Lines et al. 2017).

GEMMs of β cell conditional Men1 seem to be robust models of insulinoma; hyperinsulinemia was reported to be increased, in some cases even before tumor formation (4 months), and hypoglycemia was detected after tumor onset (8 months) (Bertolino et al. 2003c, Jiang et al. 2014).

α cell-specific Men1 deletion

Mice with α cell-specific Men1 deletion using the pre-proglucagon promoter presented unexpected results (Lu et al. 2010, Shen et al. 2010). Glu-Cre;Menf/f mice exhibit loss of menin in 35% of the α cells at 6 weeks, while its expression is preserved in the β cells. Menin-deficient α cells begin to proliferate at 2–3 months, with concomitant hyperglucagonemia, and adenoma formation occurs at 7 months. Tumor types consisted not only of glucagonomas but also insulinomas and mixed glucagon/insulin-expressing tumors. Hyperinsulinemia started at 6 months. Over time (> 12 months), the proportions of glucagonomas and mixed tumors declined, whereas pure insulinomas became the dominant tumor type. Insulinoma cells contained the engineered Men1 allele, thus excluding the possibility of insulinoma development from adaptive β cell proliferation. Menin-negative, insulin-positive cells accounted for less than 2% of all menin-negative cells at 2 months. However, the proportion increased to 20% at 4 months and to even higher rates after that. By cell lineage tracing immunofluorescence, the authors concluded that the insulin-producing cells and insulinomas were derived from transdifferentiation of the Men1-deleted α cells (Lu et al. 2010). Using a similar approach, Shen et al. reported a similar mouse model with α cell-specific Men1 gene ablation (Shen et al. 2010). Here, Men1 deletion exhibited only insulinomas at 13–14 months. However, they did not examine the pancreas at 7–8 months and thus could not prove the existence of a glucagonoma stage before insulinomas become dominant. In both models, no progression to carcinoma was noted.

Men1 inactivation in pancreatic progenitor cells

Pancreatic and duodenal homeobox 1 (Pdx1), formerly known as insulin promoter factor 1 (Ipf1), is an essential regulator of both exocrine and endocrine pancreas organogenesis. Pdx1 is initially expressed in pre-pancreatic endoderm but postnatally is active in the duodenum and pancreas, at high levels in β cells and low levels in acinar cells (Jonsson et al. 1994, Offield et al. 1996, Magnuson & Osipovich 2013). The use of Pdx1-Cre started to be explored following reports advising prudent use of RIP-Cre driver lines by leaky expression in neural tissues (Gannon et al. 2000, Magnuson & Osipovich 2013). Pdx1-Cre;Men1f/f do not express Men1 in either the exocrine or the endocrine counterpart but exhibit normal early development of the pancreas although presenting more proliferating cells in islets (Shen et al. 2009). Islet cell hyperplasia was noticed at 5–6 months, progressing to insulinoma by 10–12 months. No exocrine hyperplasia or development of other tumor types was reported. The absence of exocrine neoplasms suggests the presence of other cell-specific factors in Men1-related tumorigenesis, consistent with the lack of liver tumors in mice with homozygous liver-specific Men1 inactivation. Based on Vegf protein quantification by ELISA and IHC, these authors reported that upregulation in Vegf signaling is concurrent with the development of insulinomas caused by the loss of menin. Vegf expression in islets can thus predate tumor development after Men1 inactivation in mice. This effect could be prevented by using sunitinib, a Vegf-signaling inhibitor.

Novel PanNEN mouse models

The last decade was prolific in identifying novel genetic alterations influencing PanNEN genesis and/or progression. So far, the most representative pathways identified to be altered in PanNENs have already been addressed in GEMMs. Table 1 presents an allocation of the revisited PanNEN GEMMs by the target protein and the enumeration of the chosen gene promoters by the pancreatic target cell. This section will address three critical pathways: (1) cell cycle and apoptosis, (2) Pi3k/Akt/mTOR, and (3) Atrx/Daxx.

Table 1

Representation of the target proteins in PanNEN GEMMs and gene promoters used by the pancreatic target cell.

Target protein, count (%) Promoters used by the target cell
β cells α cells Global Totals
Rip1/Rip2 Ins1/Ins2 Pdx1 Ren Glu/Glu2 No promoter
Tag #1 2 2 20
Tag and p53 1
Tag and Rb 1
Tag and other cell cycle players #2 1 1
Tag and growth factors #3 5 2
Tag and other players #4 5
Glucagon #5 3 3
Menin 6 1 2 6 22
Menin and cell cycle players #2 3
Menin and Pten 2
Menin and other Pi3k/Akt/mTOR players 1
Menin and other players #4 1
p53 1 9
p53 and Rb 1 1
p53 and other cell cycle players #2 1
Rb 1
Other cell cycle players #4 2 1 1
Pten 2 2
Atrx/Daxx 2 3 1 6
Totals 16 19 7 1 4 15 62

#1 Tag Simian virus 40 large T antigen, #2 Other cell cycle players include (rather than p53 and Rb1): apoptosis regulator Bcl-x, cyclin-dependent kinase 2, c-Myc, p16/p19, p18, p27; polyoma middle T antigen was also included in this category because it only appears in a study that targets cell cycle players, #3 Growth factors include: colony stimulating factor 1, insulin-like growth factor 1 receptor, insulin receptor, vascular endothelial growth factors A, B, and C (Vegfa, Vegfb, and Vegfc); antiangiogenic proteins (β3 integrin, endostatin, and thrombospondin-1, and tumstatin) were also included in this category because they only appear in a study that targets Vegf, #4 Other players include: β-catenin, cathepsin H, E-cadherin, polyoma small T antigen, receptor for subgroup A avian leukosis virus, recombinase activator gene 1, #5 Glucagon includes: glucagon, glucagon receptor, and prohormone convertase-2. Row totals concern the total of GEMMs targeting each of the targeted proteins (Tag, glucagon, menin, cell cycle players, Pten, or Atrx/Daxx) regardless of the chosen gene promoter. In contrast, the column totals concern the selected gene promoters’ total (Rip, Ins, Pdx1, Ren, or Glu) regardless of the targeted protein.

Cell cycle and apoptosis players

Although p53 and Rb tumor-suppressor pathways are inactivated in RT2 GEMMs, some authors directly focused on strategies to disrupt these crucial players.

In 1995, Harvey et al. explored for the first time a global heterozygous deletion of Tp53 and/or Rb tumor suppressors in a mouse model (Harvey et al. 1995). Homozygous Rb deletion was lethal, but Tp53+/–;Rb+/– and Tp53–/–;Rb+/– combinations resulted in (non-invasive) islet carcinoma at the age of 2 months, together with multiple other NE and non-NE tumors, especially osteosarcomas, lymphomas, and soft tissue sarcomas, constituting one of the first GEMMs of Li–Fraumeni syndrome. Tp53–/– mice died more prematurely than Tp53+/–, as all Tp53–/–;Rb+/+ were dead below 12 months and all Tp53–/;Rb+/– below 7 months Tp53+/–;Rb+/+. These results indicate that p53 and Rb cooperate in accelerating NE (and non-NE) tumorigenesis.

Years later, Pelengaris et al. reported a novel model of aggressive insulin-immunoreactive PanNEN (Pelengaris et al. 2002). These transgenic mice expressed apoptosisthe apoptosis inhibitor Bcl-xL and an inducible construct of oncogene c-Myc under RIP. After induction of c-Myc expression, β cells started to proliferate and, within a week, β-cell hyperplasia was evident; within 6 weeks, multiple insulinomas arose throughout the whole pancreas. Unlike most PanNEN models, tumor progression does not occur via a distinct multistage tumorigenic pathway. Instead, it derives from switching on a powerful proliferative, undifferentiated, and antiapoptotic state by the synergistic action of c-Myc and Bcl-xL. Tumors were likely PD as they expressed reduced levels of insulin. More extended induction of c-Myc expression was associated with vascular invasion and lymph node metastasis. All the lesions caused by c-Myc expression, including the large IC, were rapidly regressed after c-Myc deactivation.

Glenn et al. developed a glucagonoma GEMM with homozygous Tp53 and Rb deletion in renin (Ren)-expressing cells (present in renal and extra-renal tissues including pancreas) (Glenn et al. 2014). These mice developed glucagon-producing IC within 4 months with complete penetrance and nodal and liver metastases.

Yamauchideveloped other insulinoma GEMM by homozygous Rb deletion under the Pdx1 promoter (Yamauchi et al. 2020). This different design led to WD IA formation by 18–20 months (graded as G1 and comparable to human pancreatic neuroendocrine microadenomas); by adding the additional Tp53 mutation to the previous model, they were able to generate IC (aggressive insulinomas) (graded as G2–G3) within a shorter period (4 months), associated with liver metastases (9 months).

Lastly, Azzopardi et al. explored the impact of the loss of p53 and the Cdk inhibitors, p16 and p19, codified by Cdkn2a, using transgenic GEMMs expressing the viral oncogene polyoma middle T antigen (PyMT) (Azzopardi et al. 2016). The conditional mutations were performed under the pancreas promoter Pdx1 or insulin promoter 7 (RIP7). Under Pdx1, only p16/p19 loss induced PanNENs, not otherwise specified (NOS), as p53 loss and concomitant p53 and p16/p19 loss resulted in aggressive pancreatic acinar cell carcinoma (PACC), a rare event in humans. Under RIP7, all p53, p16/p19, and concomitant p53 and p16/p19 mutated proteins caused PanNEN, NOS, with a crescent incidence (17%, 20%, and 40%, respectively), suggesting that p16/p19 plays an additive effect to PanNEN progression in β cells but not in Pdx1+ progenitor cells.

Pi3k/Akt/mTOR pathway

The Pi3k/Akt/mTOR pathway has been studied for decades in many solid tumors and recently received more attention in PanNEN genetic profiling. This complex pathway involves a series of downstream events via mTOR, including cell growth, apoptosis, differentiation, angiogenesis, and migration (Inoki et al. 2002, Alliouachene et al. 2008, Crabtree 2017). Half of the PanNEN patients presented decreased PTEN expression in a human clinical setting, and 35% with downregulation of TSC2; both features correlated with poor survival (Missiaglia et al. 2010). In addition, mutations in Pi3K/Akt/mTOR pathway are present in around 9–15% of PanNETs (Jiao et al. 2011, Scarpa et al. 2017, Cives et al. 2019).

Alliouachene et al. reported in 2008 that transgenic expression of the constitutively active kinase Akt1, an isoform of Akt carrying a myristoylation signal (MyrAkt1), under RIP, leads to β cell-specific oncogenic transformation (Alliouachene et al. 2008). RIP-MyrAkt1 mice harbor increased β cell mass primarily due to hypertrophy rather than marked hyperplasia. Insulinomas developed in approximately 30% of the animals and onset at 10–12 months. In older mice (age not specified), such tumors were invasive and metastatic to distant organs. The authors tried the same approach by double-expressing Akt1 and S6K1, which did not result in PanNEN formation. RIP-MyrAkt1;S6K1–/– mice did not develop islet hyperplasia, suggesting that S6K1 activity is not required for the Akt effects on β cell size but specifically affects β cell proliferation during the hyperplastic and tumoral stages (Alliouachene et al. 2008). Rachdi et al. and Shigeyama et al. assessed the results of disrupting Tsc2 in mice using a β cell-specific KO (Rachdi et al. 2008, Shigeyama et al. 2008). Although neither was able to induce tumor formation, they obtained essential data regarding the Tsc2/mTOR pathway in the regulation of β-cell mass. When the KO was performed using the RIP-Cre transgene, mice died around 3 weeks of seizures, possibly due to Cre expression in the brain (Shigeyama et al. 2008). Using other Cre lines (RIP-CreHerr), both authors reported increased islet size from 1.5 to 2 months, mainly due to augmented measures of individual β cells and concurrent hypoglycemia and hyperinsulinemia (1–7 months) (Rachdi et al. 2008, Shigeyama et al. 2008). The metabolic state was changed after 10 months; mice developed progressive hyperglycemia and hypoinsulinemia associated with a reduced β-cell number. Rapamycin treatment induced insulin resistance and reduced β-cell mass, reversing the metabolic changes. These results seem to indicate that Tsc2 regulates pancreatic β-cell mass biphasically.

In 2020, Wong et al. used the Cre-LoxP system to inactivate Men1 and/or Pten in β cells and investigated the single and concurrent effects of such mutations (Wong et al. 2020). To do so, they used the RIP-Cre tool and the recently described MIP-Cre. The insulin 1 (Ins1) gene is selectively expressed by the β cells (exclusively). The mouse Ins1 promoter (MIP), firstly described in 2015 by Thorens et al., works in the same manner as Ins2 (RIP) does, with the advantage of not having leaky expression in the hypothalamus (Gannon et al. 2000, Wicksteed et al. 2010, Thorens et al. 2015). In total, six GEMMs were generated,, and all developed WD insulin-staining tumors. RIP-Cre;Men1f/f;Ptenf/f (MPR) and MIP-Cre;Men1f/f;Ptenf/f(MPM) mice had an earlier PanNET onset (around 2 months) than Men1 or Pten single deletions (6 months) with either promoter, suggesting a cooperative role of Pten and menin in NE tumorigenesis suppression. Finally, while the MIP-Cre GEMMs only gave rise to PanNETs, mutated mice harboring RIP-Cre also exhibited enlarged pituitary due to prolactin-positive pituitary NE tumor (PitNETs) development, especially in MPR genotype, followed by RIP-Cre;Ptenf/f (PR); MPR mice started dying at 9 weeks with lethargic signs and did not live beyond 23 weeks. The authors suggested that Pten might play a more substantial role than menin in PitNETs, considering the faster pituitary growth with loss of Pten function. In contrast, single menin loss seems to play a more relevant role in PanNET tumorigenesis than Pten regarding endocrine fraction comparisons.

Atrx/Daxx pathway

GEMMs assessing the loss of function of chromatin remodeling players are the most underrepresented. ATRX and the histone chaperone DAXX mediate the deposition of the histone H3.3 into telomeric and pericentromeric regions of the genome to maintain DNA configuration (Lewis et al. 2010, Clynes et al. 2015, Amorim et al. 2016). Mutually exclusive mutations in ATRX/DAXX activate the HR-based alternative lengthening of telomere (ALT) mechanism. They are associated with poorer prognostic features in non-metastatic PanNET patients (Marinoni et al. 2014). Atrx, Daxx, and H3.3 are responsible for the epigenetic silencing of transposable elements like the endogenous retroviral elements (ERVs) in the mouse genome (Elsasser et al. 2015). It is plausible that derepression of ERV silencing may occur upon Atrx and Daxx loss, activating transposons and leading to the expression of endogenous genes with an impact on carcinogenesis (Wasylishen et al. 2020). The deposition of H3.3 is dependable on MEN1, which has acted as a transcriptional regulator integrating the histone methyltransferase complex and methylates lysine 4 of histone H3 explicitly (Hughes et al. 2004, Chan et al. 2018).

In 1999, Michaelson et al. generated mice with global homozygous deletion of Daxx (2.5-kb fragment, containing exons 1 and 2), resulting in extensive apoptosis in mutant embryos and death by embryonic day 7.5 (Michaelson et al. 1999). Following this study, two PanNET GEMMs of Daxx disruption were described (Wasylishen et al. 2018, 2020). The first model evaluated loss of function with a global Daxx deletion (2.5-kb fragment, containing exons 2 and 3). First, the authors verified that Daxx-null mice (DaxxΔ3/Δ3) were not viable and that the concomitant loss of Tp53 did not rescue such lethality, thereby ascertaining that no important interaction exists between Daxx and the p53 pathway in causing embryonic lethality of DaxxΔ3/Δ3 mice embryos. Then, mice with heterozygous Daxx deletion (DaxxΔ3/+) were subjected to ionizing radiation (IR). One out of 34 developed a WD PanNET and increased the proportion of carcinomas, compared to Daxx+/+ mice under the same conditions. Mdm2Tg and Mdm2Tg;DaxxΔ3/+ mice were also treated with IR. No PanNET was formed, and it was reported that Daxx heterozygosity did not sensitize or alter the survival or tumor phenotype of Mdm2 transgenic mice. In their second work, Wasylishen et al. developed a conditional GEMM of Daxx mutation by CRISPR-Cas9, using the Pdx1 promoter to generate a functional conditional allele (Wasylishen et al. 2020). Initially, they observed that Daxx loss was well tolerated in the developing pancreas and did not compromise normal function in vivo at least until 12 months. Then, they followed up mice of four different genotypes (WT: Pdx1-Cre, DC: Pdx1-Cre;Daxxf/f, MC: Pdx1-Cre;Men1f/f, and DMC: Pdx1-Cre;Daxxf/f;Men1f/f) during 2 years and reported that DMC mice developed cystic and metaplastic lesions; both MC and DMC mice developed WD-PanNETs which suggests that Daxx loss in combination with Men1 deficiency results in an impaired homeostatic response. Additionally, Daxx loss resulted in a permissive transcriptional state that cooperated with the cerulein-induced environmental stress and Men1 deficiency to alter gene expression and cell state. After a 4-week recovery period following cerulein withdrawal, DMC mice were the only ones with persistent metaplastic lesions, suggesting impaired pancreas recovery from inflammation. Transcriptome analysis cleared up that gene expression changes were related to ERV dysregulation, indicating a new mechanism associated with impaired tissue regeneration and tumorigenesis associated with Daxx loss that future GEMMs should aim at clarifying (Wasylishen et al. 2020).

Recently, two works on GEMMs of Atrx/Daxx pathway disruption were published. Our group with a conditional β-cell disruption of Atrx verified in an aging cohort (up to 24 months) that PanNENs were not formed; however, individuals with deletion presented increased weights, inflammaging lesions, and a strong dysglycemia, characterized by the impairment of aging-related endocrine fraction growth and glucose intolerance (Gaspar et al. 2022). Sun et al. studied Atrx and Daxx disruption under different contexts. They verified that Atrx or Daxx loss, alone or in combination with Men1 loss, did not drive or boost PanNET tumorigenesis; additionally, they observed that Daxx loss did not cooperate with environmental stress caused by cerulein-induced pancreatitis or IR nor with concomitant Pten or Tp53 disruption to PanNET formation (Sun et al. 2022). These results suggest that these genes, although commonly altered in human disease, are not potent drivers in the mouse endocrine pancreas.

Figure 2 presents the ‘bigger picture’ of the studies considering PanNEN GEMMs here revisited. Beta cell-specific conditional GEMMs are the leading fraction of the models (69%). Tag and Men1 are the most common targets used in model engineering, accountable for more aggressive fast-rising PanNENs and indolent to moderate slow-rising PanNENs, respectively.

Figure 2
Figure 2

Characteristics of the revisited genetically engineered mouse models (GEMMs) of pancreatic neuroendocrine neoplasms (PanNENs) and respective outcome. Tumor types were divided into islet adenomas or neuroendocrine neoplasms not otherwise specified (IA/NEN) and ‘invasive carcinomas’ (IC) in the cases this designation was specifically adopted by the authors. The threshold of the PanNEN onset was set at 4 months, considering the high number of reports of tumors arising before this time point. This diagram illustrates the proportion of global and conditional deletions; note that β cell-specific conditional GEMMs stand out as the largest fraction of the diagram (69%), followed by global targeting GEMMs (24%), and ɑ cell-specific conditional GEMMs (7%). Tag and Men1 are the most common targets (29% and 27%, respectively), regardless of the GEMM design. RIP-Tag (RT2) models give rise to PanNENs before 4 months (4-) (100%) and encompass the largest fraction of IC (61%). Men1 GEMMs, on the other hand, originate PanNENs solely by 4 months or after (4+): either IA/NENs in conditional models or IC in global Men1 GEMMs. n.s., not specified. Created with BioRender.com.

Citation: Endocrine-Related Cancer 29, 12; 10.1530/ERC-22-0166

Human disease recapitulation in PanNEN GEMMs

Over the last decades, PanNEN GEMMs were important for the comprehension of tumor progression and response to antiangiogenic drugs. The Gcgr–/– and Men1 constitutive and conditional KO GEMMs stand out as the most remarkable, reinforcing the importance of these genetic abnormalities (Crabtree et al. 2001, Bertolino et al. 2003b,c, Crabtree et al. 2003, Yu et al. 2011). Gcgr–/– mice closely mimic the human Mahvash disease clinically and histologically. Most PanNENs of these mice were glucagon-producing microadenomas with a typical trabecular growth pattern and a Ki-67 index of around 2%, comparable to human PanNET microadenomas (Yu et al. 2011). Mice bearing a germline heterozygous inactivation of Men1 are considered representative of human disease as tumor initiation is dictated by the loss of heterozygosity (LOH) of the Men1 gene (Crabtree et al. 2001). Mice with conditional homozygous KO of Men1 in β cells (Bertolino et al. 2003c, Crabtree et al. 2003, Jiang et al. 2014, Lines et al. 2017) develop insulinomas with earlier onset than that of mice with germline Men1 deletion (Crabtree et al. 2001, Bertolino et al. 2003b). In MEN1 patients, the loss of the second allele, by LOH, occurs in more than 90% of cases. The pancreatic tumor spectrum of these patients is mainly composed of multiple NF-PanNET microadenomas (up to 50%) (Marini et al. 2021), but insulinomas can occur in up to 30% of MEN1 patients (Jensen & Norton 2017). In contrast with the GEMMs, PanNETs of MEN1 patients manifest at a younger mean age compared to their sporadic counterparts (10–50 years vs 50–80 years) (Marini et al. 2021). Mice with the conditional homozygous KO of the Men1 gene in the α cells were also crucial for α-to-β-cell transdifferentiation identification. Rather than glucagonomas, α cell conditional KO of Men1 developed tumors derived from β cells, mainly insulinomas, suggesting that loss of menin functions is pivotal for endocrine cell transdifferentiation; another possible explanation is the release of paracrine signals from α cells that could lead to increased proliferation of β cells (Lu et al. 2010, Shen et al. 2010). Changes in the PanNEN secretory profile have only been punctually described in case reports of NF-PanNEN patients who were later diagnosed with hormonal hypersecretion syndromes (Miehle et al. 2004, Juhlin et al. 2019). However, islet cell plasticity is well reported in the context of pancreas homeostasis (Ziv et al. 2013, Yagihashi et al. 2016). Whether tumor transdifferentiation also occurs at this scale remains to be substantiated in a human setting.

RT2 models were also of great importance due to the many preclinical studies involving drug testing. These mice, lacking p53 and Rb activity, mimic the genetic profile of human PanNECs. The tumor spectrum of these GEMMs is mainly composed of highly vascularized, ICs that, by their aggressiveness, usually develop before 4 months, following a multistep carcinogenesis pathway (first islet hyperplasia, then frank tumor). The involvement of angiogenesis in hyperplasia–dysplasia/PanNEN transition has been extensively studied in RT2 mice. Peptide epitope profiling techniques revealed differences among normal, hyperplastic, and neoplastic islets, thus substantiating the vasculature changes throughout tumorigenic stages and raising the hypothesis that an angiogenic switch may be responsible for the transition from preneoplastic to neoplastic stages, as assessed in mice and in vitro studies (Folkman et al. 1989, Joyce et al. 2003). The overexpression of Vegf receptor 2 (Vegfr2) in dysplastic islets and PanNENs of the Gcgr–/– mice implied that angiogenesis could also play a similar role in the transformation of hyperplastic islets in this model (Yu et al. 2011). All these characteristics elected RT2 mice as a preferential target for studies evaluating neuroendocrine tumor progression and angiogenesis, with an emphasis on predicting the clinical efficacy of antiangiogenic therapeutic approaches (Tuveson & Hanahan 2011). For instance, Vegfr2 antibodies have been used in the RT2 mice for multiple purposes; they help suppress PanNET formation by inhibiting the angiogenic switching and impair tumor progression (Casanovas et al. 2005b, Paez-Ribes et al. 2009, Gocheva et al. 2010, Wicki et al. 2012). Many preclinical trials, performed in RT2 mice, testing angiogenesis inhibitors targeting the Vegf signaling pathway have been useful for documenting the role of angiogenesis in PanNEN progression (Bergers et al. 2003, Casanovas et al. 2005b, Paez-Ribes et al. 2009, Sennino et al. 2012).

PanNEN GEMMs arising from the disruption of cell cycle and apoptosis players, Pi3k/Akt/mTOR, or Atrx/Daxx pathways still need an in-depth characterization for human disease approximation. Li–Fraumeni syndrome GEMMs result in aggressive PACC development; so far, cases of PanNET in Li–Fraumeni are scarce or underreported (Aversa et al. 2020).

Final remarks

The ideal GEMM should recapitulate their related human disease closely. Up to date, some PanNEN GEMMs meet this condition and have explored the causal link between candidate cancer genes and PanNET tumorigenesis. They have been essential in clarifying the pathogenesis, treatment, and natural history of human PanNENs. Mouse models revealed that the pathogenesis of PanNENs follows a distinct and morphological multistage pathway comprising hyperplasia, atypia, or dysplasia, and bona fide PanNENs of varying malignancy (from adenoma to IC). In this context, GEMMs also allow the possibility of studying and decoding the phenomena occurring at each stage of tumor progression. In PanNEN GEMMs, the first hyperplasia lesions seem to happen at very young ages due to a high proliferation rate or neogenesis without showing features of dysplasia (Power et al. 1987, Efrat et al. 1988). The progression of lesions is not synchronous, as islet hyperplasia appears throughout the animals’ life; dysplastic endocrine cells and PanNENs continue to emerge as in older animals with PanNEN all stages of tumorigenesis usually coexist, as similarly reported in pancreata of patients with MEN1 syndrome, Mahvash disease, and VHL (Jensen et al. 2008, Safo & Pambuccian 2010, Ouyang et al. 2011, Ro et al. 2013, Yu 2018). The hyperplasia to dysplasia transformation has been more challenging to decipher. In the GEMM of Mahvash disease, the similar abnormal cytoplasmic presence of menin in dysplastic and neoplastic α cells indicates that this step is critical in PanNET tumorigenesis as dysplastic islets are already transformed (Yu et al. 2011). Because in sporadic human cases endocrine cell hyperplasia is only seen in a portion of patients with PanNENs, it is not clear if hyperplasia is a prerequisite for dysplasia or PanNEN development (Kloppel et al. 2007, Klöppel et al. 2021).

GEMMs have also given an invaluable contribution in a preclinical setting for the PanNEN patient treatment as already discussed, mainly concerning antiangiogenic therapeutic approaches. Nonetheless, GEMMs could also help explore the role of immune checkpoints in PanNEN patient management. Recently, mice with conditional KO of Men1 benefited from immunotherapy with a monoclonal antibody against the B7 family member, H4 (B7x). Men1/B7x double KO mice exhibited decreased islet β cell proliferation and tumor transformation accompanied by increased T-cell infiltration compared with Men1 single KO mice. This study presented the B7x family as putative targets for immunotherapy strategy in human PanNEN patients (Shen et al. 2009, Jeon et al. 2014, Yuan et al. 2021).

Besides the issue of PanNET transdifferentiation discussed in the section above, mouse models have allowed the study of islet biology, particularly tissue-specific PanNET tumorigenesis. It was reported that hypertrophy and nuclear pleomorphism are pathological features found either in the endocrine and exocrine pancreas of Gcgr–/– mice; however, the exocrine pancreas does not develop tumors even at 22 months, whereas the endocrine pancreas produces PanNETs with 100% penetrance at 12 months (Yu et al. 2011, Yu 2018). The same occurred in Men1TSM/+ and Men1ΔN3-8/+ mice that developed minor pancreatic lesions in exocrine counterparts but did not originate tumors (Crabtree et al. 2001).

The versatility of murine models, warranted by the ease of genetic manipulation, allows studying a relatively large number of animals during a short period and tissue retrieval of any organ of interest to study site-specific tumorigenesis. Nevertheless, some challenges should be considered.

First, concerning the experience designs, there is a lack of standardization in PanNET nomenclature and histopathological description and in endocrine function assessment, namely fasting times for glycemia assessment or metabolic tolerance tests (MTTs). Establishing a veterinary pathological routine will allow, through detailed necropsy study and tissue sectioning, to characterize better the developed lesions, determine more solid comparisons with the human PanNEN histotypes, and reveal subclinical or rare organ involvements. This should be based on the pioneering work of Lopez and Hanahan built on an exhaustive analysis of RT2 transgenic mice; it would be essential to apply similar standards to help stratifying lesions in other GEMMs (Lopez & Hanahan 2002). Appropriate preparation is critical to ensure the accuracy and reproducibility of MTTs, and the distinct metabolism of mice will depend on the standardization of procedures. Recent guidelines advised against overnight fasting, recommending blood glucose measurement performance in the daytime after a 6- to 7-h fasting period in animals that are used in the operator’s presence (Sun et al. 2016, Benede-Ubieto et al. 2020). To increase the translational potential, the terms of F-PanNETs such as ‘insulinoma’ and ‘glucagonoma’ should only be applied upon confirmation of clinical syndrome rather than immunophenotyping alone.

Second, the inherent limitations of GEMM generation and subsequent validation are time-consuming, laborious, and expensive (Kersten et al. 2017). The choice of animals’ backgrounds is enough to dramatically alter the viability of embryos or anticipate tumor formation and preneoplastic lesions (Bertolino et al. 2003b, Crabtree et al. 2003, Harding et al. 2009). Thus, it is thus essential to know the transgenic models for constructing conditional GEMMs (Li et al. 2021). One of the most used, the RIP-Cre line in a B6 background, has the additional side effects of driving recombination activity in the hypothalamus, spontaneously developing glucose intolerance and impaired insulin secretion before 2 months and islet hyperplasia from 9 months (Lee et al. 2006, Pomplun et al. 2007). The impact of the background strain on these phenotypic features remains to be determined. Pdx1-Cre and MIP-Cre lines are increasingly chosen for driving β cell-specific transgene expression without compromising endocrine function (Shen et al. 2009, Magnuson & Osipovich 2013, Wong et al. 2020).

Yet, the current GEMMs cannot reproduce a typical sporadic human PanNEN in an essentially normal pancreatic endocrine background, i.e. solitary, NF-PanNEN, often with hepatic metastases by diagnosis. Even so, we can still use such models to identify specific histotypes. For instance, since insulinomas are a typical phenotype of the established GEMMs, it should be helpful to address the loss of Yy1 in the context of their formation and progression. Suitable GEMMs need to be designed to deeply explore the role of ATRX and DAXX in PanNET tumorigenesis, as its relevance, in association with ALT, has continuously been helpful for prognostic assessment (Hackeng et al. 2022). It remains to be clarified whether the mutations found in human PanNETs are etiological or passenger events (Jiao et al. 2011, Scarpa et al. 2017, Cives et al. 2019, Hong et al. 2020).

Overall, in the short term and predicting the future of novel models, it is expectable that the novel GEMMs will address the role of novel discovered genetic and epigenetic players. Seeking putative synergistic effects derived from the combination of such players may be fruitful in establishing a novel generation of PanNET GEMMs with high translational value.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/ERC-22-0166.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by national funds from the FCT – Fundação para a Ciência e Tecnologia, I.P., through a PhD grant to T B G (SFRH/BD/129431/2017), a research contract to J V (CEECIND/00201/2017) and the project PTDC/MED-ONC/0531/2021 – CTRL+ALT+CEL: how ATRX controls an alternative program in the β-cell.

References

  • Agarwal SK, Kennedy PA, Scacheri PC, Novotny EA, Hickman AB, Cerrato A, Rice TS, Moore JB, Rao S & Ji Y et al.2005 Menin molecular interactions: insights into normal functions and tumorigenesis. Hormone and Metabolic Research 37 369374. (https://doi.org/10.1055/s-2005-870139)

    • Search Google Scholar
    • Export Citation
  • Akirov A, Larouche V, Alshehri S, Asa SL & Ezzat S 2019 Treatment options for pancreatic neuroendocrine tumors. Cancers 11 828. (https://doi.org/10.3390/cancers11060828)

    • Search Google Scholar
    • Export Citation
  • Alliouachene S, Tuttle RL, Boumard S, Lapointe T, Berissi S, Germain S, Jaubert F, Tosh D, Birnbaum MJ & Pende M 2008 Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation. Journal of Clinical Investigation 118 36293638. (https://doi.org/10.1172/JCI35237)

    • Search Google Scholar
    • Export Citation
  • Amorim JP, Santos G, Vinagre J & Soares P 2016 The role of ATRX in the alternative lengthening of telomeres (ALT) phenotype. Genes 7 66. (https://doi.org/10.3390/genes7090066)

    • Search Google Scholar
    • Export Citation
  • Asa SL 2011 Pancreatic endocrine tumors. Modern Pathology 24 (Supplement 2) S66S77. (https://doi.org/10.1038/modpathol.2010.127)

  • Asa SL, Lee YC & Drucker DJ 1996 Development of colonic and pancreatic endocrine tumours in mice expressing a glucagon-SV40 T antigen transgene. Virchows Archiv 427 595606. (https://doi.org/10.1007/BF00202891)

    • Search Google Scholar
    • Export Citation
  • Aversa JG, De Abreu FB, Yano S, Xi L, Hadley DW, Manoli I, Raffeld M, Sadowski SM & Nilubol N 2020 The first pancreatic neuroendocrine tumor in Li-Fraumeni syndrome: a case report. BMC Cancer 20 256. (https://doi.org/10.1186/s12885-020-06723-6)

    • Search Google Scholar
    • Export Citation
  • Azzopardi S, Pang S, Klimstra DS & Du YN 2016 p53 and p16(Ink4a)/p19(Arf) loss promotes different pancreatic tumor types from PyMT-expressing progenitor cells. Neoplasia 18 610617. (https://doi.org/10.1016/j.neo.2016.08.003)

    • Search Google Scholar
    • Export Citation
  • Babu V, Paul N & Yu R 2013 Animal models and cell lines of pancreatic neuroendocrine tumors. Pancreas 42 912923. (https://doi.org/10.1097/MPA.0b013e31827ae993)

    • Search Google Scholar
    • Export Citation
  • Bai F, Pei XH, Nishikawa T, Smith MD & Xiong Y 2007 p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors. Molecular and Cellular Biology 27 14951504. (https://doi.org/10.1128/MCB.01764-06)

    • Search Google Scholar
    • Export Citation
  • Batukbhai BDO & De Jesus-Acosta A 2019 The molecular and clinical landscape of pancreatic neuroendocrine tumors. Pancreas 48 921. (https://doi.org/10.1097/MPA.0000000000001189)

    • Search Google Scholar
    • Export Citation
  • Benede-Ubieto R, Estevez-Vazquez O, Ramadori P, Cubero FJ & Nevzorova YA 2020 Guidelines and considerations for metabolic tolerance tests in mice. Diabetes Metabolic Syndrome and Obesity 13 439450. (https://doi.org/10.2147/DMSO.S234665)

    • Search Google Scholar
    • Export Citation
  • Bergers G, Song S, Meyer-Morse N, Bergsland E & Hanahan D 2003 Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. Journal of Clinical Investigation 111 12871295. (https://doi.org/10.1172/JCI17929)

    • Search Google Scholar
    • Export Citation
  • Bertolino P, Radovanovic I, Casse H, Aguzzi A, Wang ZQ & Zhang CX 2003a Genetic ablation of the tumor suppressor menin causes lethality at mid-gestation with defects in multiple organs. Mechanisms of Development 120 549560. (https://doi.org/10.1016/s0925-4773(0300039-x)

    • Search Google Scholar
    • Export Citation
  • Bertolino P, Tong WM, Galendo D, Wang ZQ & Zhang CX 2003b Heterozygous Men1 mutant mice develop a range of endocrine tumors mimicking multiple endocrine neoplasia type 1. Molecular Endocrinology 17 18801892. (https://doi.org/10.1210/me.2003-0154)

    • Search Google Scholar
    • Export Citation
  • Bertolino P, Tong WM, Herrera PL, Casse H, Zhang CX & Wang ZQ 2003c Pancreatic beta-cell-specific ablation of the multiple endocrine neoplasia type 1 (MEN1) gene causes full penetrance of insulinoma development in mice. Cancer Research 63 48364841.

    • Search Google Scholar
    • Export Citation
  • Biondi CA, Gartside MG, Waring P, Loffler KA, Stark MS, Magnuson MA, Kay GF & Hayward NK 2004 Conditional inactivation of the MEN1 gene leads to pancreatic and pituitary tumorigenesis but does not affect normal development of these tissues. Molecular and Cellular Biology 24 31253131. (https://doi.org/10.1128/MCB.24.8.3125-3131.2004)

    • Search Google Scholar
    • Export Citation
  • Brooks JC, Shavelle RM & Vavra-Musser KN 2019 Life expectancy in pancreatic neuroendocrine cancer. Clinics and Research in Hepatology and Gastroenterology 43 8897. (https://doi.org/10.1016/j.clinre.2018.08.005)

    • Search Google Scholar
    • Export Citation
  • Cao Y, Gao Z, Li L, Jiang X, Shan A, Cai J, Peng Y, Li Y, Jiang X & Huang X et al.2013 Whole exome sequencing of insulinoma reveals recurrent T372R mutations in YY1. Nature Communications 4 2810. (https://doi.org/10.1038/ncomms3810)

    • Search Google Scholar
    • Export Citation
  • Capecchi MR 1989 Altering the genome by homologous recombination. Science 244 12881292. (https://doi.org/10.1126/science.2660260)

  • Casanovas O, Hager JH, Chun MG & Hanahan D 2005a Incomplete inhibition of the Rb tumor suppressor pathway in the context of inactivated p53 is sufficient for pancreatic islet tumorigenesis. Oncogene 24 65976604. (https://doi.org/10.1038/sj.onc.1208823)

    • Search Google Scholar
    • Export Citation
  • Casanovas O, Hicklin DJ, Bergers G & Hanahan D 2005b Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8 299309. (https://doi.org/10.1016/j.ccr.2005.09.005)

    • Search Google Scholar
    • Export Citation
  • Chan CS, Laddha SV, Lewis PW, Koletsky MS, Robzyk K, Da Silva E, Torres PJ, Untch BR, Li J & Bose P et al.2018 ATRX, DAXX or MEN1 mutant pancreatic neuroendocrine tumors are a distinct alpha-cell signature subgroup. Nature Communications 9 4158. (https://doi.org/10.1038/s41467-018-06498-2)

    • Search Google Scholar
    • Export Citation
  • Chen M, Van Ness M, Guo Y & Gregg J 2012 Molecular pathology of pancreatic neuroendocrine tumors. Journal of Gastrointestinal Oncology 3 182188. (https://doi.org/10.3978/j.issn.2078-6891.2012.018)

    • Search Google Scholar
    • Export Citation
  • Chen J, Yang Y, Liu Y & Kan H 2021 Prognosis analysis of patients with pancreatic neuroendocrine tumors after surgical resection and the application of enucleation. World Journal of Surgical Oncology 19 11. (https://doi.org/10.1186/s12957-020-02115-z)

    • Search Google Scholar
    • Export Citation
  • Cives M, Partelli S, Palmirotta R, Lovero D, Mandriani B, Quaresmini D, Pelle E, Andreasi V, Castelli P & Strosberg J et al.2019 DAXX mutations as potential genomic markers of malignant evolution in small nonfunctioning pancreatic neuroendocrine tumors. Scientific Reports 9 18614. (https://doi.org/10.1038/s41598-019-55156-0)

    • Search Google Scholar
    • Export Citation
  • Clynes D, Jelinska C, Xella B, Ayyub H, Scott C, Mitson M, Taylor S, Higgs DR & Gibbons RJ 2015 Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nature Communications 6 7538. (https://doi.org/10.1038/ncomms8538)

    • Search Google Scholar
    • Export Citation
  • Crabtree JS 2017 Clinical and preclinical advances in gastroenteropancreatic neuroendocrine tumor therapy. Frontiers in Endocrinology 8 341. (https://doi.org/10.3389/fendo.2017.00341)

    • Search Google Scholar
    • Export Citation
  • Crabtree JS, Scacheri PC, Ward JM, Garrett-Beal L, Emmert-Buck MR, Edgemon KA, Lorang D, Libutti SK, Chandrasekharappa SC & Marx SJ et al.2001 A mouse model of multiple endocrine neoplasia, type 1, develops multiple endocrine tumors. PNAS 98 11181123. (https://doi.org/10.1073/pnas.98.3.1118)

    • Search Google Scholar
    • Export Citation
  • Crabtree JS, Scacheri PC, Ward JM, McNally SR, Swain GP, Montagna C, Hager JH, Hanahan D, Edlund H & Magnuson MA et al.2003 Of mice and MEN1: insulinomas in a conditional mouse knockout. Molecular and Cellular Biology 23 60756085. (https://doi.org/10.1128/MCB.23.17.6075-6085.2003)

    • Search Google Scholar
    • Export Citation
  • Das S & Dasari A 2021 Epidemiology, incidence, and prevalence of neuroendocrine neoplasms: are there global differences? Current Oncology Reports 23 43. (https://doi.org/10.1007/s11912-021-01029-7)

    • Search Google Scholar
    • Export Citation
  • Dasari A, Shen C, Halperin D, Zhao B, Zhou S, Xu Y, Shih T & Yao JC 2017 Trends in the incidence, prevalence, and survival outcomes in patients with neuroendocrine tumors in the United States. JAMA Oncology 3 13351342. (https://doi.org/10.1001/jamaoncol.2017.0589)

    • Search Google Scholar
    • Export Citation
  • Deng C-X 2012 The use of Cre–loxP technology and inducible systems to generate mouse models of cancer. In Genetically Engineered Mice for Cancer Research. Eds Green J, Ried T. New York, NY, USA: Springer.

    • Search Google Scholar
    • Export Citation
  • Du YC, Lewis BC, Hanahan D & Varmus H 2007 Assessing tumor progression factors by somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion. PLoS Biology 5 e276. (https://doi.org/10.1371/journal.pbio.0050276)

    • Search Google Scholar
    • Export Citation
  • Efrat S, Teitelman G, Anwar M, Ruggiero D & Hanahan D 1988 Glucagon gene regulatory region directs oncoprotein expression to neurons and pancreatic alpha cells. Neuron 1 605613. (https://doi.org/10.1016/0896-6273(8890110-9)

    • Search Google Scholar
    • Export Citation
  • Elsasser SJ, Noh KM, Diaz N, Allis CD & Banaszynski LA 2015 Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells. Nature 522 240244. (https://doi.org/10.1038/nature14345)

    • Search Google Scholar
    • Export Citation
  • Folkman J, Watson K, Ingber D & Hanahan D 1989 Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 339 5861. (https://doi.org/10.1038/339058a0)

    • Search Google Scholar
    • Export Citation
  • Gannon M, Shiota C, Postic C, Wright CV & Magnuson M 2000 Analysis of the Cre-mediated recombination driven by rat insulin promoter in embryonic and adult mouse pancreas. Genesis 26 139142. (https://doi.org/10.1002/(sici)1526-968x(200002)26:2<139::aid-gene12>3.0.co;2-7)

    • Search Google Scholar
    • Export Citation
  • Gaspar TB, Macedo S, Sa A, Soares MA, Rodrigues DF, Sousa M, Mendes N, Martins RS, Cardoso L & Borges I et al.2022 Characterisation of an Atrx conditional knockout mouse model: Atrx loss causes endocrine dysfunction rather than pancreatic neuroendocrine tumour. Cancers 14 3865. (https://doi.org/10.3390/cancers14163865)

    • Search Google Scholar
    • Export Citation
  • Gaudenzi G, Carra S, Dicitore A, Cantone MC, Persani L & Vitale G 2020 Fishing for neuroendocrine tumors. Endocrine-Related Cancer 27 R163R176. (https://doi.org/10.1530/ERC-19-0437)

    • Search Google Scholar
    • Export Citation
  • Geurts JL 2020 Inherited syndromes involving pancreatic neuroendocrine tumors. Journal of Gastrointestinal Oncology 11 559566. (https://doi.org/10.21037/jgo.2020.03.09)

    • Search Google Scholar
    • Export Citation
  • Gillam MP, Nimbalkar D, Sun L, Christov K, Ray D, Kaldis P, Liu X & Kiyokawa H 2015 MEN1 tumorigenesis in the pituitary and pancreatic islet requires Cdk4 but not Cdk2. Oncogene 34 932938. (https://doi.org/10.1038/onc.2014.3)

    • Search Google Scholar
    • Export Citation
  • Glenn ST, Jones CA, Sexton S, LeVea CM, Caraker SM, Hajduczok G & Gross KW 2014 Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma. Oncogene 33 57065715. (https://doi.org/10.1038/onc.2013.514)

    • Search Google Scholar
    • Export Citation
  • Gocheva V, Chen X, Peters C, Reinheckel T & Joyce JA 2010 Deletion of cathepsin H perturbs angiogenic switching, vascularization and growth of tumors in a mouse model of pancreatic islet cell cancer. Biological Chemistry 391 937945. (https://doi.org/10.1515/BC.2010.080)

    • Search Google Scholar
    • Export Citation
  • Grant SG, Seidman I, Hanahan D & Bautch VL 1991 Early invasiveness characterizes metastatic carcinoid tumors in transgenic mice. Cancer Research 51 49174923.

    • Search Google Scholar
    • Export Citation
  • Hackeng WM, Brosens LAA, Kim JY, O'Sullivan R, Sung YN, Liu TC, Cao D, Heayn M, Brosnan-Cashman J & An S et al.2022 Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size. Gut 71 961973. (https://doi.org/10.1136/gutjnl-2020-322595)

    • Search Google Scholar
    • Export Citation
  • Hager JH, Ulanet DB, Hennighausen L & Hanahan D 2009 Genetic ablation of Bcl-x attenuates invasiveness without affecting apoptosis or tumor growth in a mouse model of pancreatic neuroendocrine cancer. PLoS ONE 4 e4455. (https://doi.org/10.1371/journal.pone.0004455)

    • Search Google Scholar
    • Export Citation
  • Hanahan D 1985 Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315 115122. (https://doi.org/10.1038/315115a0)

    • Search Google Scholar
    • Export Citation
  • Harding B, Lemos MC, Reed AA, Walls GV, Jeyabalan J, Bowl MR, Tateossian H, Sullivan N, Hough T & Fraser WD et al.2009 Multiple endocrine neoplasia type 1 knockout mice develop parathyroid, pancreatic, pituitary and adrenal tumours with hypercalcaemia, hypophosphataemia and hypercorticosteronaemia. Endocrine-Related Cancer 16 13131327. (https://doi.org/10.1677/ERC-09-0082)

    • Search Google Scholar
    • Export Citation
  • Harvey M, Vogel H, Lee EYHP, Bradley A & Donehower LA 1995 Mice deficient in both P53 and Rb develop tumors primarily of endocrine origin. Cancer Research 55 11461151.

    • Search Google Scholar
    • Export Citation
  • Hayashi Y, Yamamoto M, Mizoguchi H, Watanabe C, Ito R, Yamamoto S, Sun XY & Murata Y 2009 Mice deficient for glucagon gene-derived peptides display normoglycemia and hyperplasia of islet {alpha}-cells but not of intestinal L-cells. Molecular Endocrinology 23 19901999. (https://doi.org/10.1210/me.2009-0296)

    • Search Google Scholar
    • Export Citation
  • Herzig M, Novatchkova M & Christofori G 1999 An unexpected role for p53 in augmenting SV40 large T antigen-mediated tumorigenesis. Biological Chemistry 380 203211. (https://doi.org/10.1515/BC.1999.028)

    • Search Google Scholar
    • Export Citation
  • Hijioka S, Hosoda W, Matsuo K, Ueno M, Furukawa M, Yoshitomi H, Kobayashi N, Ikeda M, Ito T & Nakamori S et al.2017 Rb loss and KRAS mutation are predictors of the response to platinum-based chemotherapy in pancreatic neuroendocrine neoplasm with grade 3: a Japanese multicenter pancreatic NEN-G3 study. Clinical Cancer Research 23 46254632. (https://doi.org/10.1158/1078-0432.CCR-16-3135)

    • Search Google Scholar
    • Export Citation
  • Hong X, Qiao S, Li F, Wang W, Jiang R, Wu H, Chen H, Liu L, Peng J & Wang J et al.2020 Whole-genome sequencing reveals distinct genetic bases for insulinomas and non-functional pancreatic neuroendocrine tumours: leading to a new classification system. Gut 69 877887. (https://doi.org/10.1136/gutjnl-2018-317233)

    • Search Google Scholar
    • Export Citation
  • Hu W, Feng Z, Modica I, Klimstra DS, Song L, Allen PJ, Brennan MF, Levine AJ & Tang LH 2010 Gene amplifications in well-differentiated pancreatic neuroendocrine tumors inactivate the p53 pathway. Genes and Cancer 1 360368. (https://doi.org/10.1177/1947601910371979)

    • Search Google Scholar
    • Export Citation
  • Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland TD, Levine SS, Lee JC, Hayes DN, Shanmugam KS, Bhattacharjee A & Biondi CA et al.2004 Menin associates with a trithorax family histone methyltransferase complex and with the hoxc8 locus. Molecular Cell 13 587597. (https://doi.org/10.1016/s1097-2765(0400081-4)

    • Search Google Scholar
    • Export Citation
  • Hunter KE, Quick ML, Sadanandam A, Hanahan D & Joyce JA 2013 Identification and characterization of poorly differentiated invasive carcinomas in a mouse model of pancreatic neuroendocrine tumorigenesis. PLoS ONE 8 e64472. (https://doi.org/10.1371/journal.pone.0064472)

    • Search Google Scholar
    • Export Citation
  • Inoki K, Li Y, Zhu T, Wu J & Guan KL 2002 TSC2 is phosphorylated and inhibited by Akt and suppresses mTOR signalling. Nature Cell Biology 4 648657. (https://doi.org/10.1038/ncb839)

    • Search Google Scholar
    • Export Citation
  • Inoue M, Hager JH, Ferrara N, Gerber HP & Hanahan D 2002 VEGF-A has a critical, nonredundant role in angiogenic switching and pancreatic β cell carcinogenesis. Cancer Cell 1 193202. (https://doi.org/10.1016/s1535-6108(0200031-4)

    • Search Google Scholar
    • Export Citation
  • Jensen RT & Norton JA 2017 Treatment of pancreatic neuroendocrine tumors in multiple endocrine neoplasia type 1: some clarity but continued controversy. Pancreas 46 589594. (https://doi.org/10.1097/MPA.0000000000000825)

    • Search Google Scholar
    • Export Citation
  • Jensen RT, Berna MJ, Bingham DB & Norton JA 2008 Inherited pancreatic endocrine tumor syndromes: advances in molecular pathogenesis, diagnosis, management, and controversies. Cancer 113 (7 S upplement) 18071843. (https://doi.org/10.1002/cncr.23648)

    • Search Google Scholar
    • Export Citation
  • Jeon H, Vigdorovich V, Garrett-Thomson SC, Janakiram M, Ramagopal UA, Abadi YM, Lee JS, Scandiuzzi L, Ohaegbulam KC & Chinai JM et al.2014 Structure and cancer immunotherapy of the B7 family member B7x. Cell Reports 9 10891098. (https://doi.org/10.1016/j.celrep.2014.09.053)

    • Search Google Scholar
    • Export Citation
  • Jiang X, Cao Y, Li F, Su Y, Li Y, Peng Y, Cheng Y, Zhang C, Wang W & Ning G 2014 Targeting beta-catenin signaling for therapeutic intervention in MEN1-deficient pancreatic neuroendocrine tumours. Nature Communications 5 5809. (https://doi.org/10.1038/ncomms6809)

    • Search Google Scholar
    • Export Citation
  • Jiao Y, Shi C, Edil BH, de Wilde RF, Klimstra DS, Maitra A, Schulick RD, Tang LH, Wolfgang CL & Choti MA et al.2011 DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 331 11991203. (https://doi.org/10.1126/science.1200609)

    • Search Google Scholar
    • Export Citation
  • Jones HB, Reens J, Brocklehurst SR, Betts CJ, Bickerton S, Bigley AL, Jenkins RP, Whalley NM, Morgan D & Smith DM 2014 Islets of Langerhans from prohormone convertase-2 knockout mice show alpha-cell hyperplasia and tumorigenesis with elevated alpha-cell neogenesis. International Journal of Experimental Pathology 95 2948. (https://doi.org/10.1111/iep.12066)

    • Search Google Scholar
    • Export Citation
  • Jonsson J, Carlsson L, Edlund T & Edlund H 1994 Insulin-promoter-factor 1 is required for pancreas development in mice. Nature 371 606609. (https://doi.org/10.1038/371606a0)

    • Search Google Scholar
    • Export Citation
  • Joyce JA, Laakkonen P, Bernasconi M, Bergers G, Ruoslahti E & Hanahan D 2003 Stage-specific vascular markers revealed by phage display in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 4 393403. (https://doi.org/10.1016/s1535-6108(0300271-x)

    • Search Google Scholar
    • Export Citation
  • Juhlin CC, Skoglund S, Juntti-Berggren L, Karlberg M & Calissendorff J 2019 Non-functioning neuroendocrine pancreatic tumors transforming to malignant insulinomas – four cases and review of the literature. Neuro Endocrinology Letters 40 175183.

    • Search Google Scholar
    • Export Citation
  • Kawasaki K, Fujii M & Sato T 2018 Gastroenteropancreatic neuroendocrine neoplasms: genes, therapies and models. Disease Models and Mechanisms 11 dmm029595. (https://doi.org/10.1242/dmm.029595)

    • Search Google Scholar
    • Export Citation
  • Kawasaki K, Toshimitsu K, Matano M, Fujita M, Fujii M, Togasaki K, Ebisudani T, Shimokawa M, Takano A & Takahashi S et al.2020 An organoid biobank of neuroendocrine neoplasms enables genotype-phenotype mapping. Cell 183 1420 .e211435.e21. (https://doi.org/10.1016/j.cell.2020.10.023)

    • Search Google Scholar
    • Export Citation
  • Kersten K, de Visser KE, van Miltenburg MH & Jonkers J 2017 Genetically engineered mouse models in oncology research and cancer medicine. EMBO Molecular Medicine 9 137153. (https://doi.org/10.15252/emmm.201606857)

    • Search Google Scholar
    • Export Citation
  • Kim H, An S, Lee K, Ahn S, Park DY, Kim JH, Kang DW, Kim MJ, Chang MS & Jung ES et al.2020 Pancreatic high-grade neuroendocrine neoplasms in the Korean population: a multicenter study. Cancer Research and Treatment 52 263276. (https://doi.org/10.4143/crt.2019.192)

    • Search Google Scholar
    • Export Citation
  • Klein Haneveld MJ, van Treijen MJC, Pieterman CRC, Dekkers OM, van de Ven A, de Herder WW, Zandee WT, Drent ML, Bisschop PH & Havekes B et al.2021 Initiating pancreatic neuroendocrine tumor (pNET) screening in young MEN1 patients: results From the DutchMEN study group. Journal of Clinical Endocrinology and Metabolism 106 35153525. (https://doi.org/10.1210/clinem/dgab569)

    • Search Google Scholar
    • Export Citation
  • Kloppel G, Anlauf M & Perren A 2007 Endocrine precursor lesions of gastroenteropancreatic neuroendocrine tumors. Endocrine Pathology 18 150155. (https://doi.org/10.1007/s12022-007-0025-5)

    • Search Google Scholar
    • Export Citation
  • Klöppel G, Perren A, Sipos B & Klimstra DS 2021 Tumors of the endocrine pancreas. In Diagnostic Histopathology of Tumors, 5 th ed. Ed Fletcher CDM Philadelphia , PA, USA: Elsevier.

    • Search Google Scholar
    • Export Citation
  • Kobayashi S, Contractor T, Vosburgh E, Du YN, Tang LH, Clausen R & Harris CR 2019 Alleles of Insm1 determine whether RIP1-Tag2 mice produce insulinomas or nonfunctioning pancreatic neuroendocrine tumors. Oncogenesis 8 16. (https://doi.org/10.1038/s41389-019-0127-1)

    • Search Google Scholar
    • Export Citation
  • Lawrence B, Gustafsson BI, Chan A, Svejda B, Kidd M & Modlin IM 2011 The epidemiology of gastroenteropancreatic neuroendocrine tumors. Endocrinology and Metabolism Clinics of North America 40 118, vii. (https://doi.org/10.1016/j.ecl.2010.12.005)

    • Search Google Scholar
    • Export Citation
  • Lee YC, Asa SL & Drucker DJ 1992 Glucagon gene 5′-flanking sequences direct expression of simian virus 40 large T antigen to the intestine, producing carcinoma of the large bowel in transgenic mice. Journal of Biological Chemistry 267 1070510708. (https://doi.org/10.1016/S0021-9258(1950075-8)

    • Search Google Scholar
    • Export Citation
  • Lee JY, Ristow M, Lin X, White MF, Magnuson MA & Hennighausen L 2006 RIP-Cre revisited, evidence for impairments of pancreatic beta-cell function. Journal of Biological Chemistry 281 26492653. (https://doi.org/10.1074/jbc.M512373200)

    • Search Google Scholar
    • Export Citation
  • Lemos MC & Thakker RV 2008 Multiple endocrine neoplasia type 1 (MEN1): analysis of 1336 mutations reported in the first decade following identification of the gene. Human Mutation 29 2232. (https://doi.org/10.1002/humu.20605)

    • Search Google Scholar
    • Export Citation
  • Lewis PW, Elsaesser SJ, Noh KM, Stadler SC & Allis CD 2010 Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres. PNAS 107 1407514080. (https://doi.org/10.1073/pnas.1008850107)

    • Search Google Scholar
    • Export Citation
  • Li Z, Zheng W, Wang H, Cheng Y, Fang Y, Wu F, Sun G, Sun G, Lv C & Hui B 2021 Application of animal models in cancer research: recent progress and future prospects. Cancer Management and Research 13 24552475. (https://doi.org/10.2147/CMAR.S302565)

    • Search Google Scholar
    • Export Citation
  • Lines KE, Vas Nunes RP, Frost M, Yates CJ, Stevenson M & Thakker RV 2017 A MEN1 pancreatic neuroendocrine tumour mouse model under temporal control. Endocrine Connections 6 232242. (https://doi.org/10.1530/EC-17-0040)

    • Search Google Scholar
    • Export Citation
  • Loffler KA, Biondi CA, Gartside M, Waring P, Stark M, Serewko-Auret MM, Muller HK, Hayward NK & Kay GF 2007 Broad tumor spectrum in a mouse model of multiple endocrine neoplasia type 1. International Journal of Cancer 120 259267. (https://doi.org/10.1002/ijc.22288)

    • Search Google Scholar
    • Export Citation
  • Lopez T & Hanahan D 2002 Elevated levels of IGF-1 receptor convey invasive and metastatic capability in a mouse model of pancreatic islet tumorigenesis. Cancer Cell 1 339353. (https://doi.org/10.1016/s1535-6108(0200055-7)

    • Search Google Scholar
    • Export Citation
  • Lu J, Herrera PL, Carreira C, Bonnavion R, Seigne C, Calender A, Bertolino P & Zhang CX 2010 Alpha cell-specific Men1 ablation triggers the transdifferentiation of glucagon-expressing cells and insulinoma development. Gastroenterology 138 19541965. (https://doi.org/10.1053/j.gastro.2010.01.046)

    • Search Google Scholar
    • Export Citation
  • Mafficini A & Scarpa A 2018 Genomic landscape of pancreatic neuroendocrine tumours: the International Cancer Genome Consortium. Journal of Endocrinology 236 R161R167. (https://doi.org/10.1530/JOE-17-0560)

    • Search Google Scholar
    • Export Citation
  • Magnuson MA & Osipovich AB 2013 Pancreas-specific Cre driver lines and considerations for their prudent use. Cell Metabolism 18 920. (https://doi.org/10.1016/j.cmet.2013.06.011)

    • Search Google Scholar
    • Export Citation
  • Mandriota SJ, Jussila L, Jeltsch M, Compagni A, Baetens D, Prevo R, Banerji S, Huarte J, Montesano R & Jackson DG et al.2001 Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis. EMBO Journal 20 672682. (https://doi.org/10.1093/emboj/20.4.672)

    • Search Google Scholar
    • Export Citation
  • Marini F, Giusti F, Tonelli F & Brandi ML 2021 Pancreatic neuroendocrine neoplasms in multiple endocrine neoplasia type 1. International Journal of Molecular Sciences 22 4041. (https://doi.org/10.3390/ijms22084041)

    • Search Google Scholar
    • Export Citation
  • Marinoni I, Kurrer AS, Vassella E, Dettmer M, Rudolph T, Banz V, Hunger F, Pasquinelli S, Speel EJ & Perren A 2014 Loss of DAXX and ATRX are associated with chromosome instability and reduced survival of patients with pancreatic neuroendocrine tumors. Gastroenterology 146 453.e5–460.e5. (https://doi.org/10.1053/j.gastro.2013.10.020)

    • Search Google Scholar
    • Export Citation
  • McKenna LR & Edil BH 2014 Update on pancreatic neuroendocrine tumors. Gland Surgery 3 258275. (https://doi.org/10.3978/j.issn.2227-684X.2014.06.03)

    • Search Google Scholar
    • Export Citation
  • McNamara S, Wlizla M & Horb ME 2018 Husbandry, general care, and transportation of Xenopus laevis and Xenopus tropicalis. Methods in Molecular Biology 1865 117. (https://doi.org/10.1007/978-1-4939-8784-9_1)

    • Search Google Scholar
    • Export Citation
  • Metz DC & Jensen RT 2008 Gastrointestinal neuroendocrine tumors: pancreatic endocrine tumors. Gastroenterology 135 14691492. (https://doi.org/10.1053/j.gastro.2008.05.047)

    • Search Google Scholar
    • Export Citation
  • Michaelson JS, Bader D, Kuo F, Kozak C & Leder P 1999 Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development. Genes and Development 13 19181923. (https://doi.org/10.1101/gad.13.15.1918)

    • Search Google Scholar
    • Export Citation
  • Miehle K, Tannapfel A, Lamesch P, Borte G, Schenker E, Kluge R, Ott RA, Wiechmann V, Koch M & Kassahun W et al.2004 Pancreatic neuroendocrine tumor with ectopic adrenocorticotropin production upon second recurrence. Journal of Clinical Endocrinology and Metabolism 89 37313736. (https://doi.org/10.1210/jc.2003-032164)

    • Search Google Scholar
    • Export Citation
  • Missiaglia E, Dalai I, Barbi S, Beghelli S, Falconi M, della Peruta M, Piemonti L, Capurso G, Di Florio A & delle Fave G et al.2010 Pancreatic endocrine tumors: expression profiling evidences a role for AKT-mTOR pathway. Journal of Clinical Oncology 28 245255. (https://doi.org/10.1200/JCO.2008.21.5988)

    • Search Google Scholar
    • Export Citation
  • Naert T, Dimitrakopoulou D, Tulkens D, Demuynck S, Carron M, Noelanders R, Eeckhout L, Van Isterdael G, Deforce D & Vanhove C et al.2020 RBL1 (p107) functions as tumor suppressor in glioblastoma and small-cell pancreatic neuroendocrine carcinoma in Xenopus tropicalis. Oncogene 39 26922706. (https://doi.org/10.1038/s41388-020-1173-z)

    • Search Google Scholar
    • Export Citation
  • Offield MF, Jetton TL, Labosky PA, Ray M, Stein RW, Magnuson MA, Hogan BLM & Wright CVE 1996 PDX-1 is required for pancreatic outgrowth and differentiation of the rostral duodenum. Development 122 983995. (https://doi.org/10.1242/dev.122.3.983)

    • Search Google Scholar
    • Export Citation
  • Ouyang D, Dhall D & Yu R 2011 Pathologic pancreatic endocrine cell hyperplasia. World Journal of Gastroenterology 17 137143. (https://doi.org/10.3748/wjg.v17.i2.137)

    • Search Google Scholar
    • Export Citation
  • Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Vinals F, Inoue M, Bergers G, Hanahan D & Casanovas O 2009 Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15 220231. (https://doi.org/10.1016/j.ccr.2009.01.027)

    • Search Google Scholar
    • Export Citation
  • Parker JC, Andrews KM, Allen MR, Stock JL & McNeish JD 2002 Glycemic control in mice with targeted disruption of the glucagon receptor gene. Biochemical and Biophysical Research Communications 290 839843. (https://doi.org/10.1006/bbrc.2001.6265)

    • Search Google Scholar
    • Export Citation
  • Pelengaris S, Khan M & Evan GI 2002 Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. Cell 109 321334. (https://doi.org/10.1016/s0092-8674(0200738-9)

    • Search Google Scholar
    • Export Citation
  • Perl AK, Wilgenbus P, Dahl U, Semb H & Christofori G 1998 A causal role for E-cadherin in the transition from adenoma to carcinoma. Nature 392 190193. (https://doi.org/10.1038/32433)

    • Search Google Scholar
    • Export Citation
  • Perren A, Couvelard A, Scoazec JY, Costa F, Borbath I, Delle Fave G, Gorbounova V, Gross D, Grossma A & Jense RT et al.2017 ENETS consensus guidelines for the standards of care in neuroendocrine tumors: pathology: diagnosis and prognostic stratification. Neuroendocrinology 105 196200. (https://doi.org/10.1159/000457956)

    • Search Google Scholar
    • Export Citation
  • Pomplun D, Florian S, Schulz T, Pfeiffer AF & Ristow M 2007 Alterations of pancreatic beta-cell mass and islet number due to Ins2-controlled expression of Cre recombinase: RIP-Cre revisited; part 2. Hormone and Metabolic Research 39 336340. (https://doi.org/10.1055/s-2007-976538)

    • Search Google Scholar
    • Export Citation
  • Porcellini S, Asperti C, Valentinis B, Tiziano E, Mangia P, Bordignon C, Rizzardi GP & Traversari C 2015 The tumor vessel targeting agent NGR-TNF controls the different stages of the tumorigenic process in transgenic mice by distinct mechanisms. Oncoimmunology 4 e1041700. (https://doi.org/10.1080/2162402X.2015.1041700)

    • Search Google Scholar
    • Export Citation
  • Power RF, Holm R, Bishop AE, Varndell IM, Alpert S, Hanahan D & Polak JM 1987 Transgenic mouse model: a new approach for the investigation of endocrine pancreatic B-cell growth. Gut 28 (Supplement) 121129. (https://doi.org/10.1136/gut.28.suppl.121)

    • Search Google Scholar
    • Export Citation
  • Rachdi L, Balcazar N, Osorio-Duque F, Elghazi L, Weiss A, Gould A, Chang-Chen KJ, Gambello MJ & Bernal-Mizrachi E 2008 Disruption of Tsc2 in pancreatic beta cells induces beta cell mass expansion and improved glucose tolerance in a TORC1-dependent manner. PNAS 105 92509255. (https://doi.org/10.1073/pnas.0803047105)

    • Search Google Scholar
    • Export Citation
  • Raj N & Reidy-Lagunes D 2016 Systemic therapies for advanced pancreatic neuroendocrine tumors. Hematology/Oncology Clinics of North America 30 119133. (https://doi.org/10.1016/j.hoc.2015.09.005)

    • Search Google Scholar
    • Export Citation
  • Rindi G, Grant SG, Yiangou Y, Ghatei MA, Bloom SR, Bautch VL, Solcia E & Polak JM 1990 Development of neuroendocrine tumors in the gastrointestinal tract of transgenic mice. Heterogeneity of hormone expression. American Journal of Pathology 136 13491363.

    • Search Google Scholar
    • Export Citation
  • Rindi G, Efrat S, Ghatei MA, Bloom SR, Solcia E & Polak JM 1991 Glucagonomas of transgenic mice express a wide range of general neuroendocrine markers and bioactive peptides. Virchows Archiv: A, Pathological Anatomy and Histopathology 419 115129. (https://doi.org/10.1007/BF01600225)

    • Search Google Scholar
    • Export Citation
  • Ro C, Chai W, Yu VE & Yu R 2013 Pancreatic neuroendocrine tumors: biology, diagnosis, and treatment. Chinese Journal of Cancer 32 312324. (https://doi.org/10.5732/cjc.012.10295)

    • Search Google Scholar
    • Export Citation
  • Sadowski SM & Triponez F 2015 Management of pancreatic neuroendocrine tumors in patients with MEN 1. Gland Surgery 4 6368. (https://doi.org/10.3978/j.issn.2227-684X.2014.12.01)

    • Search Google Scholar
    • Export Citation
  • Safo AO & Pambuccian SE 2010 Pancreatic manifestations of von Hippel-Lindau disease. Archives of Pathology and Laboratory Medicine 134 10801083. (https://doi.org/10.5858/2009-0172-RS.1)

    • Search Google Scholar
    • Export Citation
  • Salanga MC & Horb ME 2015 Xenopus as a model for GI/pancreas disease. Current Pathobiology Reports 3 137145. (https://doi.org/10.1007/s40139-015-0076-0)

    • Search Google Scholar
    • Export Citation
  • Scarpa A, Chang DK, Nones K, Corbo V, Patch AM, Bailey P, Lawlor RT, Johns AL, Miller DK & Mafficini A et al.2017 Whole-genome landscape of pancreatic neuroendocrine tumours. Nature 543 6571. (https://doi.org/10.1038/nature21063)

    • Search Google Scholar
    • Export Citation
  • Scott AT & Howe JR 2019 Evaluation and management of neuroendocrine tumors of the pancreas. Surgical Clinics of North America 99 793814. (https://doi.org/10.1016/j.suc.2019.04.014)

    • Search Google Scholar
    • Export Citation
  • Sennino B, Ishiguro-Oonuma T, Wei Y, Naylor RM, Williamson CW, Bhagwandin V, Tabruyn SP, You WK, Chapman HA & Christensen JG et al.2012 Suppression of tumor invasion and metastasis by concurrent inhibition of c-Met and VEGF signaling in pancreatic neuroendocrine tumors. Cancer Discovery 2 270287. (https://doi.org/10.1158/2159-8290.CD-11-0240)

    • Search Google Scholar
    • Export Citation
  • Shen HC, He M, Powell A, Adem A, Lorang D, Heller C, Grover AC, Ylaya K, Hewitt SM & Marx SJ et al.2009 Recapitulation of pancreatic neuroendocrine tumors in human multiple endocrine neoplasia type I syndrome via Pdx1-directed inactivation of Men1. Cancer Research 69 18581866. (https://doi.org/10.1158/0008-5472.CAN-08-3662)

    • Search Google Scholar
    • Export Citation
  • Shen HC, Ylaya K, Pechhold K, Wilson A, Adem A, Hewitt SM & Libutti SK 2010 Multiple endocrine neoplasia type 1 deletion in pancreatic alpha-cells leads to development of insulinomas in mice. Endocrinology 151 40244030. (https://doi.org/10.1210/en.2009-1251)

    • Search Google Scholar
    • Export Citation
  • Shigeyama Y, Kobayashi T, Kido Y, Hashimoto N, Asahara S, Matsuda T, Takeda A, Inoue T, Shibutani Y & Koyanagi M et al.2008 Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice. Molecular and Cellular Biology 28 29712979. (https://doi.org/10.1128/MCB.01695-07)

    • Search Google Scholar
    • Export Citation
  • Sipos B, Sperveslage J, Anlauf M, Hoffmeister M, Henopp T, Buch S, Hampe J, Weber A, Hammel P & Couvelard A et al.2015 Glucagon cell hyperplasia and neoplasia with and without glucagon receptor mutations. Journal of Clinical Endocrinology and Metabolism 100 E783E788. (https://doi.org/10.1210/jc.2014-4405)

    • Search Google Scholar
    • Export Citation
  • Song YL, Xu J, Zhao DC, Zhang TP, Jin KZ, Zhu LM, Yu S & Chen YJ 2021 Mutation and expression of gene YY1 in pancreatic neuroendocrine tumors and its clinical significance. Endocrine Practice 27 874880. (https://doi.org/10.1016/j.eprac.2021.02.016)

    • Search Google Scholar
    • Export Citation
  • Sun CX, Li XZ, Liu L, Canet MJ, Guan Y, Fan YY & Zhou YF 2016 Effect of fasting time on measuring mouse blood glucose level. International Journal of Clinical and Experimental Medicine 9 41864189.

    • Search Google Scholar
    • Export Citation
  • Sun C, Estrella JS, Whitley EM, Chau GP, Lozano G & Wasylishen AR 2022 Context matters – Daxx and Atrx are not robust tumor suppressors in the murine endocrine pancreas. Disease Models and Mechanisms 15 dmm049552. (https://doi.org/10.1242/dmm.049552)

    • Search Google Scholar
    • Export Citation
  • Takano Y, Kasai K, Takagishi Y, Kikumori T, Imai T, Murata Y & Hayashi Y 2015 Pancreatic neuroendocrine tumors in mice deficient in proglucagon-derived peptides. PLoS ONE 10 e0133812. (https://doi.org/10.1371/journal.pone.0133812)

    • Search Google Scholar
    • Export Citation
  • Thakker RV 2010 Multiple endocrine neoplasia type 1 (MEN1). Best Practice and Research: Clinical Endocrinology and Metabolism 24 355370. (https://doi.org/10.1016/j.beem.2010.07.003)

    • Search Google Scholar
    • Export Citation
  • Thorens B, Tarussio D, Maestro MA, Rovira M, Heikkila E & Ferrer J 2015 INS1(Cre) knock-in mice for beta cell-specific gene recombination. Diabetologia 58 558565. (https://doi.org/10.1007/s00125-014-3468-5)

    • Search Google Scholar
    • Export Citation
  • Tuveson D & Hanahan D 2011 Translational medicine: cancer lessons from mice to humans. Nature 471 316317. (https://doi.org/10.1038/471316a)

  • Vitale G, Gaudenzi G, Dicitore A, Cotelli F, Ferone D & Persani L 2014 Zebrafish as an innovative model for neuroendocrine tumors. Endocrine-Related Cancer 21 R67R83. (https://doi.org/10.1530/ERC-13-0388)

    • Search Google Scholar
    • Export Citation
  • Wang S, Zhang J, Liu S & Zhang J 2019 The prognostic analysis of different metastatic patterns in pancreatic neuroendocrine tumors patients: a population based analysis. Medicine 98 e17773. (https://doi.org/10.1097/MD.0000000000017773)

    • Search Google Scholar
    • Export Citation
  • Wasylishen AR, Estrella JS, Pant V, Chau GP & Lozano G 2018 Daxx functions are p53-independent in vivo. Molecular Cancer Research 16 15231529. (https://doi.org/10.1158/1541-7786.MCR-18-0281)

    • Search Google Scholar
    • Export Citation
  • Wasylishen AR, Sun C, Moyer SM, Qi Y, Chau GP, Aryal NK, McAllister F, Kim MP, Barton MC & Estrella JS et al.2020 Daxx maintains endogenous retroviral silencing and restricts cellular plasticity in vivo. Science Advances 6 eaba8415. (https://doi.org/10.1126/sciadv.aba8415)

    • Search Google Scholar
    • Export Citation
  • Webb GC, Akbar MS, Zhao C, Swift HH & Steiner DF 2002 Glucagon replacement via micro-osmotic pump corrects hypoglycemia and alpha-cell hyperplasia in prohormone convertase 2 knockout mice. Diabetes 51 398405. (https://doi.org/10.2337/diabetes.51.2.398)

    • Search Google Scholar
    • Export Citation
  • Wicki A, Rochlitz C, Orleth A, Ritschard R, Albrecht I, Herrmann R, Christofori G & Mamot C 2012 Targeting tumor-associated endothelial cells: anti-VEGFR2 immunoliposomes mediate tumor vessel disruption and inhibit tumor growth. Clinical Cancer Research 18 454464. (https://doi.org/10.1158/1078-0432.CCR-11-1102)

    • Search Google Scholar
    • Export Citation
  • Wicksteed B, Brissova M, Yan W, Opland DM, Plank JL, Reinert RB, Dickson LM, Tamarina NA, Philipson LH & Shostak A et al.2010 Conditional gene targeting in mouse pancreatic ss-Cells: analysis of ectopic Cre transgene expression in the brain. Diabetes 59 30903098. (https://doi.org/10.2337/db10-0624)

    • Search Google Scholar
    • Export Citation
  • Wong C, Tang LH, Davidson C, Vosburgh E, Chen W, Foran DJ, Notterman DA, Levine AJ & Xu EY 2020 Two well-differentiated pancreatic neuroendocrine tumor mouse models. Cell Death and Differentiation 27 269283. (https://doi.org/10.1038/s41418-019-0355-0)

    • Search Google Scholar
    • Export Citation
  • Yachida S, Vakiani E, White CM, Zhong Y, Saunders T, Morgan R, de Wilde RF, Maitra A, Hicks J & Demarzo AM et al.2012 Small cell and large cell neuroendocrine carcinomas of the pancreas are genetically similar and distinct from well-differentiated pancreatic neuroendocrine tumors. American Journal of Surgical Pathology 36 173184. (https://doi.org/10.1097/PAS.0b013e3182417d36)

    • Search Google Scholar
    • Export Citation
  • Yagihashi S, Inaba W & Mizukami H 2016 Dynamic pathology of islet endocrine cells in type 2 diabetes: beta-cell growth, death, regeneration and their clinical implications. Journal of Diabetes Investigation 7 155165. (https://doi.org/10.1111/jdi.12424)

    • Search Google Scholar
    • Export Citation
  • Yamauchi Y, Kodama Y, Shiokawa M, Kakiuchi N, Marui S, Kuwada T, Sogabe Y, Tomono T, Mima A & Morita T et al.2020 Rb and p53 execute distinct roles in the development of pancreatic neuroendocrine tumors. Cancer Research 80 36203630. (https://doi.org/10.1158/0008-5472.CAN-19-2232)

    • Search Google Scholar
    • Export Citation
  • Yang Z, Zhang L, Serra S, Law C, Wei A, Stockley TL, Ezzat S & Asa SL 2016 Establishment and characterization of a human neuroendocrine tumor xenograft. Endocrine Pathology 27 97103. (https://doi.org/10.1007/s12022-016-9429-4)

    • Search Google Scholar
    • Export Citation
  • Yao JC, Hassan M, Phan A, Dagohoy C, Leary C, Mares JE, Abdalla EK, Fleming JB, Vauthey JN & Rashid A et al.2008 One hundred years after ‘carcinoid’: epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. Journal of Clinical Oncology 26 30633072. (https://doi.org/10.1200/JCO.2007.15.4377)

    • Search Google Scholar
    • Export Citation
  • Yu R 2016 Animal models of spontaneous pancreatic neuroendocrine tumors. Molecular and Cellular Endocrinology 421 6067. (https://doi.org/10.1016/j.mce.2015.08.004)

    • Search Google Scholar
    • Export Citation
  • Yu R 2018 Mahvash disease: 10 years after discovery. Pancreas 47 511515. (https://doi.org/10.1097/MPA.0000000000001044)

  • Yu R, Dhall D, Nissen NN, Zhou C & Ren SG 2011 Pancreatic neuroendocrine tumors in glucagon receptor-deficient mice. PLoS ONE 6 e23397. (https://doi.org/10.1371/journal.pone.0023397)

    • Search Google Scholar
    • Export Citation
  • Yu R, Ren SG & Mirocha J 2012 Glucagon receptor is required for long-term survival: a natural history study of the Mahvash disease in a murine model. Endocrinologia y Nutricion 59 523530. (https://doi.org/10.1016/j.endonu.2012.06.006)

    • Search Google Scholar
    • Export Citation
  • Yuan F, Shi M, Ji J, Shi H, Zhou C, Yu Y, Liu B, Zhu Z & Zhang J 2014 KRAS and DAXX/ATRX gene mutations are correlated with the clinicopathological features, advanced diseases, and poor prognosis in Chinese patients with pancreatic neuroendocrine tumors. International Journal of Biological Sciences 10 957965. (https://doi.org/10.7150/ijbs.9773)

    • Search Google Scholar
    • Export Citation
  • Yuan Z, Gardiner JC, Maggi EC, Huang S, Adem A, Bagdasarov S, Li G, Lee S, Slegowski D & Exarchakis A et al.2021 B7 immune-checkpoints as targets for the treatment of neuroendocrine tumors. Endocrine-Related Cancer 28 135149. (https://doi.org/10.1530/ERC-20-0337)

    • Search Google Scholar
    • Export Citation
  • Zhou C, Dhall D, Nissen NN, Chen CR & Yu R 2009 Homozygous P86S mutation of the human glucagon receptor is associated with hyperglucagonemia, alpha cell hyperplasia, and islet cell tumor. Pancreas 38 941946. (https://doi.org/10.1097/MPA.0b013e3181b2bb03)

    • Search Google Scholar
    • Export Citation
  • Ziv O, Glaser B & Dor Y 2013 The plastic pancreas. Developmental Cell 26 37. (https://doi.org/10.1016/j.devcel.2013.06.013)

 

  • Collapse
  • Expand
  • View in gallery
    Figure 1

    Advantages and limitations of the main PanNEN models. GEP, gastroenteropancreatic; NE, neuroendocrine; PanNEN, pancreatic neuroendocrine neoplasm; PDX, patient-derived xenograft; TFs, transcription factors; ZF, zebrafish. Created with BioRender.com. A full color version of this figure is available at https://doi.org/10.1530/ERC-22-0166.

  • View in gallery
    Figure 2

    Characteristics of the revisited genetically engineered mouse models (GEMMs) of pancreatic neuroendocrine neoplasms (PanNENs) and respective outcome. Tumor types were divided into islet adenomas or neuroendocrine neoplasms not otherwise specified (IA/NEN) and ‘invasive carcinomas’ (IC) in the cases this designation was specifically adopted by the authors. The threshold of the PanNEN onset was set at 4 months, considering the high number of reports of tumors arising before this time point. This diagram illustrates the proportion of global and conditional deletions; note that β cell-specific conditional GEMMs stand out as the largest fraction of the diagram (69%), followed by global targeting GEMMs (24%), and ɑ cell-specific conditional GEMMs (7%). Tag and Men1 are the most common targets (29% and 27%, respectively), regardless of the GEMM design. RIP-Tag (RT2) models give rise to PanNENs before 4 months (4-) (100%) and encompass the largest fraction of IC (61%). Men1 GEMMs, on the other hand, originate PanNENs solely by 4 months or after (4+): either IA/NENs in conditional models or IC in global Men1 GEMMs. n.s., not specified. Created with BioRender.com.

  • Agarwal SK, Kennedy PA, Scacheri PC, Novotny EA, Hickman AB, Cerrato A, Rice TS, Moore JB, Rao S & Ji Y et al.2005 Menin molecular interactions: insights into normal functions and tumorigenesis. Hormone and Metabolic Research 37 369374. (https://doi.org/10.1055/s-2005-870139)

    • Search Google Scholar
    • Export Citation
  • Akirov A, Larouche V, Alshehri S, Asa SL & Ezzat S 2019 Treatment options for pancreatic neuroendocrine tumors. Cancers 11 828. (https://doi.org/10.3390/cancers11060828)

    • Search Google Scholar
    • Export Citation
  • Alliouachene S, Tuttle RL, Boumard S, Lapointe T, Berissi S, Germain S, Jaubert F, Tosh D, Birnbaum MJ & Pende M 2008 Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation. Journal of Clinical Investigation 118 36293638. (https://doi.org/10.1172/JCI35237)

    • Search Google Scholar
    • Export Citation
  • Amorim JP, Santos G, Vinagre J & Soares P 2016 The role of ATRX in the alternative lengthening of telomeres (ALT) phenotype. Genes 7 66. (https://doi.org/10.3390/genes7090066)

    • Search Google Scholar
    • Export Citation
  • Asa SL 2011 Pancreatic endocrine tumors. Modern Pathology 24 (Supplement 2) S66S77. (https://doi.org/10.1038/modpathol.2010.127)

  • Asa SL, Lee YC & Drucker DJ 1996 Development of colonic and pancreatic endocrine tumours in mice expressing a glucagon-SV40 T antigen transgene. Virchows Archiv 427 595606. (https://doi.org/10.1007/BF00202891)

    • Search Google Scholar
    • Export Citation
  • Aversa JG, De Abreu FB, Yano S, Xi L, Hadley DW, Manoli I, Raffeld M, Sadowski SM & Nilubol N 2020 The first pancreatic neuroendocrine tumor in Li-Fraumeni syndrome: a case report. BMC Cancer 20 256. (https://doi.org/10.1186/s12885-020-06723-6)

    • Search Google Scholar
    • Export Citation
  • Azzopardi S, Pang S, Klimstra DS & Du YN 2016 p53 and p16(Ink4a)/p19(Arf) loss promotes different pancreatic tumor types from PyMT-expressing progenitor cells. Neoplasia 18 610617. (