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).
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
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
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.
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.
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
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
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.
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