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Pancreatic Neuroendocrine Tumors: Molecular Mechanisms and Therapeutic Targets

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Pancreatic neuroendocrine tumors (pNETs) are unique, slow-growing malignancies whose molecular pathogenesis is incompletely understood. With rising incidence of pNETs over the last four decades, larger and more comprehensive ‘omic’ analyses of patient tumors have led to a clearer picture of the pNET genomic landscape and transcriptional profiles for both primary and metastatic lesions. In pNET patients with advanced disease, those insights have guided the use of targeted therapies that inhibit activated mTOR and receptor tyrosine kinase (RTK) pathways or stimulate somatostatin receptor signaling. Such treatments have significantly benefited patients, but intrinsic or acquired drug resistance in the tumors remains a major problem that leaves few to no effective treatment options for advanced cases. This demands a better understanding of essential molecular and biological events underlying pNET growth, metastasis, and drug resistance. This review examines the known molecular alterations associated with pNET pathogenesis, identifying which changes may be drivers of the disease and, as such, relevant therapeutic targets. We also highlight areas that warrant further investigation at the biological level and discuss available model systems for pNET research. The paucity of pNET models has hampered research efforts over the years, although recently developed cell line, animal, patient-derived xenograft, and patient-derived organoid models have significantly expanded the available platforms for pNET investigations. Advancements in pNET research and understanding are expected to guide improved patient treatments.
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cancers
Review
Pancreatic Neuroendocrine Tumors: Molecular Mechanisms
and Therapeutic Targets
Chandra K. Maharjan 1, Po Hien Ear 2, Catherine G. Tran 2, James R. Howe 2, Chandrikha Chandrasekharan 3
and Dawn E. Quelle 1,4,5,*


Citation: Maharjan, C.K.; Ear, P.H.;
Tran, C.G.; Howe, J.R.;
Chandrasekharan, C.; Quelle, D.E.
Pancreatic Neuroendocrine Tumors:
Molecular Mechanisms and
Therapeutic Targets. Cancers 2021,13,
5117. https://doi.org/10.3390/
cancers13205117
Academic Editor: Murray Korc
Received: 17 September 2021
Accepted: 9 October 2021
Published: 12 October 2021
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Attribution (CC BY) license (https://
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4.0/).
1Department of Neuroscience and Pharmacology, Carver College of Medicine, University of Iowa,
Iowa City, IA 52242, USA; cmaharjan@ufl.edu
2Department of Surgery, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA;
pohien-ear@uiowa.edu (P.H.E.); catherine-tran@uiowa.edu (C.G.T.); james-howe@uiowa.edu (J.R.H.)
3
Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA;
chandrikha-chandrasekharan@uiowa.edu
4Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA
5Holden Comprehensive Cancer Center, University of Iowa, Iowa City, IA 52242, USA
*Correspondence: dawn-quelle@uiowa.edu
Simple Summary:
Pancreatic neuroendocrine tumors (pNETs) are rare, indolent cancers whose cau-
sation is only partly understood. An increasing number of studies have uncovered molecular changes
associated with pNETs, helping to identify common disease mechanisms. This knowledge has guided
current pNET therapies that can effectively slow progression of the disease. However, tumors often
become resistant to available therapies, necessitating a deeper understanding of mechanisms driving
disease progression in order to develop new treatments. Here, we provide a comprehensive review
of pNET-associated molecular alterations and existing pNET models to illustrate potential areas for
advancement in research and therapy.
Abstract:
Pancreatic neuroendocrine tumors (pNETs) are unique, slow-growing malignancies whose
molecular pathogenesis is incompletely understood. With rising incidence of pNETs over the last
four decades, larger and more comprehensive ‘omic’ analyses of patient tumors have led to a clearer
picture of the pNET genomic landscape and transcriptional profiles for both primary and metastatic
lesions. In pNET patients with advanced disease, those insights have guided the use of targeted
therapies that inhibit activated mTOR and receptor tyrosine kinase (RTK) pathways or stimulate
somatostatin receptor signaling. Such treatments have significantly benefited patients, but intrinsic
or acquired drug resistance in the tumors remains a major problem that leaves few to no effective
treatment options for advanced cases. This demands a better understanding of essential molecular
and biological events underlying pNET growth, metastasis, and drug resistance. This review exam-
ines the known molecular alterations associated with pNET pathogenesis, identifying which changes
may be drivers of the disease and, as such, relevant therapeutic targets. We also highlight areas
that warrant further investigation at the biological level and discuss available model systems for
pNET research. The paucity of pNET models has hampered research efforts over the years, although
recently developed cell line, animal, patient-derived xenograft, and patient-derived organoid models
have significantly expanded the available platforms for pNET investigations. Advancements in
pNET research and understanding are expected to guide improved patient treatments.
Keywords: pancreatic neuroendocrine tumors; molecular mechanisms; pNET models
1. pNET Introduction, Pathological Features and Classification
The incidence and prevalence of NETs have risen more than 6-fold in the United
States over the past 40 years, largely reflecting an increase in the diagnosis of early stage
disease [
1
]. NETs are a heterogenous group of slowly growing neoplasms arising in the
Cancers 2021,13, 5117. https://doi.org/10.3390/cancers13205117 https://www.mdpi.com/journal/cancers
Cancers 2021,13, 5117 2 of 51
neuroendocrine tissues of mainly the gastrointestinal (GI) tract (small intestine, appendix,
and large intestine), lungs, and pancreas. Primary NETs can also develop in the thyroid,
adrenal, pituitary glands, and ovaries. Notably, some NETs, particularly those in the
small bowel, display a spectrum of symptoms (called carcinoid syndrome) that include
flushing, diarrhea, and bronchospasm, which are associated with tumoral hypersecretion
of vasoactive amines, such as serotonin and histamine [2].
Pancreatic NETs (pNETs) constitute only 1–2% of all pancreatic neoplasms [
3
]. Al-
though these originate primarily from aberrantly proliferating cells of the endocrine pan-
creas, they can also develop from pluripotent cells of the exocrine pancreas [
4
,
5
]. The
annual incidence of pNETs is less than 1 per 100,000 individuals; however, their rate has
significantly increased over the past few decades [
1
]. Most pNETs occur sporadically, but
some occur in association with hereditary multi-tumor predisposition syndromes, such
as multiple endocrine neoplasia type 1 (MEN1), von-Hippel-Lindau (VHL), neurofibro-
matosis type 1 (NF-1), tuberous sclerosis complex (TSC), and Cowden syndrome (CS) [
6
].
Based on symptoms manifested by tumor-secreted hormones, pNETs are categorized as
functional or non-functional. Insulinoma, gastrinoma, and rare pNET subtypes such as
glucagonoma, VIP (vasoactive intestinal peptide)-oma, and somatostatinoma are some ex-
amples of functional pNETs, which manifest hormone-related clinical symptoms that may
help in diagnosing these tumors at an earlier stage [
4
,
7
]. In contrast, non-functional pNETs,
which represent 60–90% of all pNETs, are often asymptomatic and likely to remain undi-
agnosed until they are advanced and unresectable. As such, patients with non-functional
pNETs tend to have worse outcomes compared to those with functional tumors [2,4].
The 2019 World Health Organization (WHO) tumor grading system defines pNETs
as well-differentiated (WD) pancreatic neuroendocrine neoplasms (pNENs) and hence,
a pNEN subset [
8
]. The second subset comprises pancreatic neuroendocrine carcinomas
(pNECs), which are poorly differentiated pNENs. Mixed neuroendocrine-non-neuroendocrine
neoplasms (miNENs) are grouped as the third pNEN subset to represent rare, poorly
defined pancreatic tumors possessing specific molecular and pathological features of
neuroendocrine and non-neuroendocrine components [9].
A pNET cell displays minimal to moderate atypia, lacks necrosis, and exhibits intense
staining of NE differentiation markers, synaptophysin and chromogranin [
10
]. In contrast,
a pNEC consists of highly atypical cells of varying sizes and exhibits faint staining of
synaptophysin and chromogranin A. pNETs are further subdivided into grade 1 (G1),
grade 2 (G2), and grade 3 (G3) based on increasing order of their proliferation index
(
Ki-67 index <3,
3–20, and >20, for G1, G2, and G3 pNETs, respectively), whereas pNECs
are invariably G3. Common alterations in pNECs, such as genetic mutations in the RB1
and TP53 genes, are distinct from those of pNETs (e.g., MEN1,ATRX,DAXX) suggesting
pNECs originate de novo and not via pNET dedifferentiation [
11
,
12
]. miNENs could be
well or poorly differentiated and usually possess an aggressive phenotype by virtue of its
NEC component [
8
,
9
]. On the other hand, they mimic adenocarcinomas histologically and
at the molecular level by exhibiting high rates of TP53,BRAF, and KRAS mutations.
To supplement the WHO classification system with additional criteria refining prog-
nostic stratification and therapeutic decision-making for pNENs, the European Neuroen-
docrine Tumor Society (ENETS) and American Joint Committee on Cancer/Union for
International Cancer Control (AJCC/UICC) introduced the TNM-staging system [
13
,
14
].
‘T’ stages (1–4) describe tumor size and invasion into the surrounding tissues and blood
vessels; ‘N’ indicates the presence of regional lymph node metastases; ‘M’ indicates the
presence of distant metastases [
3
,
13
,
14
]. Accurate grading and staging of pNENs guides
prognostication, choice of treatment modality, and clinical decision making [
15
]. Interest-
ingly, a recent multicenter retrospective study of pNEN susceptibility identified gender
differences in age at diagnosis, associated co-morbidities (such as type II diabetes), and
potential risk factors [
16
]. Although females were slightly more likely to be diagnosed at a
younger age compared to males, in females the presence of type II diabetes was associated
Cancers 2021,13, 5117 3 of 51
with higher tumor grade and metastatic disease. The authors suggest a gender-tailored
approach could be important in improving pNEN clinical management.
There are several effective therapies against pNETs that have improved patient out-
comes, although surgical resection is the only curative treatment for localized tumors [
15
].
Approximately 40% of patients present with advanced, metastatic disease [
1
], requiring
systemic pharmacological or radiation-based therapies. The Food and Drug Administra-
tion (FDA) approved drug therapies used in the management of advanced, unresectable
or metastatic pNETs include somatostatin analogs such as octreotide LAR (long acting
repeat) and lanreotide; mTOR inhibitor, everolimus; multitargeted tyrosine kinase inhibitor,
sunitinib; and chemotherapeutic agents, 5 fluorouracil, and streptozotocin. Although
the combination of 5 fluorouracil and streptozotocin is approved, the capecitabine and
temozolamide combination is often used in clinical practice for their better side effect
profile and more reliable efficacy. Peptide receptor radionuclide therapy (PRRT) using
radiolabeled somatostatin analogue,
177
Lu-Dotatate (Lutathera), is also FDA approved for
the management of advanced pNETs [
17
,
18
]. For pNET patients with carcinoid syndrome
(less than 1%) [
19
], telotristat ethyl is an oral tryptophan hydroxylase 1 (TPH1) inhibitor
that can provide relief from symptoms, particularly by decreasing the number of daily
bowel movements [
20
]. Together, the broadly relevant therapies described above and
context dependent use of telotristat ethyl have improved outcomes and quality of life of
pNET patients.
Despite these advances, a significant percentage of pNET patients are non-responsive
to existing anti-tumor therapies or they develop acquired drug resistance. This highlights
an urgent need to develop more effective therapies, which can only be achieved through a
better understanding of the molecular mechanisms driving pNET pathogenesis.
Several molecular profiling studies have revealed important pNET-signature genes
and pathways. However, which molecular alterations are essential for pNET genesis and
progression is not clearly understood. Our review provides a comprehensive discussion of:
(i) genes and signaling pathways shown to be frequently altered in pNETs by molecular
profiling studies, as well as their prognostic and therapeutic significance, (ii) genes associ-
ated with familial pNETs with a focus on the downstream mechanisms underlying pNET
development, (iii) additional proteins and signaling pathways, whose genes or expression
might remain unchanged yet whose altered activities are functionally essential for pNET
development and progression, (iv) interplay between these various signaling pathways,
(v) preclinical and clinical studies evaluating pNET targeted therapies, and
(vi) in vitro
and
in vivo
pNET models used for biological studies and drug screenings. Figure 1pro-
vides a consolidated diagram of important signaling pathways discussed herein whose
dysregulation drives pNET pathogenesis.
Cancers 2021,13, 5117 4 of 51
Figure 1.
Diagrammatic illustration of important signaling proteins and pathways that drive pNET cell survival, prolifer-
ation, angiogenesis, and metastasis. Proteins having oncogenic roles in pNETs are shown in pink boxes whereas pNET
suppressors are shown in green. Numbers highlighted in blue indicate FDA approved drugs for therapy that target the
corresponding proteins: 1-everolimus, 2-sunitinib, and 3-somatostatin analogues. Asterisks in red denote additional targets
being evaluated for pNET therapy. Arrows, activating events; perpendicular bars, inhibitory events. Schematic developed
using BioRender software (Toronto, Canada).
2. pNET-Associated Genes and Signaling Pathways
The molecular pathogenesis of pNETs is only partially understood. By comparison,
much more is known about pancreatic ductal adenocarcinomas (PDACs), the most common
type of pancreatic cancer, which arise from exocrine cells and are primarily driven by KRAS
activation [
21
] and other major genetic changes, including TP53 mutation [
22
]. Several
groups have conducted whole genome and exome sequencing of patient-derived pNETs
to gain more insight into genetic alterations driving their development. One of the major
conclusions has been that pNETs, unlike PDACs, have a low mutational burden [
23
,
24
].
These and other molecular profiling studies, along with functional investigations performed
in pNET cells and mouse models, have identified the most frequently altered, biologically
relevant genes and pathways underlying the pathogenesis of familial and sporadic pNETs
(Figure 2).
2.1. Menin
The MEN1 gene encodes a 610-amino acid nuclear scaffold protein called menin, which
plays an important role in chromosomal remodeling and gene transcription. Inheritance of
germline mutations in one MEN1 allele from either parent leads to a familial autosomal
dominant tumor syndrome called multiple endocrine neoplasia type 1 (MEN 1). Of all the
different familial causes of pNETs, MEN1 syndrome remains the most frequent. Historically,
Cancers 2021,13, 5117 5 of 51
linkage analysis and loss of heterozygosity analysis of tumors from affected individuals
mapped the gene to the chromosomal region 11q13 [
25
,
26
], which led to positional cloning
studies identifying the MEN1 gene [
27
,
28
]. Several studies suggest that oncogenesis in
MEN1 individuals involves unmasking of the mutated MEN1 allele at the disease locus by
loss of the remaining wild-type copy, in agreement with Knudson’s two-hit mutation model
for tumor suppressor genes originally proposed for RB1 loss in retinoblastoma [2931].
Figure 2.
Schematic of frequently altered genes and pathways in familial and sporadic pNETs. Genes
(italics) and pathways (non-italics).
Over 90% of MEN1 patients exhibit one or more different types of endocrine tumors
by the age of 50 [
6
]. This mainly includes parathyroid (with a frequency of 95–100%),
pancreatic (80–100%), and pituitary (54–64%) tumors [
6
,
32
35
]. Nearly all MEN1 patients
(80–100%) develop NF-pNETs, which remain small and asymptomatic in most individuals.
Insulinomas are the most common functional pNETs in MEN1, observed in 18% of MEN1
patients [
6
]. While more than half of MEN1 individuals also develop gastrinomas, the vast
majority (greater than 80%) are duodenal, and only a small percentage of these tumors
are pancreatic. Glucagonomas (3%), VIPomas (3%), GRF (growth hormone-releasing
factor)-omas (3%), and somatostatinomas (<1%) are other less frequent functional pNETs
associated with MEN1 [6].
In addition to its prominent role in familial pNETs, somatic MEN1 mutations are
arguably the most frequent genetic event found in sporadic pNETs (Table 1). Whole exome
sequencing revealed MEN1 somatic mutations are present in 40–56% of sporadic pNETs, far
exceeding the incidence of alterations in any other single gene within these tumors [
36
39
].
This finding builds upon earlier studies showing MEN1 mutations in 5–30% of patient
pNETs [
29
,
30
,
32
,
36
,
37
]. Notably, several allelotyping and loss of heterozygosity (LOH)
analyses have found allelic loss of MEN1 or the chromosomal segment encompassing the
gene on 11q13 in 30–70% of sporadic pNETs [
31
,
33
35
,
38
,
39
]. Since allelic deletions of
MEN1 may be 2–3 times more common than MEN1 gene mutations, it has been suggested
that other tumor suppressor genes on 11q13 may factor into tumorigenesis of these neo-
Cancers 2021,13, 5117 6 of 51
plasms [
36
]. On the other hand, MEN1 mutations are usually co-incident with deletion of
the other functional allele, ultimately leading to complete loss of menin activity [
40
42
].
Such results suggest that pathogenesis of non-familial, sporadic pNETs may recapitulate
tumor development seen in MEN1 patients.
Table 1. Molecular profiling studies revealing MEN1 alterations in pNETs.
Technique Reference Key Findings
Exome/genome
sequencing
[24]Somatic MEN1 mutations in 44.1% of 68 sporadic pNETs.
MEN1 mutations correlated with poor patient survival.
[23]Somatic MEN1 mutations in 41% of 102 primary pNETs.
Abnormal telomere length observed in MEN1-mutated tumors.
[39]
In total, 26% of 57 sporadic well differentiated pNETs had recurrent LOH of 10
specific chromosomes and biallelic MEN1 inactivation.
Another 40% had chromosome 11 LOH and biallelic MEN1 inactivation. The first
patient group had worse clinical outcomes compared to the second.
[40]MEN1 mutations in 43% of 65 pNETs.
[43]Somatic MEN1 mutations in 56% of 80 patient pNETs. In total, 1 of 17 patients
carried a germline MEN1 mutation.
Allelotyping/LOH
analysis
[31] Loss of chromosomal segment 11q (where MEN1 is located) in >60% cases.
[34]LOH of 11q13 in 70% and MEN1 mutations in 27% of 11 advanced pNETs (9 NF
and 2 glucagonomas).
[29]MEN1 mutatons in 6 (mostly pNETs) of 43 sporadic GEPNETs.
[37]MEN1 allelic deletions in 93% of gastrinomas and 50% of 12 insulinomas;
mutations in 33% gastrinomas and 17% insulinomas.
Microarray [35]
Consensus cluster analysis of microarray results showed clustering of 5 out of 9
sporadic NF-pNETs with MEN1-associated familial pNETs. In total, 4 of those 5
sporadic pNETs had MEN1 LOH.
Exactly how menin suppresses pNET development is still unfolding although it clearly
plays a critical role in the nucleus. Menin is a 68 kilodalton (kDa) protein that contains two
carboxy-terminal nuclear localization sequences (NLS) and normally resides in the nucleus
of all pancreatic cells [
41
,
44
,
45
]. Tumor-associated frameshift and nonsense mutations in
MEN1 encode truncated forms of the protein that lack one or both NLS and protein-protein
interaction domains [
30
,
44
]. This correlates with abnormally high cytoplasmic expression
of menin in the majority (80%) of sporadic pNETs, whereas the wild-type protein is almost
exclusively nuclear in normal islets [
30
]. Interestingly, cytoplasmic mis-localization of
menin is not dependent on MEN1 mutation, possibly reflecting loss of nuclear interactions,
unmasking of a functional nuclear export signal (NES) in menin, and/or altered localization
of its partners that may physically mobilize menin into the cytoplasm [30].
In the nucleus, menin interacts with a variety of proteins involved in transcription and
DNA damage repair. Its partners include JunD, ASK, FANCD2, Smad3, Pem, Nm23, NF-kB,
and replication protein A (RPA2) [
30
]. Menin either inhibits the activities of its oncogenic
partners or promotes the function of its tumor suppressive partners. For example, menin
association with JunD and NF-kB proteins (p50, p52 and p65) blocks their transcriptional
activities [
46
,
47
], whereas its binding and activation of Smad3 promotes growth inhibitory
signaling by transforming growth factor
β
(TGF
β
) [
42
]. FANCD2 is a DNA repair protein
in the BRCA1 DNA repair pathway that is mutated in patients with Fanconi anemia, an
inherited cancer-prone syndrome. Jin et al., showed that menin forms complexes with
FANCD2, which are enhanced by gamma irradiation and associated with reduced DNA
damage [
48
]. These data suggest menin helps repair damaged DNA and maintain genomic
stability, in agreement with prior studies showing increased chromosomal breaks and
instability in MEN1 mutant pancreatic tumors [49].
Cancers 2021,13, 5117 7 of 51
Additional studies revealed that menin is a member of histone methyltransferase com-
plex that promotes histone methylation [
50
] as well as gene expression of cyclin dependent
kinase inhibitors, p27
KIP1
and p18
INK4C
, to suppress pancreatic islet
growth [51,52].
In islet
cells, loss of MEN1 enhances proliferation by accelerating S-phase entry [
53
]. Recently, a
genome-wide CpG methylation profiling study showed MEN1-associated pNETs have a
higher rate of hypermethylated CpG sites compared to VHL- or other pNET types, indicat-
ing menin contributes significantly to the epigenetic control of DNA methylation [54].
Overexpression of menin inhibits the growth of rat insulinoma cells, whereas its
loss promotes their proliferation, supporting the tumor suppressor role of this gene [
55
].
These
in vitro
results were corroborated by studies showing development of pNETs in
genetically engineered mouse models lacking Men1 (Table 2). Menin can cooperate with
other molecules to suppress pNET development. For instance, menin directly interacts with
death-domain-associated protein (DAXX), another commonly mutated tumor suppressor
gene in pNETs [
23
,
24
], to repress matrix metalloendopeptidase (MME), a zinc-dependent
metalloprotease required for pNET cell proliferation, leading to pNET suppression [
55
].
Menin also cooperates with PTEN (Phosphatase and Tensin homolog), a tumor suppressive
phosphatase that negatively regulates PI3K-Akt-mTOR signaling. The importance of menin-
PTEN crosstalk in limiting pNET formation was demonstrated in conditional knockout
mice lacking both Men1 and Pten in pancreatic islet
β
cells. These mice had elevated
PI3K-Akt-mTOR activity in tumors and developed pNETs at a much shorter latency than
mice lacking either gene alone, suggesting menin-PTEN inhibition of the PI3K-Akt-mTOR
pathway is crucial in preventing pNET pathogenesis [56].
Table 2. Genetically engineered pNET mouse models.
Model Strain pNET Type Key Findings
RIP-Tag2 C57BL/6 Insulinoma
SV40 large T-antigen inactivates p53 and Rb in
islet-βcells and promotes insulinoma development
in a multi-stage, synchronized fashion [57]
Men1f/f Ptenf/f;
MIP-Cre or RIP-Cre
(MPM or MPR)
C57BL/6J Insulinoma Loss of Pten co-operates with that of Men1 to
develop well differentiated G1/G2 pNETs [56]
RIP-Tag2 AB6F1
AB6F1 (hybrids from
A/J dam and RT2
C57BL/6 sire)
Non-functional (NF)
pNETs
RT2 mice develop NF pNETs with higher rate of
liver metastases on AB6F1 genetic background,
which is attributed to low expression of Insm1, a
β-cell specific differentiation factor required for
insulin secretion [58]
RIP-MyrAkt1 C57/BL6 Insulinoma
β-cell specific expression of constitutively active
Myr-Akt leads to formation of insulinomas in S6K1
(a mTOR downstream target) dependent
manner [59]
Avp-Tag C57B1/10; CBA/J Insulinoma
Mice bearing vasopressin promoter (1.2 kb 50
sequence)-SV40 hybrid transgene
uncharacteristically transformed pancreatic β-cells
and anterior pituitary cells with no effect in
hypothalamus and other organs where vasopressin
is normally expressed [60]
Men1TSM/+ NIH Black Swiss;
129/Sv Insulinoma
Mutation of one Men1 allele by homologous
recombination leads to insulinoma development by
9 months and other tumors involving parathyroid,
thyroid, adrenal cortex, and pituitary by 16 months
mimicking human MEN1 syndrome [61]
Men1+/T 129
Insulinoma and
glucagonoma that
dedifferentiated into
advanced NF-pNETs
Disruption of one Men1 allele by gene targeting
resulted in tumors of pancreatic, parathyroid,
thyroid, pituitary, and adrenal glands exhibiting
multistage progression and metastatic potential [
62
]
Cancers 2021,13, 5117 8 of 51
Table 2. Cont.
Model Strain pNET Type Key Findings
Men1f/f; RIP-Cre C57BL/6 Insulinoma
RIP-Cre mediated conditional knockout of Men1
gene leads to insulinomas and pituitary
prolactinomas by 9 months [63]
Men1f/f; Glu-Cre Not specified Insulinoma
Surprisingly, loss of Men1 in
α
-cells resulted in
β
-cell
insulinomas rather than glucagonomas suggesting
the role of intercellular talk between islet cells [64]
Men1f/f; Pdx1-Cre FVB; 129Sv Insulinoma
Although Men1 is lost in both pancreatic exocrine
and endocrine cells, only endocrine cells developed
into highly angiogenic tumors suggesting the role of
tissue-specific menin modulators and surrounding
microenvironment during tumorigenesis [65]
Men1f/f; RIP2-CreER 129; (C57BL/6 X CBA) Insulinoma
Temporally controlled β-cell specific loss of Men1
led to insulinomas. Moreover, the model helps
elucidate early stage events such as β-cell
hyperproliferation [66].
Men1f/f; RIP-Cre B6; FVB; 129Sv Insulinoma
Conditional knockout of both Men1 alleles promoted
islet cell tumor development much faster than that
of one allele [67]
Men1f/f; RIP-Cre 129 Insulinoma
Disruption of Men1 gene directly in β-cells led to
insulinoma development by 6 months in a
multistage fashion, exhibiting angiogenesis and
altered E-cadherin and β-catenin expression [68]
Men1f/f; Glu-Cre 129; B6/CBAJ-F1
Insulinoma,
glucagonoma, and
mixed islet cell tumor
α-cell specific loss of Men1 leads to α-cell
hyperplasia that grow into glucagonomas, however,
majority of the hyperplastic
α
-cells transdifferentiate
into insulinomas and mixed islet tumors [69]
Glu2-Tag C57BL/6 Glucagonoma
Expression of Tag under preproglucagon promoter
drives hyperproliferation of alpha cells and
formation of glucagonomas by 9–12 months.
Promiscuous expression of T-antigen in hind brain
neurons is not sufficient for their hyperplasia or
tumorigenesis [70].
Gcgr/DBA/1 Glucagonoma
Inhibition of glucagon signaling by glucagon
receptor mutation causes α-cell hyperplasia that
progress into islet dysplasia and solid tumors. A few
animals develop mixed tumors or NF-pNETs [71].
Pc2/C57BL/6 Glucagonoma
Loss of prohormone convertase 2 required for
glucagon synthesis leads to α-cell hyperplasia that
develop glucagonomas and mixed islet tumors by
6–8 months [72]
RIP7-rtTA; tet-o-MT;
p48-Cre; Ink4a/Arf f/f C57BL6; FVB; ICR Not determined
Loss of Ink4a/Arf tumor suppressor locus
cooperates with overexpression of PyMT in
pancreatic progenitor cells to induce pNET
formation however at low incidence rate of 20% [
73
]
RIP7-rtTA; tet-o-MT;
p48-Cre; Trp53f/f;
Ink4a/Arf f/f C57BL6; FVB; ICR Not determined
Overexpression of oncogenic PyMT in β-cells
together with deletion of P53 and Ink4a/Arf loci
results in pNET incidence in 40% mutant mice [73]
pIns-c-MycERTAM/
RIP-Bcl-xLCBAxC57BL/6 Insulinoma
Conditional expression of transgenic Myc and Bcl-x
L
to suppress Myc-induced apoptosis in islet βcells
causes islet tumor development in a reversible
fashion [74]
Cancers 2021,13, 5117 9 of 51
Table 2. Cont.
Model Strain pNET Type Key Findings
Pdx1-Cre;
Trp53R172H;Rbf/f FVB/N; J1;
Well differentiated,
metastatic insulinoma
and glucagonoma
Pancreas-specific p53 mutation and Rb deletion
caused islet dysplasia that progressed to indolent
and metastatic pNET in stepwise fashion [75]
Men1
f/f
Rb1
f/f
RIP-Cre;
Men1f/f Ptenf/f
RIP-Cre; Trp53f/f
Rb1f/f RIP-Cre;
Men1f/f (129S, FVB)
Rb1f/f(FVB;129)
Ptenf/f(C;129S4)
Trp53f/f(B6.129P2)
RIP-Cre (C57BL/6)
Well differentiated G1,
G2, and G3 pNETs
(insulinoma)
Demonstrated the cooperative role of tumor
suppressor genes, Men1,Rb1,Pten, and Trp53 in
pNET suppression [76]
RIP-Tag2; Rabl6m/m C57BL/6N Insulinoma Loss of oncogenic RABL6A attenuates pNET
progression and angiogenesis in RIP-Tag2 mice [77]
Pdx1-Cre; Men1f/f;
B7x KO C57BL/6 Insulinoma
Loss of B7x, an immune-checkpoint ligand, reduces
islet β-cell proliferation and pNET formation
consistent with increased T-cell infiltration [78]
(Abbreviations: RIP = rat insulin promoter, MIP = mouse insulin promoter, Tag = T-antigen).
2.2. PI3K-Akt-mTOR Pathway
Aberrant activation of oncogenic PI3K-Akt-mTOR pathway is implicated in both
familial and sporadic pNETs. Inherited mutations in TSC2 (Tuberous Sclerosis Complex 2)
and PTEN tumor suppressors, key negative regulators of this oncogenic pathway, lead to
autosomal dominant, multisystem disorders in which a small percentage of individuals
develop pNETs [
6
,
79
]. The TSC2 gene encodes a protein called tuberin that drives Rheb-
GTP hydrolysis, thereby inhibiting mTORC1 activation [
80
]. Germline mutations in the
TSC2 gene leads to an autosomal dominant tumor predisposition syndrome called tuberous
sclerosis (TSC), which is characterized by formation of benign tumors in almost every organ
in the body [
6
]. In rare cases of TSC, patients develop functional (primarily gastrinomas
and insulinomas) and non-functional pNETs [
81
,
82
]. LOH analysis of malignant pNETs
reveal loss of the wild type TSC2 copy with only the non-functional copy of the gene
present at the disease locus [
83
]. A similar familial disease, called Cowden’s syndrome, is
also associated with pNET development. Most patients with this syndrome inherit loss-
of-function mutations in the PTEN gene, which results in Akt activation [
84
]. Although
Cowden’s syndrome is characterized by tumors in the breast, thyroid, and endometrium, a
minority of these patients develop pNETs driven by PTEN gene mutations [79,85].
The importance of PI3K-Akt-mTOR pathway activation in sporadic pNETs is well
established and clinically relevant. Whole genome sequencing studies have revealed
mutations in mTOR pathway genes including PTEN,TSC2,PIK3CA, and DEPDC5 in
12–25% sporadic pNETs (Table 3) [
36
39
]. Moreover, allelic loss of chromosomal segments
containing TSC2 (16p) and PTEN (10q23) are observed in 25–36% of human pNETs [
86
,
87
].
Although mTOR pathway-related genetic mutations are less common, the percentage of
sporadic pNETs with altered expression of the pathway members is remarkably high.
mRNA expression profiling and IHC of PTEN and/or TSC2 display downregulation of
these genes in 75% sporadic pNETs, which correlates with disease progression and worse
survival [
88
]. Aberrant cytoplasmic distribution of PTEN, which is primarily nuclear in
normal islets, has also been reported in sporadic pNETs [
87
]. Mutations in mTOR pathway
genes have been associated with poor prognosis in pNET patients [
23
], and hyperactivation
of Akt and mTOR kinases has also been found in a subset of pNETs [89,90].
Findings from the tumor molecular profiling studies have been functionally validated
using both
in vitro
and
in vivo
pNET models. Conditional loss of Pten in pancreatic islet
cells leads to neuroendocrine tumor formation in mice (Table 2) [
56
]. Conversely, inhibitors
targeting PI3K, Akt, or mTOR, either used alone or in combination, impede pNET cell
growth and tumor development and metastasis in mice, suggesting pNET cells require
Akt-mTOR signaling for their survival, growth, and migration [
91
94
]. Moreover, treat-
Cancers 2021,13, 5117 10 of 51
ment of RIP-Tag2 mice (the first developed and prototypic transgenic pNET model) with an
mTORC1 inhibitor, rapamycin, increases their average life span by 7 weeks
(i.e., 16 weeks
for males) [
95
]. Consistently, a rapamycin analogue—everolimus (RAD001)—was found
to have potent anti-tumor activity in human pNET cell lines [
96
98
] and mouse mod-
els [
12
]. This provided a strong rationale for its use in clinical trials for advanced pNET
patients [99,100].
In a phase III study (the RADIANT-3 trial) comprising 410 patients with
advanced, low- or intermediate grade pNETs, everolimus treatment was associated with
improved median PFS of patients compared to the placebo group (median PFS 11.0 vs.
4.6 months, p< 0.001), representing a 65% lower risk of disease progression or death in
the everolimus treatment group [
100
]. A critical review of everolimus therapy for pNETs
concluded it should be a first line therapy for patients with symptomatic, unresectable,
insulin-secreting pNETs to control endocrine syndrome regardless of tumor growth [101].
Importantly, sustained inhibition of mTORC1 eliminates the ribosomal S6 kinase 1
(S6K1)/insulin receptor substrate-1 (IRS1) feedback loop, resulting in unwanted mTORC2-
promoted Akt activation, which paradoxically enhances pNET growth and acquired re-
sistance to mTORC1 inhibitors such as everolimus [
102
104
]. Therefore, the combination
of an mTORC1 inhibitor with agents that prevent Akt activation, such as inhibitors of
PI3K, Akt, mTORC2, or receptor tyrosine kinases, have been tested in various contexts and
shown to have better outcomes [102,105].
Table 3. Molecular profiling studies revealing PI3K-Akt-mTOR signaling pathway alterations in pNETs.
Technique Reference Key Findings
Exome sequencing
[24]Somatic PTEN,TSC2, and PIK3CA mutations in 7.3%, 8.8%, and 1.4% of 68 sporadic
pNETs, respectively.
[23]
Somatic mutations in mTOR pathway genes observed in 102 primary pNETs: PTEN (7%),
DEPDC (2%), TSC1 (2%), and TSC2 (2%).
mTOR pathway gene mutations associated with poor survival.
[40] 11% of 65 pNETs had mTOR pathway gene mutations: TSC2 (6%) and PTEN (5%).
[43]TSC2 mutations in 25% of 80 patient pNETs. In total, 1 of 17 patients carried a germline
TSC2 mutation.
Allelotyping/LOH
analysis
[87]LOH of 10q23 (where PTEN is located) in >50% of 22 pNETs. PTEN mutations
rarely observed.
[86] Allelic deletions of 16p13 (where TSC2 is located) in 36% of 28 pNETs
Microarray [88]
TSC2, PTEN or both downregulated in 85% of primary PNETs, subsequently validated by
qRT-PCR and IHC studies.
Reduced expression of TSC2 and PTEN correlated with poor patient prognosis.
RNA sequencing [106]
Ingenuity pathway analysis (IPA) and Connectivity Map (CMap) analysis of 626
metastatic gene signatures obtained from 39 primary tumors, 21 lymph node metastases
and 17 liver metastases predicted mTOR and PI3K as top pNET pharmacological targets.
[92] Alteration of Akt signaling genes revealed by sequencing of 20 primary pNETs.
IHC [107] Low PTEN expression in 48% of 21 pNETs.
2.3. INK4A/ARF Locus and RB1 Pathway
Mounting evidence implicates the role of the INK4A/ARF (originally called CDKN2A)
gene locus and retinoblastoma 1 (RB1) tumor suppressor pathway in pNET pathogenesis.
The INK4A/ARF locus encodes two different proteins, p16
INK4a
and p14
ARF
(mouse homo-
logue p19
ARF
), derived from transcripts having distinct first exons but shared downstream
exons that are translated in alternative reading frames [
108
,
109
]. As a result, the two protein
products share no amino acid identity. The first product discovered, p16
INK4a
(Inhibitor of
CDK4/6), is a cyclin dependent kinase (CDK) inhibitor that enforces RB1 tumor suppres-
sive activity by specifically inhibiting CDK4 and CDK6 [
108
]. By comparison, the other
product, p14
ARF
(Alternative Reading Frame protein), has numerous targets that it inter-
Cancers 2021,13, 5117 11 of 51
acts with to prevent cancer [
110
]. ARF primarily activates the p53 tumor suppressor via
MDM2 degradation, however, it also possesses many p53 independent tumor suppressive
functions [110,111].
Inactivating p16
INK4a
gene alterations are common in pNETs (Table 4). Several studies
reveal homozygous deletions of the INK4A gene or hypermethylation of its promoter
5
0
CpG island in the vast majority (up to 92%) of pNETs comprising gastrinomas and
non-functional PNETs [
103
,
104
,
112
]. In general, insulinomas have a low frequency (17%)
of INK4A genetic inactivation suggesting a pNET subtype-specific role of the gene [
113
].
Studies focusing on the mutational status of INK4a/ARF suggest mutations at this locus
are absent in pNETs [
24
,
112
]. Nuclear staining of the p16
INK4a
protein demonstrated
loss of its expression in half of the analyzed pNETs [
114
], although interpreting loss of
p16INK4a in tumors is always difficult since it is not expressed in most normal tissues due
to tight transcriptional repression [
115
]. That said, silencing of INK4A in patient pNETs has
clinical relevance as its hypermethylation occurs more commonly in malignant pNETs with
metastases than in benign tumors [
116
]. Moreover, low p16
INK4a
protein expression was
associated with reduced survival in patients with G2-grade GEPNETs [117]. Mice lacking
both p16
INK4a
and p19
ARF
, or just p16
INK4a
alone, spontaneously develop a spectrum of
tumors but do not form NETs [
118
120
]. The likely reason is because the animals die
of other, more aggressive tumors before NETs have time to develop. In neuroendocrine
tissues, loss of p16
INK4a
could be compensated by other CDK inhibitors that can enforce
RB1 activity, such as p27KIP1 [113].
Unlike p16
INK4a
, promoter hypermethylation at the ARF gene is extremely rare in
pNETs, although it is more commonly observed in other gastro-intestinal NETs [
113
].
Likewise, loss of ARF mRNA expression has been observed in a low percentage of non-
functional pNETs [
104
]. However, the tumor suppressive role of p19
ARF
has been demon-
strated in a pNET mouse model of aggressive insulinomas. Ulanet et al., showed that loss
of p19
ARF
promotes pNET initiation and angiogenesis in RIP-Tag2 mice (Table 2) [
121
].
Since the tumors are driven by SV40 T antigen-mediated inactivation of RB1 and p53, the
role of p19
ARF
in this setting is independent of both tumor suppressors. Most recently,
a tumor suppressive role for p19
ARF
was implicated in a study of one of its interacting
proteins, RABL6A, in pNET progression [
77
]. Genetic loss of Rabl6 in RIP-Tag2 mice
slowed pNET development of angiogenesis, which correlated with increased expression of
p19
ARF
protein in RABL6A-deficient tumors. RABL6A inactivation had no effect on Arf
mRNA expression, suggesting part of RABL6A’s oncogenic effects may involve p19
ARF
downregulation at the protein level.
Unlike advanced G3 pNECs (which have a high rate of RB1 mutation), most well
differentiated pNETs have an intact RB1 gene [
11
]. Nevertheless, loss of RB1 tumor sup-
pressor activity is critical for pNET development. Besides loss of p16
INK4a
expression, RB1
activity can be impaired in pNETs via aberrant expression of upstream CDKs.
Tang et al.,
showed that overexpression of CDK4 and its binding partner, cyclin D1, occurs in a ma-
jority of human pNETs, which correlated with elevated inactivating phosphorylation of
RB1 [
122
]. Upregulated CDK expression and activity coincided with 19% of pNETs ex-
hibiting amplification of both CDK4 and CDK6 genes. A more recent study of 267 patients
demonstrated that high expression of cyclin D1, CDK4, and CDK6 proteins was associated
with significantly increased Ki-67 in tumors [123].
Preclinical studies of CDK inhibitors in pNET cell lines and xenograft models have
supported their use in treating pNET patients. Early studies with a non-selective kinase
inhibitor targeting multiple CDKs (1/2/4/7), VEGFR and PDGFR, called ZK 304709,
displayed significant anti-tumor activity (G2 phase cell cycle arrest and apoptosis) in BON1
xenograft tumors [
124
]. Others used a specific CDK4/6 inhibitor, palbociclib (PD0332991),
and showed it induced a strong G1 phase arrest in cultured BON1 and QGP-1 cells as well
as QGP-1 xenografts [
122
]. Another CDK4/6 inhibitor, ribociclib (LEE01), decreased BON1
and QGP-1 cell viability in a time and dose dependent manner [
125
]. That study found
enhanced cell killing when ribociclib was combined with 5-fluorouracil or everolimus.
Cancers 2021,13, 5117 12 of 51
Table 4. Molecular profiling studies revealing INK4a/ARF and RB1 pathway alterations in pNETs.
Technique Reference Key Findings
Sequencing and
mutational analysis
[24] No INK4A/ARF mutation observed in 68 pNETs.
[11]
Inactivating mutations in the RB1 gene identified in 75% (3/4) small cell pNECs and
66.67% (2 of 3) large cell pNECs, however, absent in 11 well-differentiated
pNETs analyzed.
No CDKN2A mutations observed in 7 pNECs and 11 pNETs.
Methylation Specific
PCR (MSP)
[112]
In total, 91.7% of 12 gastrinomas and non-functioning pNETs demonstrated INK4a
homozygous gene deletions (41.7%) or 50CpG island hypermethylation. However, no
mutations were found by single-strand conformation polymorphism (SSCP) analyses.
[103]INK4a 50-CpG island hypermethylation in 52% of 44 gastrinomas, along with
homozygous gene deletions.
[126]
In total, 17% of 17 insulinomas exhibited INK4a gene alterations: homozygous deletion in
5.9% and promoter hypermethylation in 11.8%. No INK4a mutations were identified by
SSCP. IHC confirmed loss of the p16 protein expression in samples harboring
gene alterations.
[116]INK4a CpG island hypermethylation in 17% of 12 pNETs, more frequently in malignant
(29%) than in benign (0%) tumors.
PCR (MSP or gene
specific and LOH) [114]
INK4a and ARF CpG island hypermethylation in 9% (1 out of 11) pNETs each vs. 44% and
31%, respectively, in carcinoid NETs. Chromosome 9p loss identified in
18% (2 of 11) pNETs.
RT-PCR
[104]
Absent expression of INK4a,INK4b, and ARF in 28% (2/7), 57% (4/7), and 43% (3/7)
NF-pNETs. Loss of INK4b observed in 26% insulinomas and gastrinomas (N = 19),
however, INK4a and ARF found to be expressed.
[35]Overexpression of CDK4 and CDK6 in MEN1 NF-pNETs (N = 10) compared to VHL
(N = 9) and sporadic (N = 9) NF-pNETs and normal islets (N = 4).
Tissue microarray,
qPCR, and FISH [122]
IHC revealed high CDK4, cyclin D1 and phospho-RB1 levels in 58–68% of total pNETs
(N = 92) in contrast to negative staining in the normal pancreas.
qRT-PCR revealed marked upregulation of CDK4 in 19% of 26 pNETs, which were found
to have amplified CDK4 or CDK6 genes by qPCR and FISH.
IHC [11]
RB1 protein expression intact in well-differentiated pNETs, however, lost in 88.9% of
9 small cell
and 60% of large cell pNECs. Loss of p16 expression observed in pNECs with
intact RB1 suggest p16/Rb pathway is disrupted in virtually all pNECs.
Clinical trials of CDK4/6 inhibitors in GEPNETs have yielded unsatisfying results. In
a phase II (PALBONET) study, single agent palbociclib treatment failed to show beneficial
outcomes in patients with metastatic grade 1 and 2 pNETs [
127
]. Patients in this study were
not selected for molecular alterations and were heavily pretreated, with resistance linked
to high levels of CCNE1 and somatic RB1 inactivating mutations. Establishing CDK4/6
and RB1 status as predictive biomarkers prior to treatment with these pathway inhibitors
may be essential. Indeed, Keutgen et al., showed that non-functional pNETs associated
with MEN1 and VHL syndromes have highly upregulated CDK4/6, which provides some
rationale for selecting molecular targets for therapy based on pNET subtype [
35
]. In a pilot
study by Dasari et al., (presented at ENETS conference, 2018), treatment of 18 patients
having foregut NETs (55% of the cohort had pNETs) with ribociclib yielded no radiographic
responses, no reduction in Ki-67 or phosphorylated RB1 in tumors, but did slightly improve
the PFS of those patients. An ongoing, phase II trial of a more potent CDK4/6 inhibitor,
abemaciclib, is currently being conducted in patients with advanced and refractory well-
differentiated GEPNETs (NCT03891784). Most recently, combination of everolimus and
ribociclib was found to have insufficient clinical activity to warrant further investigation in
foregut well differentiated NETs [128].
While largely unremarkable, the clinical results with CDK4/6 inhibitors in pNET
patients have highlighted several important points. Nearly all the trials were conducted
Cancers 2021,13, 5117 13 of 51
as monotherapies. It is well established that
in vivo
inhibition of CDK4/6 alone using
monotherapy approaches has cytostatic antitumor activity that can lead to acquired drug re-
sistance through numerous mechanisms, including upregulation of CDK4, CDK6,
cyclin E,
CDK2 and/or activation of MEK [
129
]. This suggests that rational combination therapies
simultaneously targeting known mediators of resistance may have greater success. In that
regard, mechanistic studies providing deeper insight into factors controlling RB1 activity
are warranted. Recent studies of a novel oncoprotein, RABL6A, showed it suppresses RB1
function in pNETs and other tumors by promoting CDK4/6 activity [129131]. Mechanis-
tic studies suggest RABL6A regulates the CDK4/6-RB1 pathway via multiple effectors,
thereby providing novel options for combination therapies inhibitors that directly target
CDK4/6 [
132
]. RABL6A was discovered as a binding partner of p14
ARF
and subsequently
shown to promote pNET cell survival and proliferation by inhibiting the RB1 pathway and
activating Akt-mTOR oncogenic signaling [
92
,
130
]. Identification of new pNET targets,
such as RABL6A and/or its effectors, may pave the way for developing combination
therapies that will overcome poor pNET responses to CDK inhibitor monotherapy.
Genetically engineered mouse models have helped establish the functional role of the
RB1 pathway in pNET pathogenesis. Loss of one Rb1 allele caused moderate hyperplasia
of the islet of Langerhans but was insufficient to drive islet cell tumorigenesis in mice [
133
].
However, when monoallelic loss of the Rb1 gene is combined with mono- or bi-allelic loss
of Trp53, pNETs are formed [
134
]. Those results suggest that loss of RB1 and p53 tumor
suppressors cooperate to drive pNET pathogenesis. This theory is supported by the rapid
development of islet cell tumors in RIP-Tag2 mice and Glu2-Tag mice (Table 2), both of
which have silenced p53 and Rb1 by SV40 large T-antigen [57,70].
2.4. ATRX/DAXX
Mutations in ATRX (
α
-thalassemia/mental retardation syndrome X-linked) and
DAXX (death domain associated protein) have been frequently observed in pNETs
(Table 5) [
11
,
36
,
37
]. The ATRX protein is a component of heterochromatin bearing an
ATPase/helicase-like domain characteristic of the SNF2 (sucrose non-fermentable 2) family
of chromatin remodeling proteins [
135
,
136
]. Inactivating mutations in the ATRX gene
lead to an X-linked condition called ATRX syndrome manifested as intellectual disability,
α
-thalassemia, genital abnormalities, and facial malformation [
137
,
138
]. Although ATRX
is known to regulate gene expression by modifying chromatin, its exact function remains
unclear [139].
Table 5. Molecular profiling studies revealing ATRX/DAXX alterations in pNETs.
Technique Reference Key Findings
Exome sequencing and
mutational analysis
[24]
Somatic ATRX and DAXX inactivating-to-missense mutations in 25% and 17.6%
of 68 sporadic pNETs, respectively.
IHC showed complete loss of ATRX or DAXX in pNETs harboring the corresponding
gene mutations.
ATRX/DAXX mutations with or without MEN1 mutations correlated with improved
patient survival.
[23]Somatic mutations in ATRX (10%) and DAXX (22%) of 102 primary pNETs.
ATRX/DAXX mutations linked with longer telomere length and poor patient survival.
[40]DAXX and ATRX mutations in 28% and 11% of 65 pNETs, respectively. These mutations
were mutually exclusive.
[43] Somatic alterations in DAXX and ATRX observed in 40% and 25% of 80 patient pNETs.
IHC [11] ATRX or DAXX immunolabeling lost in 45% of 11 pNETs, but intact in all of 19 pNECs.
DAXX is a pro-apoptotic protein that promotes c-Jun NH2-terminal kinase (JNK)
pathway-induced apoptotic cell death by interacting with FAS and TGF-
β
(transforming
growth factor-
β
) in the cytoplasm [
136
,
140
]. DAXX is primarily nuclear and associates with
Cancers 2021,13, 5117 14 of 51
a tumor suppressor protein, promyelocytic leukemia (PML), at chromatin-bound PML-
nuclear bodies (PML-NBs) that are involved in transcriptional regulation, cell apoptosis,
and cell cycle [
135
,
136
]. In PML-NBs, DAXX has also been shown to interact with ATRX
and inhibit its transcriptional repression activity [
135
,
141
]. The ATRX/DAXX complex
orchestrates deposition of histone H3.3 and constitutes a novel chromatin remodeling
complex at the pericentromeric and telomeric heterochromatin [139,142].
Whole exome sequencing of non-familial pNETs revealed inactivating-to-missense
mutations in either ATRX or DAXX in up to 65% of tumors, predicting tumor suppressive
roles in pNET pathogenesis [
36
39
]. The ATRX and DAXX mutations were mutually
exclusive in those pNETs, in keeping with their functional involvement in the same path-
way [
24
,
40
]. Immunohistochemical staining confirmed complete loss of ATRX or DAXX
protein in the pNET samples harboring the corresponding gene mutations. Homozygous
ATRX/DAXX mutations were observed only in a small fraction of the pNETs, however,
the majority of tumors carrying heterozygous mutations are believed to lose the other
non-mutant allele through gene deletion or epigenetic silencing [24].
Considering the role of ATRX/DAXX in modulating telomeric chromatin, Heaphy et al.,
performed telomere-specific fluorescence in situ hybridization (FISH) in 41 pNETs con-
firmed to have ATRX/DAXX mutations [
143
]. The majority (61%) of those pNETs displayed
large, ultrabright telomere FISH signals, indicative of telomerase independent telomere
modulation called alternative lengthening of telomeres (ALT). This positive correlation
between ATRX/DAXX mutations and ALT in pNETs was similarly observed by others with
one study suggesting these are late events in MEN1-associated pNET development [
23
,
144
].
Key pathways dysregulated by ATRX or DAXX loss in pNETs remain poorly un-
derstood. Feng et al., showed DAXX can directly interact with menin to epigenetically
repress the expression of membrane metallo-endopeptidase (MME, also called CD10), a
zinc dependent metalloprotease required for pNET cell proliferation [
55
]. Earlier studies in
other cell types suggested DAXX negatively regulates p53, in apparent contradiction to its
pro-apoptotic and tumor suppressive activities. Specifically, DAXX can inhibit p53 activity
though direct interactions with p53 itself to block its transactivation of target genes or with
a HAUSP-MDM2 complex to promotes MDM2 stabilization and downregulation of p53
expression [
145
,
146
]. Mouse modeling revealed no significant regulation of p53 signaling
by DAXX
in vivo
but did solidify its role as a tumor suppressor since Daxx-deficient mice
displayed more radiation-induced carcinomas than controls [147].
More recently, the same group developed mice with conditional Pdx1-Cre driven Daxx
inactivation in the pancreas to better evaluate its function in pNETs (Table 2) [
148
]. Daxx
loss alone had no effect on pancreas development and function, which remained normal,
and surprisingly did not cooperate with Men1 loss to enhance pNET pathogenesis. This
suggested that, in mice, Daxx is not a strong endocrine tumor suppressor. On the other
hand, loss of Daxx altered the transcriptome in association with derepression of endogenous
retroviral elements (ERVs), which the authors found mirrors a similar dysregulation of
genes near ERVs in patient pNETs with mutated DAXX. This creates a permissive chromatin
state that cooperates with tissue stress, caused by inflammation or Men1 loss for instance, to
impair pancreas recovery following stress and potentially promote tumorigenesis. Future
studies of ERV adjacent genes in human pNETs may provide meaningful insights into
important mechanisms mediating effects of DAXX loss.
Human tumor studies have highlighted the pathological and prognostic significance of
ATRX/DAXX mutations. pNETs having ATRX/DAXX or MEN1 sporadic mutations were
found to have an
α
-cell gene signature including high ARX (
α
cell-specific transcription
factor) and low PDX (
β
cell-specific transcription factor) expression, which predicted worse
recurrence-free survival of the patients [
149
]. Epigenomic and transcriptomic profiling
was used to classify pNETs based on their ARX or PDX gene expression and found that
ARX-positive pNETs with the ALT phenotype, characteristic of ATRX/DAXX mutation,
have the shortest recurrence-free survival [
150
]. ATRX/DAXX mutation and the ALT phe-
notype also correlated with increased chromosomal instability, advanced tumor stage and
Cancers 2021,13, 5117 15 of 51
metastasis, and reduced relapse-free survival [
151
]. In contrast to those studies associating
ATRX/DAXX mutations with poor prognosis, Jiao et al., found that pNET patients having
mutations in ATRX/DAXX (regardless of MEN1 status) had better overall survival relative
to others with the wild type genes [
24
]. Additional survival studies with larger and more
diverse patient pools are warranted to clarify this discrepancy.
2.5. p53 Pathway
The TP53 (Trp53 in mice) gene, which encodes the p53 tumor suppressor, is rarely
mutated in pNETs. However, evidence suggests loss of p53 activity via alterations in its
regulators is critical for pNET genesis. p53 is a transcription factor involved in transac-
tivation of genes that mediate DNA damage repair, cell growth arrest, cell senescence
and apoptosis, among other antitumor processes. TP53 gene alterations that limit p53
protein expression and activity contribute to the development and progression of a high
proportion of human cancers [
152
,
153
]. Several key proteins are known to tightly regulate
p53 expression and activity. Murine double minute 2 (MDM2) is an E3 ubiquitin ligase that
catalyzes p53 ubiquitination leading to its proteasomal degradation, whereas its structural
homologue, MDM4, inhibits p53 transcriptional activity via direct interaction [154]. Wild-
type p53-induced phosphatase 1 (WIP1) is a serine threonine phosphatase that antagonizes
p53. WIP1 dephosphorylates and stabilizes MDM2 and inhibits upstream activators of
p53 such as ataxia telangiectasia mutated kinase (ATM), checkpoint kinase 1 (CHK1) and
CHK2 [155,156].
Hu et al., showed that a high percentage of well-differentiated sporadic pNETs harbor
amplifications of MDM2 (22%), MDM4 (30%), and WIP1 (51%) genes, consistent with
higher expression of their corresponding mRNAs and proteins (Table 6) [
157
]. Seventy
percent of the pNETs in the study displayed amplifications of one or more of the above p53-
inactivating genes. Moreover, these genetic alterations positively correlated with increased
pNET progression to metastatic disease [
157
]. PHLDA3 is a p53-transcriptional target and
repressor of Akt activation found to have a tumor suppressive role in pNETs [
158
]. The
same group observed LOH at the PHLDA3 locus and aberrant promoter hypermethylation
at the gene in over 70% pNETs, which correlated with poor prognosis in patients. Loss
of PHLDA3 overlapped with MEN1 loss in a high proportion of pNETs in their study,
suggesting they potentially regulate two different pathways that cooperate to suppress
pNET development. Together, these studies demonstrate that p53 pathway inactivation,
rather than TP53 genetic mutation, is frequent in pNETs.
Table 6. Molecular profiling studies revealing p53 pathway alterations in pNETs.
Technique Reference Key Findings
Sequencing and
mutational analysis
[11]Inactivating TP53 mutations found in 4 of 7 pNECs, however, in none of
11 well-differentiated pNETs.
[24]TP53 gene mutations identified in 3% of 68 pNETs.
PCR and IHC [157]
Amplification of the MDM2 gene in 22% (38 of 169), MDM4 in 30% (45 of 150), and WIP1
in 76% (34 of 45) well differential pNETs, which correlated with corresponding changes in
mRNA and protein expression.
IHC [11]11 well differentiated pNETs exhibited normal p53 labeling, however, abnormal labeling
observed in 9 (100%) small cell pNECs and 9 of 10 (90%) large cell pNECs.
ATM is a tumor suppressor and activator of p53, whose low expression in pNETs has
been associated with higher incidence of metastasis and poor prognosis [
155
]. Although
TP53 genetic mutation is reported in only 1–3% of pNETs [
24
,
159
], it is a regular event
in poorly differentiated endocrine carcinomas of the gastrointestinal systems including
pNECs [
11
]. Several groups report inactivating mutations in the TP53 gene in over 90% of
pNECs [
11
] or GEPNECs in general [
160
,
161
]. G3 pNETs and pNECs are both aggressive
pNENs with an elevated Ki-67 index. In that regard, examining their TP53 and RB1
Cancers 2021,13, 5117 16 of 51
gene status could serve as an important distinguishing factor in their clinical diagnoses
and prognosis [12].
The importance of reduced p53 tumor suppressive activity in driving pNET patho-
genesis is supported by several genetically engineered pNET animal models (see
Table 2).
Combined inactivation of p53 and RB1 in pancreatic islet cells via SV40 large T antigen
expression provided early evidence that their loss drives aggressive insulinoma devel-
opment in mice [
66
,
79
,
144
]. In a
β
-cell polyoma middle T antigen (PyMT) transgenic
model, Trp53 deletion yielded primary pNETs in ~20% of mice while concurrent deletion of
Ink4a/Arf doubled tumor incidence relative to loss of either tumor suppressor alone [
73
].
Since PyMT stimulates multiple oncogenic pathways including mitogen-activated protein
kinase (MAPK), PI3K signaling and the Hippo pathway [
162
,
163
], and SV40 large T antigen
inhibits both p53 and Rb, none of the above studies addressed the individual role of p53
loss in driving pNET formation. That was examined in a recent study, in which mice with
pancreas-specific mutant p53 expression (Pdx1-Cre; Trp53
R172H
) failed to develop pNETs,
although Trp53 mutation greatly accelerated pNET progression to G3 pNETs when com-
bined with Rb1 deletion [
75
]. Likewise, Xu et al., found Trp53 loss alone yielded no pNETs
whereas its deletion with Rb1 in the pancreas resulted in aggressive pNETs [
76
]. These
findings show p53 inactivation alone is insufficient for pNET development; however, it
cooperates with the loss of other tumor suppressors, particularly the p16
INK4A
/p19
ARF
-RB1
pathway, in promoting the disease.
2.6. VHL and Growth Factor Signaling
Germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene located
on chromosome 3p25.5 cause an autosomal dominant tumor syndrome called VHL dis-
ease [
164
]. Patients with VHL develop diverse neoplasms that include hemangioblastoma
of the central nervous system, retinal angiomas, renal cell carcinomas, and pheochromocy-
tomas [
165
]. The common VHL-associated pancreatic lesions are cysts and cystadenomas,
which are benign in nature and found in 35–75% individuals [
165
,
166
]. About 12–17%
of VHL patients also develop pNETs, which are almost invariably non-functional and
have metastatic potential correlating to the primary tumor size [
167
169
]. Lubensky et al.,
observed allelic deletion (LOH) of the wild type VHL gene in all the VHL pNETs in their
study, bolstering the importance of this tumor suppressor in pNET pathogenesis [
165
,
170
].
VHL encodes a 232 amino acid protein, pVHL, which interacts with hypoxia inducible
factor 1-alpha (HIF1-
α
) under normoxic conditions, targeting it for polyubiquitination
and proteasomal degradation [
171
]. During hypoxia, HIF1-
α
translocates to the nucleus
and conjugates with HIF-1
β
to form the heterodimeric HIF-1 transcription factor, which
transactivates genes involved in cell proliferation, angiogenesis (e.g., vascular endothelial
growth factor, VEGF), erythropoiesis, glucose homeostasis, and metastasis [
172
174
]. In
VHL disease, the absence of pVHL causes constitutive stabilization and activation of
HIF-1
α
irrespective of oxygen availability, leading to upregulation of HIF-1 target genes,
essentially driving multi-organ tumorigenesis [
175
]. Of note, strong expression of HIF-1
α
and its transcriptional targets, carbonic anhydrase 9 and VEGF, was observed in early
pNET stages suggesting these could be critical to pNET genesis in VHL patients [
175
].
Spiesky et al., reported that upregulation of several angiogenic genes, including VEGF
and other HIF1-
α
targets, is required for cell cycle progression and cancer metastasis in
VHL pNETs [
176
]. Exaggerated VEGF signaling caused by pVHL loss is consistent with
the hyper-angiogenic phenotype of cancers arising in VHL individuals. Belzutifan (aka
MK-6482), a HIF inhibitor, has recently been approved by FDA for use in adult VHL
patients requiring treatment for associated pNETs, renal cell carcinoma, or central nervous
system hemangioblastomas, however, not requiring immediate surgery (NCT03401788).
Moreover, the efficacy and safety of Belzutifan monotherapy in patients with advanced
pNETs or pheochromocytoma/paraganglioma are being studied in a new phase II clinical
trial (NCT04924075).
Cancers 2021,13, 5117 17 of 51
Although the VHL gene per se is unaltered in sporadic pNETs, the genes and pathways
downstream of pVHL or HIF1
α
play important roles in pNET pathogenesis. Immunos-
taining studies show positive expression of the VEGF (aka VEGF-A) protein in up to 85%
of pNETs although intestinal NETs reportedly display higher VEGF expression per cell
than pNETs (Table 7) [
177
179
]. Even when positive, the expression pattern of VEGF in
pNETs is sparse, with only a small percentage of scattered tumor cells expressing the
protein [
178
,
180
]. VEGF-C, another member of the VEGF protein family, is moderately to
highly expressed in the majority of pNETs, with more than half of the cells in tumors stain-
ing for the protein [
180
]. Strikingly, VEGF-C expression was higher in pNET metastases
than the primary tumors [
180
]. High expression of VEGF receptor-2 (VEGFR-2), the recep-
tor for VEGF and VEGF-C, was observed in pNET endothelial cells suggesting VEGFR-2
mediates the angiogenic role of these specific VEGF proteins in pNETs [
180
]. Apart from
growth signaling in endothelial cells, the VEGF family proteins can transduce autocrine
signals required for proliferation, survival, and cell migration by binding to specific VEGFR
sub-types expressed by the tumor cells [
181
]. In this regard, immunostaining studies have
shown variable expression of VEGFR-2 and VEGFR-3 (which binds VEGF-C and -D) in
pNET cells, implying autocrine signaling via these receptor tyrosine kinases could help
promote pNET cell survival, proliferation, and metastasis [180].
Findings from pNET xenograft and genetic mouse models support the oncogenic role
of VEGF in pNET development and progression. One group demonstrated that administra-
tion of bevacizumab, a VEGF monoclonal antibody, reduces endothelial cell proliferation
and tubulogenesis
in vitro
and suppresses angiogenesis and growth of BON-1 cell derived
xenografts in mice [
177
]. VEGF and its receptors are highly expressed throughout islet cell
tumorigenesis in RIP-Tag2 mice, however, their levels are not elevated compared to normal
islets [
182
]. Thus, it was suggested that VEGFR signaling was selectively increased in the
hyper-proliferative, angiogenic islets, potentially via cross talk with other angiogenic HIF-1
targets [
182
]. Indeed,
β
-cell specific knockout of VEGF or blockade of VEGF receptor 2
(VEGFR2) disrupted initiation and progression of angiogenesis as well as tumor growth
demonstrating the critical role of VEGF in pNET genesis [
183
,
184
]. Inhibition of VEGF
signaling in RIP-Tag2 mice, however, is associated with rapid development of ‘adaptive
resistance’ mediated by several mechanisms. One of these mechanisms is upregulation
of other pro-angiogenic HIF-1 targets such as fibroblast growth factors (FGFs), as evi-
denced by sustained inhibition of tumor growth and angiogenesis by concomitant FGF
and VEGF targeting [183].
Table 7. Molecular profiling studies on growth factor signaling in pNETs.
Technique Reference Key Findings
FISH [185]EGFR copy number found to be elevated in 38% of 44 pNET cases.
qRT-PCR [186]
Positive VEGF expression in 5 of 8 pNETs and EGFR in 4 pNETs of which 2 had overexpressed EGFR
compared to the normal pancreatic tissue.
IHC
[178] Positive expression of VEGF in 16 (11 mild, 3 moderate, and 2 strong) out of 20 pNETs.
[187]
Of 38 malignant pNETs, 100% expressed PDGFRαtumor cells, 57% in stromal cells; 74% expressed
PDGFRβin tumor cells, 97% in stromal cells; 92% of tumors expressed c-kit and 55%
expressed EGFR.
[180]
Positive VEGF-A expression in all 19 primary well-differentiated pNETs and 7 liver metastases. Mild
to moderate VEGF-C immunostaining in the majority of primary pNETs with significantly increased
expression in liver metastases. High immunoreactivity for VEGFR2 observed in all 8 primary pNETs
and 3 liver metastases examined.
[179]Positive expression of VEGF in pNETs 73% (33/45) pNETs which correlated negatively with WHO
disease stage. In total, 91% (41/45) pNETs showed high HIF-1αstaining.
[188]
Positive expression of EGFR or p-EGFR in 25–50% of 48 primary pNETs or their metastases. Higher
percentage of carcinoid NETs were found to be EGFR- or p-EGFR positive. Activated p-EGFR in
primary NETs correlated with poor prognosis.
Cancers 2021,13, 5117 18 of 51
Table 7. Cont.
Technique Reference Key Findings
[189]
Positive PDGFRβexpression observed in 18 of 21 (86%) primary tumors and all of 19 pNET
metastases. PDGFRβshown to be more frequently expressed in primary pNETs and metastases as
compared to normal endocrine pancreas.
[177] In total, 80% of 15 pNETs showed mild to moderate VEGF staining.
[185]
High IHC staining (score 3) of VEGFR1, TGFBR1, PDGFRA, SSTR5, SSTR2A, and IGF1R in 80%, 69%
65%, 55%, 55%, and 47% of 44 pNETs, respectively.
The prognostic significance of VEGF expression levels and angiogenic status of pNETs
seems to be context dependent. Zhang et al., showed that expression of VEGF correlates
with micro-vessel density (MVD), a morphological gold standard of neo-vascularization,
as well as increased metastasis and reduced PFS in GEPNETs [
177
]. This is consistent
with other studies showing VEGF is a marker of progression and poor outcomes in GEP-
NECs [
190
]. However, a pNET-focused study by Couvelard et al., showed that although
pNETs maintain high vasculature throughout their progression, they are associated with
gradual decrease in VEGF expression and MVD as they progress [
179
]. In their immuno-
histochemical analysis, the low-grade benign pNETs unexpectedly displayed higher VEGF
expression and MVD than the high-grade advanced pNETs. Moreover, low tumor MVD
correlated with reduced survival in pNET patients, suggesting poor angiogenesis could
be a marker of disease progression and poor prognosis, particularly in pNETs [
179
]. It
is noteworthy that Zhang et al., analyzed low-grade, well-differentiated GEPNETs while
Pavel et al., studied GEPNECs. By comparison, Couvelard et al., examined pNETs of
varying histologic grade [
185
,
187
,
191
]. Therefore, observed discrepancies in the prognostic
role of VEGF and angiogenesis could be greatly affected by differences in pNET types
being analyzed.
Besides VEGF, other growth factors and their receptor tyrosine kinases have also been
implicated in pNET development. Receptors of platelet derived growth factor (PDGF),
namely PDGFR-
α
and -
β
, which transduce signals promoting angiogenesis and tumor cell
proliferation, are elevated in VHL and sporadic pNET within both the tumor cells and the
surrounding stroma [
48
,
192
,
193
]. In RIP-Tag2 mice, PDGFRs are exclusively expressed
by the perivascular cells of the tumor vasculature and their pharmacological inhibition
disrupts late-stage tumor growth [
194
]. However, PDGFR inhibition alone was less effective
in preventing early stage angiogenic switch and tumorigenesis in RIP-Tag2 mice [
194
].
To that end, combined inhibition of PDGFRs and VEGFRs in RIP-Tag2 mice was more
efficacious as it suppressed both angiogenic islet formation at early stages and tumor
growth at later stages [194].
Consistent with the above findings, sunitinib (SU11248) maleate, a multitargeted
receptor tyrosine kinase (RTK) inhibitor blocking VEGFRs, PDGFRs and stem-cell factor
receptor (aka c-kit, expressed by more than 90% pNETs), displayed remarkable anti-tumor
activity in RIP-Tag2 mice by disrupting angiogenesis and pericyte coverage of tumor
vasculature [
187
,
195
]. The combination of sunitinib with a ‘chemo-switch’ regimen (i.e.,
initial phase of maximum tolerated dose followed by low maintenance dose) of cyclophos-
phamide induced remarkable tumor regression (97%) and improved median survival by
>24 weeks in RIP-Tag2 mice [
196
]. Following successful phase I and II trials of sunitinib
in pNET patients, a randomized, double-blind, placebo-controlled phase III trial was con-
ducted in patients with advanced, well-differentiated pNETs [
195
]. Patients treated with
a daily dose of 37.5 mg sunitinib had improved median PFS (11.4 vs. 5.5 months in the
placebo group) and objective response rate (10% vs. 0% in the placebo group). Furthermore,
a 10% death rate was observed in the sunitinib-treated patient group during the trial period
compared to 25% in the placebo group.
Pazopanib is another multitargeted RTK inhibitor with activity against VEGFRs,
PDGFRs, c-kit, and FGFRs. The clinical utility of this drug in patients with pNETs or
Cancers 2021,13, 5117 19 of 51
metastatic GEPNETs has been tested in several phase II clinical trials, showing an overall
response rate of 20% [
197
]. Surufatinib is a novel multitargeted RTK inhibitor that has
recently been approved by FDA under ‘Fast Track Designation’ for the treatment of patients
with advanced and progressive pNETs and non-pancreatic NETs (referred to as extra-
pNETs/epNETs in the clinical trial) [
192
]. Surufatinib inhibits VEGFR1, VEGFR2, VEGFR3,
FGFR1, and CSF1R (colony stimulating factor 1 receptor) and as such, disrupts tumor
angiogenesis and promotes immune invasion. Two independent phase III clinical trials,
SANET-p (NCT02589821) and SANET-ep, examined the effect of surufatinib in advanced
pNET (n = 172) and extra-pNETs, respectively [
193
,
198
]. In these studies, surufatinib was
found to significantly improve the median PFS of both pNET (10.9 vs. 3.7 months in
the placebo group) and extra-pNET patients (9.2 vs. 3.8 months in the placebo group).
Additional RTK inhibitors including Lenvatinib, axitinib, and sorafenib have also shown
activity in phase II clinical trials [191,199].
In RIP-Tag2 mice treated with drugs targeting VEGF or VEFGR2, tumor cells rapidly
develop ‘evasive resistance’ marked by increased invasiveness with peri-pancreatic lymph
node and liver metastases [
200
]. This phenomenon is accompanied by upregulation of
HIF-1α
and its transcriptional targets due to vascular pruning, leading to intra-tumoral
hypoxia plus upregulation and activation of hepatocyte growth factor (HGF) receptor,
c-Met [
201
]. Combined inhibition of c-Met and VEGF using their selective inhibitors or a
multi-targeted RTK inhibitor, such as cabozantinib, reduced pNET invasion and metastases.
These data suggest exaggerated HGF-c-Met signaling drives malignant transformation
of pNETs in RIP-Tag2 mice following VEGF-targeted treatments [
201
,
202
]. Activation
of HGF-c-Met promotes tumor survival, proliferation, invasion, and metastasis in many
cancers [
202
]. Krampitz et al., revealed c-Met is highly expressed in patient pNETs and
is dependent on the paracrine action of its ligand, HGF, which is expressed only in the
peripheral, normal tissues [
203
]. Because human c-Met cannot be activated by mouse
HGF, growth of pNET patient derived xenografts (PDXs) in NSG mice required continuous
administration of an exogenous c-Met agonist. This highlighted a critical role of c-Met
stimulation in pNET cell engraftment and proliferation. The prognostic significance of
c-Met expression was demonstrated by tissue microarray analysis showing that high
expression of c-Met in pNETs correlates with poor patient survival [203].
Cabozantinib is a potent non-selective RTK inhibitor with activity against VEGFRs 1, 2,
3, and c-Met, along with other RTKs such as fms-like tyrosine 3 (FLT-3), RET, and AXL [
197
].
Because cabozantinib reduces pNET burden, invasion, and metastasis in RIP-Tag2 mice and
promotes their survival [
201
], a two-cohort phase II clinical trial of this drug was carried out
in patients with progressive, well-differentiated, grade 1–2 carcinoid NETs or pNETs [
204
].
In total, 3 of 20 pNET patients achieved partial response and the median survival of this
cohort was 21.8 months. A randomized phase III clinical trial of cabozantinib in advanced
or metastatic NETs is ongoing [197].
Exaggerated activity of epidermal growth factor receptor (EGFR) drives various can-
cers. pNETs and other GEPNETs generally lack activating mutations in the EGFR kinase
domain [
205
]. Immunohistochemistry and qRT-PCR analyses revealed positive expression
of EGFR or activated EGFR (p-EGFR) in 25–50% of primary pNETs or their metastases,
which correlated with reduced survival [
186
188
]. EGFR inhibition by erlotinib alone atten-
uates aggressive pNET development and progression in RIP-Tag2 mice, eliciting a modest
survival benefit [
95
]. When erlotinib is combined the mTOR inhibitor, rapamycin, the bene-
fit is more profound. The adaptive resistance observed with rapamycin monotherapy is
overcome by erlotinib and the survival of RIP-Tag2 mice is increased almost two-fold [
95
].
Downstream to RTK receptor signaling is the activation of multiple oncogenic path-
ways, including mTOR and MAPK signaling [
94
]. Insulin like growth factor-I (IGF-I)
is another growth factor implicated in pNETs. von Wichert et al., revealed that BON-1
pNET cells release insulin-like growth factor-I (IGF-I) and express the IGF-I receptor [
206
].
Autocrine IGF-I signaling in these cells was found to promote chromogranin A release and
mTOR and MAPK-dependent proliferation.
Cancers 2021,13, 5117 20 of 51
2.7. NF1 and RAS-RAF-MEK-ERK Pathway
Neurofibromatosis type I (NF1) is a genetic tumor predisposition syndrome with
an incidence of 1 in 3000 individuals globally. All patients with NF1 almost invariably
develop benign cutaneous neurofibromas [
207
]. Additionally, one-third of patients also
develop enlarged benign plexiform neurofibromas, a subset of which progress into lethal
nerve sarcomas called malignant peripheral nerve sheath tumors (MPNSTs) [
207
,
208
]. Less
frequent tumor types in NF1 patients are juvenile myelomonocytic leukemia, pheochromo-
cytoma (NET of the adrenal medulla), gastrointestinal stromal tumors, and rhabdomyosar-
coma [
207
]. Up to 10% of NF1 individuals have also been found to develop pNETs, almost
exclusively as periampullary duodenal somatostatinomas or in the form of pancreatic
somatostatinomas, gastrinomas, insulinomas, or NF-pNETs in rare cases [6].
NF1 is an autosomal dominant disease caused by inheritance of one mutated copy
of the NF1 gene located on chromosome 17q11.2. NF1 encodes a RAS GTPase-activating
protein called neurofibromin [
209
211
], and tumorigenesis in NF1 individuals involves
biallelic NF1 inactivation [207]. Since neurofibromin negatively regulates RAS activity, its
loss constitutively activates RAS GTPases, HRAS, NRAS, and KRAS, leading to aberrant
activation of multiple downstream effector pathways including the oncogenic RAF-MEK-
ERK (aka RAS-MAPK) signaling cascade.
Genetic alterations in signal transducers constituting or directly regulating the RAS-
MAPK pathway are reportedly rare in pNETs. KRAS, one of the most commonly mutated
genes in PDACs, was found to be unaltered in pNETs (Table 8) [
24
]. Likewise, related
oncogenes HRAS, NRAS, and BRAF were mutated in less than 1% of pNETs [
155
]. Zakka
et al., investigated specific gene mutations in circulating tumor DNA (ctDNA) present in
liquid biopsies by next generation sequencing [
212
]. In their analysis of 165 pNET patients,
KRAS mutations were seen in 63 (38%) patients and NF1 mutations in 46 (28%) patients,
which is remarkably higher than was previously reported from sequencing of primary
tumor DNA. Comparative analysis of ctDNA vs. matched tissue DNA had exhibited
variable concordance in the past [
213
,
214
]. Thus, Zakka et al., admitted the importance of
validating their findings by studying the DNA of matched patient tumor tissues.
Table 8. Molecular profiling studies on Ras-MAPK signaling in pNETs.
Technique Reference Key Findings
Sequencing and
mutational analysis
[24]KRAS mutation observed in none of the 68 pNETs studied.
[155]HRAS,NRAS, or BRAF mutations in less than 1% pNETs.
ctDNA sequencing [212]
ctDNA NGS of 280 NET/NEC patient plasma samples revealed KRAS mutations in
22% (n = 61) and NF1 mutations in 7% (n = 19) samples.
Mutational analysis [185] No pNETs (n = 35) showed KRAS exon 2 mutations.
Regardless of mutational status, intact RAS-MAPK signaling is critical to pNET cell
survival and growth. Treatment with inhibitors of RAF [
215
,
216
] or MEK [
217
,
218
] induced
potent anti-tumoral effects in pNET cells. Moreover, combined inhibition of RAS-MAPK
and PI3K-mTOR pathways synergized in reducing pNET cell viability and proliferation
compared to targeting either pathway alone, suggesting cross talk between these pathways
promotes pNET pathogenesis [
219
221
]. Interestingly, RAF activation with ZM336372 has
also been shown to attenuate pNET cell growth [
103
,
222
,
223
]. In BON-1 pNET cells and
other NET cell lines, ZM336372 promoted phosphorylation of RAF and its downstream
effectors, MEK and ERK, but also caused GSK-3
β
inactivation and upregulation of the
CDK inhibitors, p21 and p18 [
224
]. As such, ZM336372 treatment resulted in reduced cell
proliferation and downregulation of NET markers, CgA, and achaete-scute complex-like 1
(ASCL1). Based on these findings, the anti-tumoral effects of RAF activation are believed
to be independent of the RAF-MAPK pathway [225].
Cancers 2021,13, 5117 21 of 51
2.8. Somatostatin Receptor Signaling
Somatostatin receptor (SSTR) signaling has anti-secretory and anti-proliferative roles
in NETs. Somatostatin (SST) is an endogenous cyclic peptide hormone concentrated in the
central nervous system, pancreas, and GI tract [
226
,
227
]. In the pancreas, SST is secreted by
the islet
δ
cells and inhibits the release of insulin (from
β
cells), glucagon (from
α
cells), and
pancreatic amylase (from exocrine cells). In the GI tract, SST inhibits the release of several
enzymes and hormones including serotonin, gastric acid, gastrin, cholecystokinin, vasoac-
tive intestinal peptide, and secretin. Moreover, SST has demonstrated anti-proliferative
functions in tumors arising from the pancreas and the GI tract [
219
,
226
228
]. This antitumor
activity is key to the development of several NET therapies.
There are two active biological forms of SST, one with 14 amino acids (SST-14) and
the other 28 (SST-28), both produced by the enzymatic cleavage of the precursor protein
prosomatostatin [
226
]. SST-14 is more commonly produced whereas SST-28 is more potent,
although their functions greatly overlap. Five different SST receptors, SSTR1-5, bind these
SSTs. SST-14 binds with higher affinity to SSTRs 1-4 while SST-28 is more selective for
SSTR5 [
220
]. SSTRs are coupled to the Gi-protein, as such their anti-secretory action is
primarily mediated by the inhibition of adenylate cyclase leading to decreased cAMP
production [
220
,
226
]. SSTR2 is widely considered the most important mediator of SST
antiproliferative functions, which include induction of cell cycle arrest and apoptosis as
well as inhibition of tumor angiogenesis and growth factor (e.g., VEGF and IGF-1) expres-
sion [
226
]. Mechanistically, SSTRs bind and activate several protein tyrosine phosphatases
(PTPs), small heterodimer partner 1 (SHP1), SHP2 and PTP
η
, via cytosolic src
homology 2
(SH2) domains in the PTPs. Activation of PTPs causes dephosphorylation of tyrosine
kinase receptors and their substrates and subsequent inhibition of important oncogenic
pathways, such as Ras-MAPK and PI3K-Akt-mTOR signaling (Figure 1). Furthermore,
SSTR2 activation can cause tumor cell arrest by upregulating the cyclin dependent kinase
inhibitor, p27, by a mechanism involving SHP-1 activation [
221
,
222
]. SSTR2 also induces
tumor cell apoptosis by several mechanisms including upregulation of death receptor 4 and
tumor necrosis receptor 1, and downregulation of the anti-apoptotic protein, Bcl-2 [223].
SSTRs, mainly 2 and 5, are abundantly expressed in up to 90% of GEPNETs
(Table 9) [
97
,
196
,
229
,
230
], including increased SSTR2 and SSTR5 levels in the vast ma-
jority of pNETs [
88
,
185
]. This SSTR upregulation in NET cells facilitates SSTR targeting
by SST analogues (SSAs) to limit tumor cell hormone secretion and proliferation while
additionally enabling use of radiolabeled SSAs for NET imaging and therapy [15].
In vitro
mechanistic studies have associated SST-SSTR signaling with other estab-
lished pNET pathways. For instance, tumor suppressive TGF-
β
signaling induces SST
expression in BON-1 cells, which promotes the SST-SSTR2 anti-proliferative autocrine
loop [
231
]. Intriguingly, inhibition of oncogenic HDACs in pNET cells that express low
endogenous SSTR2 levels (e.g., BON-1) functionally upregulates SSTR2 at the cell surface
through increased transcription and translation of the receptor, potentially via Notch1
activation [
232
234
]. These clinically relevant findings may help establish new strategies
to improve SSTR2-based imaging and therapy in pNETs. Notably, SSTR2 expression has
prognostic significance in GEPNETs. Nodal and hepatic metastases were found to exhibit
significantly lower SSTR2 expression compared to the primary pNETs [
235
]. Conversely,
higher SSTR2 expression is a predictor of better patient overall survival and correlates with
longer PFS following SSA therapy [229,236238].
Table 9. Molecular profiling studies on SSTR signaling in pNETs.
Technique Reference Key Findings
RT PCR and IHC [230]
mRNA amplification of SSTR1 in 90.1%, SSTR2 in 84.8%, SSTR3 in 78.8%, SSTR4 in 24.2%
and SSTR5 in 42.4% of pNET cases.
Positive SSTR2 immunoreactivity in 15 of 22 tumors (68.2%),
SSTR3 in 8 of 22 (36.4%), and SSTR5 in 14 of 22 (63.6%).
Microarray and IHC [88] SSTR2 expression is significantly upregulated in NF-pNETs compared to insulinomas.
Cancers 2021,13, 5117 22 of 51
Table 9. Cont.
Technique Reference Key Findings
IHC
[239] Mild to strong immunoreactivity for SSTR2A observed in 15 out of 16 (94%) pNETs.
[238]Positive (IHC score 1 to 4) SSTR2A immunostaining in 63% of 79 pNETs. Negative
staining correlated with poor outcomes.
[185] Positive SSTR2A and SSTR5 staining in 68% and 58% of 44 pNETs, respectively.
[240]Immunoreactivity for SSTR1 in 40%, SSTR2A in 90%, SSTR2B in 39%, SSTR3 in 51% and
SSTR5 in 76% of 71 NETS of the GI tract and lungs.
[241]Positive immunostaining of SSTR1 and SSTR2 in 100% of 11 G1 and G2 NETs of the GI
tract and lungs.
[229]Positive SSTR2A and SSTR5 staining in 86% and 35%, respectively, of 99 pNETs.
Positive SSTR2A expression correlated with better overall survival.
Since somatostatin has a very short half-life of 3 min [
242
], synthetic SSAs
(e.g., octreotide and lanreotide) have been developed for clinical use in the first line
treatment of unresectable, metastatic pNETs [
243
,
244
]. SSAs are effective in controlling
the hormonal secretion and growth of functional pNETs and small bowel (SB)/carcinoid
NETs, thereby alleviating the clinical symptoms precipitated by tumor-related hormone
hypersecretion. In the PROMID (placebo-controlled, prospective, randomized study in
patients with metastatic neuroendocrine midgut tumors) trial, octreotide significantly
prolonged the PFS from 6.0 months in the placebo group to 14.3 months, validating the
clinical benefit of SSTR targeting [
243
,
244
]. The subsequent CLARINET (controlled study
of lanreotide antiproliferative response in GEPNETs including those in the pancreas) trial
showed that lanreotide significantly prolongs the PFS in patients with advanced, G1/G2 dif-
ferentiated, non-functioning, SSTR-positive NETs (PFS at 24 months 65.1% in the lanreotide
group vs. 33%
in the placebo group) [
245
]. Furthermore, the combination of everolimus and
octreotide has been shown to improve the median PFS (from 9.7 to
16.7 months
) and clinical
benefit rates compared to monotherapy with everolimus or octreotide alone [
110
,
246
,
247
].
A new broader spectrum SSA, pasireotide, binds SSTR1, 2, 3, and 5 [
227
]. Pasireotide
possesses more potent anti-proliferative activity compared to octreotide in cultured cells
and in mice [
248
250
]. When used as a first line therapy in patients with advanced NETs,
pasireotide displayed long-lasting anti-tumor control efficacy (PFS 11 months) [
251
] and
provided an improved tumor control rate at 6 months compared to octreotide (though
not statistically significant) [
252
]. The use of pasireotide is, however, limited by higher
incidence of hyperglycemia in patients compared to octreotide. The COOPERATE trial
showed that the combination of pasireotide with everolimus in patients with progressive
G1 through G2 pNETs provides higher response rates compared to everolimus alone;
however, no significant differences in overall survival and PFS were observed between the
two experimental cohorts [253].
Beyond cold (non-radiolabeled) SSAs, peptide receptor radionuclide therapy (PRRT)
using radioisotope-labeled (“hot”) SSAs (e.g., DOTA peptides-DOTATATE and DOTA-
TOC) has emerged as a highly effective strategy to image and treat metastatic, well-
differentiated G1 and G2 GEPNETs [
227
]. Administration of
111
In-pentetreotide or
68
Ga-
DOTATATE/DOTATOC has facilitated the highly sensitive single photon emission com-
puted tomography (SPECT) or positron emission tomography (PET) imaging of GEP-
NETs [
246
,
254
,
255
]. The first prospective randomized study treating patients with progres-
sive metastatic midgut NETs by PRRT, NETTER-1, reported better PFS (20-month PFS at
65.2% vs. 10.8%) and improved response rate (18% vs. 3%) with
177
Lu-DOTATATE (admin-
istered with or without octreotide LAR) compared to high-dose octreotide alone [
247
]. How-
ever, pNET patients were not included in this study. Several other studies have observed
significant tumor control rates in pNET patients treated with PRRT [
15
].
Zandee et al.,
reported that
177
Lu-DOTATATE treatment in 34 patients with metastatic, functional G1-G2
Cancers 2021,13, 5117 23 of 51
pNET patients resulted in partial or complete response in 59% of patients, disease control
in 78% and reduction of symptoms in more than 80% [
17
]. A retrospective study assessing
the efficacy of PRRT (using
68
Ga-,
111
In-, or
99m
Tc-based SSAs) in
149 GEPNEN
G3 patients
(including 89 with primary pNETs) demonstrated promising response rates (complete
or partial response in ~42% patients), PFS (14 months), and overall survival
(29 months)
in patients, with comparatively better outcomes noticed in those having progressive dis-
ease [
256
]. Considering PRRT can cause adverse effects including nephrotoxicity and bone
marrow suppression, such therapy requires careful monitoring of patients during and
after treatment.
2.9. Miscellaneous Genes and Pathways
2.9.1. Myc
Myc (or cellular Myc, c-Myc) is a potent oncogene known to drive various cancers.
Functionally, Myc is a transcription factor that regulates the expression of numerous
genes involved in cell proliferation, cell cycle progression, differentiation, angiogenesis,
metabolism and apoptosis [
257
259
]. Although the MYC gene is rarely altered in patient
pNETs, several studies highlight the role of Myc protein in pNET development. Patient
tumor analyses show that almost all pNETs (80–100%) exhibit strong immunoreactivity for
Myc (Table 10) [107,260].
Expression or activation of transgenic Myc can induce pNET development in animal
models [
83
,
261
,
262
]. Pelengaris et al., designed a reversibly switchable pNET mouse model
(plns-c-MycER
TAM
) in which transgenic Myc is expressed under an insulin promoter (plns)
and its activation can be induced by tamoxifen (TAM) administration (Table 2). Activation
of Myc leads to hyperproliferation of pancreatic
β
cells in these mice, although it is rapidly
counterbalanced by Myc-induced apoptosis [
74
]. Co-expression of an antiapoptotic Bcl-2
family protein-Bcl-x
L
, however, causes mice to progressively develop angiogenic, invasive
islet tumors [
74
]. Dependence on Myc for the tumor phenotype was shown by the fact that
tumors undergo regression as a result of vascular degeneration and
β
-cell apoptosis when
Myc is deactivated. Others showed that targeted expression of Myc in pancreatic progenitor
and islet cells achieved through somatic delivery of the oncogene at post-natal day 2 (P2)
induces pNET development by 7 months in p16
INK4a
/p14
ARF
null mice, but not in mice
which are wild-type for both p16
INK4a
/p14
ARF
[
263
]. In zebrafish, targeted expression of
MYCN in pancreatic
β
-cells induces pNECs that resemble the human disease, suggesting
other members of the Myc oncogene family may be involved in pNET pathogenesis [
264
].
Myc cross-talks with several key pNET pathways, in part because it is an impor-
tant downstream target of mTOR in many cancers, including pNETs [
107
,
265
]. In that
regard, inhibition of Myc through shRNA or pharmacologic (10058F4, CPI-203) approaches
enhances the sensitivity of pNETs to mTOR inhibitors and reverses pNET resistance to
mTOR inhibition by suppressing Akt activation [
107
,
266
]. Myc also drives tumor angio-
genesis by upregulating VEGF (one of its transcriptional targets) and other angiogenic
proteins [
267
,
268
]. Consistent with those observations, Myc inhibition disrupts the pNET
vasculature and causes tumor regression in Myc-driven pNET mouse models [
74
] and in
RIP-Tag2 mice [269].
Table 10. Molecular profiling studies on miscellaneous pNET-associated proteins and pathways.
Technique Reference Key Findings
IHC [107] In total, 17 out of 21 patients (81%) pNETs showed strong immunoreactivity for Myc.
IHC [260] Positive Myc expression in all 39 benign or metastatic pNETs.
Microarray [270]
Upregulation of LCK, a Src family kinase, in primary (n = 8) and metastatic pNETs (n = 5),
and pNET cell lines compared to normal islets. Microarray results were validated by
qRT-PCR and IHC.
qPCR and IHC [130]RABL6A amplification in 6 of 11 primary pNETs and their matched metastases. High
RABL6A protein expression observed by IHC in all pNETs (n = 5).
Cancers 2021,13, 5117 24 of 51
Table 10. Cont.
Technique Reference Key Findings
IHC [271]Significant upregulation of all HDACs (I, IIa, IIIb, III and IV) in pNETs whose expression
was found correlate with tumor grading and predict disease outcomes.
RNA seq [261]
Transcriptome analysis of 212 patient GEP-NETs complemented by systematic drug
perturbation assays identified HDAC class I inhibitor, entinostat, as a potent agent to treat
42% GEP-NET patients.
RNA Seq [106]
IPA and cMAP analysis of differentially expressed genes in 43 primary pNETs vs. their
matched metastases predicted HDAC as one of the top pharmacological targets to treat
metastatic pNETs.
IHC [185]Positive immunoreactivity for HSP90 and TGF-βRI in 75% of 67 primary and
metastatic pNETs.
IHC [262]
Positive aurora kinase A expression in 8 of 10 insulinomas, in all of 13 nonfunctional pNETs
and 20 SBNETs.
IHC [272]
Ptch1, the sonic hedgehog receptor, expressed in 12 of 22 sporadic pNETs and 4 of 5 MEN-1
pNETs with no significant correlation with clinical outcomes.
IHC [273]
Nuclear
β
-catenin immunostaining in higher percentage of stage III/IV pNETs (2/13, 15%)
vs. stage I/II pNETs (0/74). Negative APC expression in 70% (57/81) of the cases.
IHC [274] Positive expression of TGF-β, TGF-βRI, and TGF-βRII in 75–100% patient pNETs.
LOH and
sequencing [275] LOH of Smad3 in 20% of 20 pNETs; no inactivating Smad3 mutations observed.
PCR and SSCP
mutational analysis
[276]DPC4 mutation or deletion detected in 55% (5 of 9) non-functional pNETs in contrast to
none of the 16 functional tumors-insulinomas, gastrinomas, and VIPnomas.
2.9.2. Src Family Kinases
Src family kinases (SFKs) promote DNA synthesis, cell cycle progression, angiogenesis,
and cell motility by activating Myc, Ras, VEGF, and mTOR driven pathways [277].
Although the oncogenic role of SFKs has been well established in several solid cancers
including breast, prostate, colon, and pancreatic cancers, studies demonstrating their
relevance in pNET pathogenesis are limited [
277
]. Early studies showed that lymphocyte
specific protein tyrosine kinase (LCK), which is a SFK, is highly upregulated at both the
mRNA and protein level in primary pNETs, pNET metastases, and cell lines, BON-1 and
QGP-1 (Table 10) [
270
]. c-Src, which is the prototype and most-studied SFK, activates
the mTOR pathway in pNETs [
278
]. Conversely, loss of endogenous Src activity using an
inhibitor (PP2) or RNAi decreases the phosphorylation of mTOR downstream targets, rpS6
and 4-EBP1, in BON-1 and QGP-1 pNET cells [
278
]. Concomitant inhibition of c-Src (by PP2)
and mTOR (by everolimus) significantly impairs pNET cell growth compared to targeting
either protein alone. Of note, this combination therapy also blocks feedback activation of the
PI3K-Akt loop responsible for pNET resistance to mTOR monotherapy [
278
], highlighting
the potential benefit of co-targeting Src and mTOR signaling in these tumors. More studies
investigating the
in vivo
efficacy of SFK inhibitors alone or in combination with mTOR
inhibitors in pNET models could have high clinical relevance but warrant deeper analyses
of SFK status in patient pNETs [277].
2.9.3. RABL6A
RABL6A is a novel oncogene first discovered as a binding partner of the p14
ARF
tumor suppressor [
279
]. It is a Rab-like GTPase that has several different synonyms in
the literature, including C9orf86, PARF, and RBEL1 [
279
281
]. Expression of RABL6A
correlates with poor survival in breast cancer [
282
], pancreatic ductal adenocarcinoma [
283
]
and non-small cell lung cancer [
284
,
285
]. RABL6A is highly expressed at the protein
and genetic level in patient pNETs (Table 10) [
106
,
130
]. Functional studies demonstrated
that RABL6A promotes pNET cell survival and proliferation by regulating important
Cancers 2021,13, 5117 25 of 51
pNET pathways including inhibition of RB1 [
130
] as well as activation of Akt-mTOR via
inhibition of protein phosphatase 2A (PP2A) [
92
]. Notably, pathway analysis of microarray
results in BON-1 cells suggests RABL6A upregulates Myc and VEGFR signaling genes in
pNETs [
130
]. Most recently, the role of RABL6A as a pNET driver has been corroborated by
in vivo
studies revealing that RABL6A promotes tumor growth and the angiogenic switch
in a transgenic (RIP-Tag2) pNET mouse model [77].
2.9.4. HDACs
Histone deacetylases (HDACs) promote chromatin compaction and inaccessibility of
the promoter regions leading to gene silencing [
271
]. Epigenetic silencing of numerous
tumor suppressor genes due to exaggerated activity of HDACs have been implicated in
the carcinogenesis of multiple organs, including the exocrine and endocrine pancreas [
286
].
A comprehensive immunohistochemical analysis of HDACs in 57 pNETs resected between
1997 and 2013 revealed significant upregulation of all 5 HDAC classes (I, IIA, IIB, III, and
IV) in pNETs compared to the corresponding pancreatic tissues (Table 10) [
271
]. The study
also found a correlation between specific HDAC expression with pNET grade and patient
survival, highlighting a predictive role HDACs can play in determining pNET therapy and
outcome [
271
]. The same group also showed that the expression of HDAC-3 (class I) and -4
(class IIa) significantly correlates with that of miRNA449a, which was found to be a maker
of proliferation status and patient survival in pNETs [287].
Alvarez et al., employed RNA-seq followed by MARINa (Master Regulator Inference
algorithm) and VIPER (Virtual Proteomics by Enriched Regulon analysis) algorithms on
a cohort of 212 patient GEP-NETs to identify master regulator proteins driving neuroen-
docrine lineage progenitor state and immune-evasion in these tumors [
261
]. Their model
predicted the class I HDAC inhibitor, entinostat, to be a potent inhibitor of master regulator
activity in 42% of metastatic pNETs. This led the authors to validate the growth suppressive
activity of entinostat in a mouse xenograft model of pNETs although its anti-metastatic
activity was not assessed.
Scott et al., performed RNA-seq on primary pNETs and their matched liver and lymph
node metastases to identify metastatic gene signatures. Ingenuity pathway analysis (IPA)
and Connectivity Map (cMAP) of 902 differentially expressed genes identified HDAC as
one of the top metastasis-specific pNET drug targets [
106
].
In vitro
testing of class I HDAC
inhibitors, entinostat and mocetinostat, on BON-1 and QGP-1 cell lines showed moderate
anti-proliferative activity [
106
]. Functional inhibition of class IIA HDACs, HDAC 4 and 5 by
LMK-235 has also been shown to reduce the viability and promote apoptosis of BON-1 and
QGP-1 cells [
233
]. At this point, assays measuring the anti-metastatic activities of HDAC
inhibitors in pre-clinical pNET models are needed. It will be important to compare and
contrast different HDAC inhibitors since some can promote metastasis of other tumor cell
types via PKC activation and HDAC11 inhibition [
288
,
289
], underscoring the importance
of thorough drug testing and validation.
Valproic acid (VPA) is another example of an HDAC inhibitor that can reduce BON-1
and H727 (human pulmonary carcinoid) NET growth, biomarker expression, and hormone
secretion [
290
]. Its activity was linked to activated Notch1 signaling. VPA also disrupted
the growth of mouse tumor xenografts derived from these carcinoid cell lines [
290
]. A
pilot phase II study of VPA in patients with low-grade carcinoid and pancreatic NETs
demonstrated stable disease as the best response in only half patients [
291
]. A phase II
trial (NCT00985946) of panobinostat, a pan-HDAC inhibitor, in 15 patients with low-grade
NETs also resulted in a low response rate although high rate of stable disease and median
PFS of 9.9 months were achieved [
292
]. Entinostat is currently being tested in phase II trials
in patients with relapsed or refractory abdominal NETs (NCT03211988). As described in a
prior section on somatostatin receptor (SSTR) signaling, combination therapies targeting
both HDACs (where inhibitors upregulate SSTR levels) and SSTRs together may have
greater efficacy than either therapy alone.
Cancers 2021,13, 5117 26 of 51
2.9.5. Heat Shock Protein (HSP) 90
Heat shock protein 90 is a molecular chaperone required to maintain stability and
activity of diverse proteins regulating cell signaling, proliferation, survival, and carcinogen-
esis [
293
]. HSP90 is highly expressed in non-pancreatic carcinoid NETs [
294
], and in 75%
of primary pNETs and their metastases (Table 10) [
185
]. Several HSP90 inhibitors exhibit
growth inhibitory effects in pNET cell lines [
185
,
294
296
]. HSP90 inhibitors, 17-AAG [
185
],
IPI-504 [
296
], AUY922, and HSP990 [
295
] not only induced apoptosis and inhibited prolifer-
ation of human pNET cell lines
in vitro
and
in vivo
, but also downregulated the expression
of important pNET growth factors such as EGFR, IGF1R, and VEGFR2. This correlated with
reduced activity of downstream ERK and Akt-mTOR oncogenic pathways. Furthermore,
combined inhibition of HSP90 and Akt or mTOR produced an additive anti-neoplastic
effect in pNET cells and overcame IGF1R-dependent feedback activation of Akt associated
with sustained mTOR inhibition [296].
2.9.6. Aurora Kinase
Aurora kinases A, B, and C are serine-threonine kinases that regulate chromosomal
assembly and segregation during mitosis [
297
]. Overexpression of aurora kinases A and
B has been reported in a variety of solid cancers, including prostate, colon, pancreas (ex-
ocrine), breast and thyroid cancers [
155
]. Of all members in this kinase family, aurora
kinase A has the most well-characterized oncogenic role. It is characterized by downreg-
ulation of the p53 tumor suppressor via MDM2 activation [
298
], inhibition of GSK3-
β
,
and activation of
β
-catenin and SRC [
299
]. In an IHC analysis of patient NETs, aurora
kinase A was expressed in 8 of 10 insulinomas, 100% of 13 nonfunctional pNETs, and
20 SBNETs
while being absent in the surrounding non-cancerous tissue and control normal
pancreas (Table 10) [
262
]. That study and others have shown that both BON-1 and QGP-1
pNET cell lines express aurora kinase A and B [
262
,
300
], and treatment with a pan-aurora
kinase inhibitor, danusertib, inhibits pNET cell proliferation at nanomolar concentrations.
Danusertib also disrupted primary tumor growth in a murine xenograft model and halted
development of pNET cell derived liver metastases following intrasplenic injections [
262
].
In agreement, Hofving et al., showed that aurora kinase inhibitors strongly attenuate pNET
cell viability [
301
]. Briest et al., (2018) found that nuclear immunoreactivity against aurora
kinase B in GEPNETs correlates positively with tumor grade and size, albeit negatively
with their differentiation and functionality [
302
]. A selective aurora B inhibitor, ZM447439,
potently suppresses proliferation and induces apoptosis of BON-1 and QGP-1 pNET cells
and exhibits potentiated activity when combined with cisplatin or streptozotocin [300].
Although pNET expression of aurora kinases and their requirement for pNET cell
growth
in vitro
and
in vivo
have been demonstrated in multiple studies, the exact mecha-
nisms by which these kinases promote pNET pathogenesis and their interplay with other
canonical pNET pathways is yet to be fully understood.
2.9.7. Developmental Pathways
(i) Hedgehog signaling
Hedgehog (Hh) signaling is a highly conserved developmental pathway in vertebrates
with important roles in cell cycle regulation and tumorigenesis [
303
]. In Hh signaling,
binding of Desert, Indian, and Sonic Hh ligands with Patched (Ptch-1), a transmembrane
receptor, relieves the inhibition of Smoothened (Smo) by Ptch-1 [
304
]. Activated Smo
cooperates with Gli transcription factors, Gli1 and 2, to transactivate pro-proliferative
genes including Cyclin D, Cyclin E, Myc, and Gli1 [
303
,
304
]. Hh signaling also induces
expression of transcription factors such as Snail, Snug and Twist, which mediate epithelial
mesenchymal transition (EMT) of metastatic tumor cells [303].
Several reports have highlighted the role of Hh signaling in NETs and NECs. Studies
based on qRT-PCR and immunohistochemical analyses showed that Gli1, Ptch1, Sonic Hh
(Shh), and Snail are highly expressed in gastrointestinal (GI) NECs [
305
] and SBNETs [
306
]
while modestly expressed or absent in GI carcinoids and adenocarcinomas [
305
]. Dis-
Cancers 2021,13, 5117 27 of 51
ruption of Hh signaling by a Smo inhibitor, cyclopamine, suppresses NEC cell survival,
proliferation, and invasion in a manner that correlates with downregulation of Gli1, Ptch1,
hAsh (a bHLH transcription factor regulating neuroendocrine differentiation), and Snail1
as well as upregulation of E-cadherin [305,307].
Hh signaling is critical to normal embryonic development and functioning of the
endocrine pancreas [
308
]. Nevertheless, limited studies have investigated the expression or
activation status of Hh pathway in patient pNETs. Fendrich et al., revealed that pancreatic
gastrinomas and their lymph node metastases, unlike primary and metastatic duodenal
gastrinomas, lack detectable expression of Shh, undermining the potential importance of
Shh signaling in this pNET subtype [
309
]. Gurung et al., found expression of Ptch1 in over
50% of sporadic or MEN-1 associated patient pNETs without clear prognostic significance
(Table 10) [
272
]. Gli transcription factors (1, 2, and 3) and Ptch are expressed in BON-1
cells, which exhibit reduced growth upon cyclopamine treatment, suggesting Hh signaling
promotes pNET cell proliferation
in vitro
[
306
]. In RIP-Tag2 pNET mice, administration
of cyclopamine by intraperitoneal injection [
310
] or of a new, orally bioavailable Smo
antagonist LDE225 [
311
] induces pNET cell apoptosis and reduces cell proliferation and
tumor burden, improving their survival by 1–3 weeks.
Evidence suggests Hh signaling is negatively regulated by menin and that this molec-
ular crosstalk could be exploited to treat MEN-1 tumor syndrome [
312
]. Menin recruits
protein arginine methyltransferase 5 (PRMT5) to the Gas1 gene promoter and downregu-
lates Gas1 via PRMT5-catalyzed repressive histone arginine symmetric demethylation [
313
].
Gas1 is critical to the binding of Shh ligand with its receptor Ptch1. Thereby, loss of menin in
MEN-1 disease promotes Gas1 upregulation and subsequent Hh pathway activation [
313
].
Consistent with those findings, increased expression of Gas1, Gli1, and Ptch1, which is
indicative of activated Hh signaling, was observed in primary islets from Men1
l/l
;RIP-Cre
mice compared to the WT controls [
313
]. Furthermore, inhibition of Hh signaling by a Smo
antagonist, GC-0449 (aka vismodegib), decreases proliferation and insulin secretion of in-
sulinomas developed by Men1
l/l
;RIP-Cre mice [
312
,
313
]. These studies suggest that agents
targeting Hh signaling, some of which are FDA approved for other cancers (vismodegib
for basal cell carcinoma), could hold a significant promise in MEN-1 therapy.
(ii) Notch1 signaling
Notch signaling comprises four transmembrane Notch receptors (Notch 1–4) activated
by five Notch ligands (Delta-like ligand 1 [DLL1], DLL3 and DLL4, and Jagged1 and
Jagged2) [
314
,
315
]. Notch signaling has an evolutionarily conserved role in cell-fate-
determination and is known to regulate the differentiation of stem cells and progenitor
cells in the development of pancreas [314].
Notch signaling in cancer can have either oncogenic or tumor suppressive outcomes
depending on the tissue type [
155
]. Activation of Notch is required for cancer stem cell
maintenance in ovarian cancer, colon cancer, lung adenocarcinoma, breast cancer, and
pancreatic cancer [
314
]. However, in GEPNETs the Notch pathway putatively acts as
a tumor suppressor based on the evidence that Notch activity or expression is greatly
reduced whereas its reactivation induces the anti-tumoral effects [
316
,
317
]. Notch sup-
presses neuroendocrine differentiation of the developing endoderm by inhibiting the
expression of bHLH transcription factors (e.g., Ascl1), known to drive the process [
318
,
319
].
Nakakura et al.,
(2005) showed that Ascl1 is upregulated in GI NETs and BON-1 cells.
Exogenous overexpression [
318
,
320
] or pharmacological activation [
223
,
299
,
321
,
322
] of
Notch1 induces loss of Ascl1, reduction in neuroendocrine markers (synaptophysin and
chromogranin), decreased serotonin production by repression of tryptophan hydroxylase
(TPH1), and growth inhibition of BON-1 and H727 cells. The HDAC inhibitor, VPA, can
also upregulate Notch1 in NET cells through unclear mechanisms [
323
]. In a phase II
pilot trial conducted in eight patients with low grade NETs (six carcinoid and two pan-
creatic), VPA increased Notch1 mRNA (which was absent prior to treatment), decreased
chromogranin A levels and induced stable disease as best response in four patients [291].
(iii) Wnt/β-catenin signaling
Cancers 2021,13, 5117 28 of 51
The canonical Wnt signaling cascade is a tumorigenic pathway that involves: (1)
binding of Wnt ligands with a receptor complex consisting of the primary receptor Frizzled
(Fz) and a co-receptor lipoprotein receptor-related protein 5/6 (LRP5/6); (2) subsequent
activation of a cytoplasmic protein called dishevelled homologue (Dv1); and (3) inhibition
of the
β
-catenin destruction complex comprising axin, adenomatous polyposis coli (APC)
and glycogen synthase kinase 3
β
(GSK3
β
) by activated Dv1, leading to
β
-catenin stabiliza-
tion, its nuclear localization, and formation of
β
-catenin/T-cell-specific transcription factor
(TCF)/lymphoid-enhancer binding factor (LEF) transcriptional complex [
315
]. Target genes
of the
β
-catenin/TCF/LEF complex (e.g., Myc, Cyclin D, gastrin, VEGF, Met) are involved
in promoting carcinogenesis, tumor invasion, and metastasis.
Wnt/
β
-catenin signaling regulates normal development of the pancreas [
324
]. Several
groups have thereby investigated its role in pNET genesis. Weiss et al., observed strong
membranous
β
-catenin immunostaining in 100% of 13 stage III/IV pNETs (as per TNM
staging) and in only 47% (35/74) of stage I/II tumors (Table 10) [
273
]. Moreover, 15% of
stage III/IV pNETs and none of stage I/II tumors immunostained for nuclear
β
-catenin.
Although the
β
-catenin expression pattern was influenced by tumor staging, no correlation
was found with tumor grade and disease-specific survival (DSS). The authors also found
loss of APC, a member of
β
-catenin degradation complex, in 70% of total pNETs without
any association with tumor stage, grade, or DSS [
273
]. Downregulation of several other
Wnt/
β
-catenin pathway inhibitors has been reported. Kim et al., demonstrated that
reduced expression of SFRP-1,Axin-2,DKK-1,DKK-3, and WIF-1 in BON-1 cells is mediated
by CpG hypermethylation or H2K9 dimethylation of the corresponding genes, highlighting
the importance of epigenetic regulation in pNET Wnt signaling [325].
Pharmacological inhibition of Wnt/
β
-catenin signaling or overexpression of its en-
dogenous inhibitors reduces viability, proliferation, and tumorigenicity of pNET cells
in vitro
and
in vivo
[
273
,
326
]. Some novel Wnt/
β
-catenin signaling activators or down-
stream transcriptional targets have been implicated in pNET pathogenesis. Tenascin-C
(TNC), an extracellular matrix molecule protein, activates Wnt/
β
-catenin signaling via
downregulation of DKK1, a Wnt pathway inhibitor, thereby promoting pNET cell survival,
angiogenic switch, tumor progression, and lung micrometastasis in RIP-Tag2 mice [
327
].
Consistent with those data, TNC expression in human insulinomas was found to correlate
with metastasis [
327
]. Kim et al., showed Neurotensin is a direct Wnt/
β
-catenin signaling
transcriptional target that promotes pNET cell proliferation, anchorage-dependent growth,
and the expression of growth-related proteins, cyclin-D1 and Myc [328].
Wnt/
β
-catenin signaling is regulated by menin in pNETs. Jiang et al., reported
that menin promotes
β
-catenin phosphorylation and degradation in mouse and human
pNETs [
329
]. They also showed that genetic ablation or pharmacological inhibition of
β-catenin
attenuates pNET growth and the hypoglycemic phenotype in
β
-cell specific Men-
1knockout mice. This coincided with downregulation of pro-proliferative
β
-catenin target
genes, Ccnd1,Myc, and Mcm2 in the tumors and improved mouse survival. These results
suggested that suppression of oncogenic
β
-catenin signaling partly accounts for the tumor
suppressive role of menin in pNETs. In contrast, Chen et al., showed that menin directly
interacts with
β
-catenin, promotes
β
-catenin transcriptional activity in TGP61 mouse islet
tumor cells, and selectively upregulates Axin2 (but no other Wnt/
β
-catenin signaling
targets tested), consistent with their observation of increased H3K4 trimethylation at the
Axin2 gene promoter following menin overexpression [
330
]. Taken together, these results
suggest that menin regulation of Wnt/
β
-catenin signaling is complex. It is likely context-
dependent and may affect the expression of Wnt target genes in a biased fashion [330].
(iv) TGF-βand SMADs
Transforming growth factor-
β
(TGF-
β
) superfamily proteins, TGF-
β1
,
β2
, and
β3
,
interact with transmembrane receptor serine threonine kinases, TGF-
β
RI and TGF-
β
RII, to
phosphorylate and activate key downstream transcription factors, SMAD2-4 [
321
,
331
]. Tar-
get genes of SMADs regulate cell differentiation (including that of the endocrine pancreas)
and proliferation [
322
,
331
]. Activation of TGF-
β
signaling can induce or inhibit cell pro-
Cancers 2021,13, 5117 29 of 51
liferation depending on the state of cell differentiation or transformation [
331
]. In pNETs,
however, TGF-
β1
signaling has been shown to inhibit cell growth through upregulation of
the p21WAF1/CIP1 tumor suppressor, which is a SMAD-target gene [274,331].
Immunohistochemical studies revealed positive expression of TGF-
β1
and its recep-
tors, TGF-
β
RI and TGF-
β
RII, in most (75–100%) patient pNETs as well as in BON-1 and
QGP-1 pNET cell lines (Table 10) [
185
,
274
]. TGF-
β1
treatment inhibits BON-1 cell growth
by promoting cell cycle arrest, concomitant with p21
WAF1/CIP1
induction and Myc down-
regulation [
274
]. Conversely, treatment with anti-TGF-
β1
antibody augments BON-1 cell
growth suggesting that the low proliferative index of pNET cells could be in part mediated
by paracrine and autocrine TGF-
β1
signaling [
274
]. Shattuck et al., observed LOH of the
SMAD3 gene in 20% of patient pNETs although no SMAD3 inactivating mutations were
found in those tumors [
275
]. SMAD4/DPC4, a putative tumor suppressor in pancreatic
carcinoma, was mutated or deleted in 55% (5 of 9) of non-functioning pNETs but remained
intact in 100% of the less aggressive, functional pNETs (insulinomas, gastrinomas, and
VIPnomas) [
276
]. Notably, SMAD7, an inhibitor of SMAD2/3 and thus TGF-
β1
signaling,
promotes islet
β
cell proliferation in adult mice, supporting the tumor suppressive role of
TGF-β/Smad signaling in pNETs [322].
A functional interplay between TGF-
β
signaling and other pNET regulators has
been seen. Kaji et al., showed that menin promotes TGF-
β
-induced growth inhibitory
and transcriptional activity by directly interacting with SMAD3 and inducing SMAD3
translocation at specific transcriptional regulatory sites [
42
]. This observation supports the
attenuation of TGF-
β
/SMAD3 signaling as one of the potential mechanisms leading to
multi-organ tumorigenesis in MEN1 syndrome. Leu et al., on the other hand, revealed that
TGF-
β
inhibits BON-1 pNET cell proliferation by inducing somatostatin (SST) upregulation
and secretion, thus activating the growth inhibitive autocrine loop of SST [231].
(v) Underexplored areas
Recent studies have examined the significance of non-coding RNAs, microRNAs
(miRNAs), and long-non-coding mRNAs (lncRNAs), in pNET pathogenesis. miRNAs are
a class of non-coding RNAs that regulate gene expression via mRNA degradation and
translational repression. They often serve as biomarkers in specific cancers due to their
abundance, cell-type and disease stage specificity and stability [
332
]. A focused analysis of
tumor miRNA expression profiles in GEP-NETs (pancreatic, ileal, rectal, and appendiceal)
revealed elevated levels of miR-375 across all samples [
332
]. Distinct NET types were
able to be classified with high accuracy based on differential miRNA expression, and
low-versus intermediate-grade pNETs could be discriminated from each other based on
varied expression of miR-328. Examining the biological roles of these miRNAs in pNET
pathogenesis is an exciting area that warrants further investigation.
A few studies have examined circulating miRNA levels. Hellberg et al., found that
NET patients displayed significantly reduced levels of miR-223 in their serum compared to
healthy individuals, but found no associations between miR-223 expression and clinico-
pathologic characteristics of the samples [
333
]. Another study evaluated circulating levels
of miR-29b in 45 patients with NETs (versus 19 healthy controls) and likewise observed
downregulation of the miRNA in NET patient serum [
334
]. Similarly, miR-29b serum levels
did not correlate with tumor stage or clinical features, such as tumor relapse or overall
survival. These studies suggest that serum levels of miR-223 and miR-29b may be useful
diagnostic biomarkers of NET but lack prognostic value.
Although initially ignored as ‘transcriptional noise’ lacking any biological function,
lncRNAs have now been shown to interact with DNA, RNA, and transcription factors. In so
doing, lncRNAs regulate various biological processes including DNA methylation, histone
modification, and chromatin remodeling [
335
]. Modali et al., showed that lncRNA Meg3
has tumor-suppressive activity in pNET cells [
336
]. They found that Meg 3 is activated by
menin and its expression is lower in mouse or human MEN1-associated pNETs compared
to the normal islets. Functionally, Meg3 overexpression in insulinoma cells significantly re-
duces their proliferation, migration, and invasion by downregulating the c-Met (hepatocyte
Cancers 2021,13, 5117 30 of 51
growth factor receptor) oncogene. A microarray-based study of patient tumors identi-
fied 280 lncRNAs that were differentially expressed in pNETs compared to the adjacent
normal tissues. Subsequent pathway analyses associated these differences with multiple
tumorigenic processes and signaling pathways, such as cell projection morphogenesis, cell
adhesion, PI3K-Akt signaling pathway and Ras signaling [337].
pNETs have low mutational burden and as such, are traditionally perceived to be ‘cold’
tumors offering limited scope for immunotherapy. Notably, a recent study by Yuan et al.,
demonstrates that B7x, an immune-checkpoint ligand expressed by tumor cells, suppresses
anti-tumor immunity in pNETs and as such, could be an attractive target for (p)NET
immunotherapy [
78
]. The authors demonstrated that genetic ablation or administration
of B7x antibody in Men1-deficient mice with insulinomas results in reduced islet cell
proliferation and pNET development accompanied by increased T-cell infiltration
(Table 2).
This study encourages further investigation into mechanisms and approaches that will
modulate immune cell activity and infiltration in pNETs.
3. In Vitro and Vivo pNET Models
3.1. Human pNET Cell Lines
Cell lines are extensively used in cancer research to better understand the underlying
molecular mechanisms of tumorigenesis and facilitate identification of novel drug targets.
In cancer drug discovery, cell cultures are used to screen large pools of molecules to select
the most promising ones for in vivo testing.
Despite numerous efforts, only 3 pNET cell lines have been established and authen-
ticated to date (Table 11). These pNET cell lines include QGP-1, BON-1, and NT-3. They
were generated from patients with metastatic tumors and have been shown to recapitulate
the parent tumors when transplanted into mice to form xenografts. The first developed
and most commonly used pNET cell lines are QGP-1 and BON-1. QGP-1 was derived
from a non-functional tumor in the tail of the pancreas and secretes carcinoembryonic
antigen (CEA) like the parent tumor [
338
]. BON-1 represents a non-functional pNET and
comes from a peripancreatic lymph node metastasis from a patient [
339
]. NT-3 is a human
insulinoma cell line established from a lymph node metastasis that proliferates very slowly
and exhibits a well-differentiated phenotype [340].
Table 11. In vitro models: cell lines.
Cell Type Source Key Features
QGP-1 Metastatic human islet cell carcinoma Production of carcinoembryonic antigen and absence of hormonal
secretion recapitulating the parent tumor [338].
BON-1 Peripancreatic lymph node metastasis of
pancreatic carcinoid tumor
Express gastrin and somatostatin receptors; synthesize serotonin and
chromogranin [339].
NT-3
Lymph node metastasis of an insulinoma
Model of well-differentiated insulinoma, highly expresses
somatostatin receptors and neuroendocrine markers, exhibit slow
growth in contrast to BON-1 and QGP-1 cells. Gene sequencing
revealed a homozygous missense mutation in MEN1 gene with other
pNET-associated genes intact [340].
Genetic profiling revealed both BON-1 and QGP-1 harbor homozygous TP53 mu-
tations with possible loss of function [
341
]. That is consistent with those lines being
derived from advanced metastatic pNETs, which in patients often display TP53 and RB1
mutations [
11
]. Notably, mechanistic studies using those cell lines have shown that both
express functional RB1 protein and that p53 has wild-type transcriptional activity in BON-1
cells [
130
]. By comparison, QGP-1 are null for p53 protein [
92
,
130
]. Evidence for other
pNET genetic mutations in those two lines are also somewhat conflicting. Vandamme
et al., detected mutations in ATRX in both BON-1 and QGP-1, TSC2 and NRAS in BON-1,
and KRAS in QGP-1 [
341
]. However, none of these pNET genetic signatures were found
by Boora et al., in their whole exome sequencing of BON-1 and QGP-1, which prompted
Cancers 2021,13, 5117 31 of 51
them to question usage of these cells lines as pNET models [
342
]. These differences may
reflect analyses of different subclones of the lines, arising through extensive passaging in
culture and distribution across labs, yielding apparent differences from the parental lines
or from one research group to another. The most definitive study of GEPNET cell lines
was conducted by Hofving et al., in 2018 [
301
]. Using immunophenotyping, copy number
profiling, whole-exome sequencing and drug screening, BON-1 and QGP-1 were validated
as being authentic pancreatic NET cells. NT-3 cells were not evaluated in the Hofving study
but were separately characterized and shown to lack mutations in TP53, RAS, or other
genes associated with high grade neuroendocrine carcinoma [
340
]. However, targeted
sequencing of some pNET genes revealed a homozygous missense mutation of MEN1.
Molecular characteristics of cell lines guide their application in specific drug testing.
BON-1 cells have activated Akt-mTOR signaling and as such are a good preclinical model
to test mTOR pathway inhibitors. Indeed, everolimus potently inhibited BON-1 cell growth
in a dose-dependent manner, providing a strong basis for a clinical trial testing it in
patients with aberrant Akt-mTOR signaling [
98
]. Moreover, both BON-1 and NT-3 express
somatostatin receptors and have been demonstrated to be highly sensitive to somatostatin
analogues [
339
,
340
]. Recently, luciferase-expressing derivatives of BON-1 and QGP-1 cells
were developed to enable bioluminescent imaging of pNET metastasis in vivo [343].
The lack of pNET cell lines representing all different pNET types reflects the challenge
in deriving stable neuroendocrine cell lines from patient samples. Exceptionally slow
growth, fastidious nutrient conditions for survival, poor adherence to substrate, and inter-
ference by fibroblasts are some of the difficulties in deriving a pNET cell line. Nevertheless,
there is an obvious need in the field to establish more diverse pNET cell lines, particularly
those representing low grade, well-differentiated non-functional pNETs since those are the
majority of tumors seen in patients.
3.2. 3D Cultures
Three-dimensional (3D) tissue culture models, organoids and spheroids, are believed
to preserve the
in vivo
tumor architecture and microenvironment to some degree (Table 12).
As such, they are sometimes preferred over cell cultures for drug screening. Wong et al.,
demonstrated a method to form 3D spheroids from BON-1 and QGP-1 cell lines for the first
time. These cells start forming spheroids on the 3rd day of plating on an agarose-coated
plate under low agitation conditions. Immunohistochemistry of spheroid sections reveal
the pNET spheroids contain a highly proliferative periphery and apoptotic core, moreover,
drug testing results were found to be reproducible [
344
]. Ear et al., established a protocol
to generate SBNET spheroid cultures in extracellular matrix using surgically removed
tumors [
345
]. Moreover, they successfully employed their SBNET spheroid model to test
the cytotoxicity of rapamycin [
345
]. Spheroids from pNETs have also been successfully
established as 3-D cultures and used for drug testing against sunitinib, everolimus, and
temozolomide [346].
Table 12. In vitro models: 3D cell cultures.
Type Source Cell/Tissue Key Features
pNET
spheroids
pNET cell lines: BON-1 and QGP-1
Human lung neuroendocrine cell
line: H727
Three-dimensional spheroids start forming by day 3 of cell plating and contain
highly proliferative cells at the periphery and a necrotic center, proposed to be
useful models for in vitro drug screening [344].
SBNET
spheroids Patient SBNETs
Cultured with the help of extracellular matrix (Matrigel); doubling time was
14 days; expressed synaptophysin, chromogranin and SSTR2; undergo
apoptosis with rapamycin treatment [345].
pNET
spheroids Patient pNETs
Primary tumor cells isolated from pNETs and cultured in vitro to form
islet-like tumoroids that retain a neuroendocrine phenotype and are viable for
at least 2 weeks in culture for drug testing [346].
Cancers 2021,13, 5117 32 of 51
3.3. Patient Derived Xenografts
Patient-derived xenograft (PDX) tumors are increasingly being developed and used
as the most authentic models of human cancer since they have been shown to faithfully
replicate disease complexity [
347
,
348
]. They are developed from the transplantation of
fresh or cryopreserved human tumor samples directly into an immunocompromised
mouse [
349
,
350
]. Unlike xenografts derived from the pre-existing cell lines, PDXs are
directly obtained from the primary patient tumor or cells without intermediate
in vitro
processing steps and they recapitulate the genetic and histological make-up of the original
patient tumor. Since PDXs replicate the disease with high fidelity, they are good preclinical
models for understanding the disease and conducting drug screenings. High throughput
drug screenings performed on PDX models are felt to have higher reproducibility and
translational potential compared to other pre-clinical cancer models [351].
Nevertheless, PDX models for pNETs or NETs in general are almost non-existent,
despite wide-ranging attempts to generate them from different groups for over thirty
years. The first reported pancreatic PDX model was developed in 2016 (Table 13) [
203
].
The authors subcutaneously transplanted tumor fragments from a pNET patient’s lymph
node metastasis into NOD scid gamma (NSG) mice, which formed PDX tumors with an
average of 80% engraftment efficiency. The successful implantation required exogenous
activation of MET in the tumor cells by continued administration of a humanized anti-
body agonist of the receptor. The group also tried to generate PDXs from an additional
39 well-differentiated,
WHO grade 1 and 2 patient pNETs, but they were only successful
with one. That illustrates the remarkably challenging and unpredictable nature of the
task. Nevertheless, PDXs in this study were not validated for their genetic resemblance
to the primary patient tumor and no follow-up studies utilizing this model have been
reported. It is possible that propagation of the PDXs over time and maintenance of the
original phenotype were difficult to maintain.
Table 13. Patient-derived xenografts (PDXs).
Reference Source Tissue Key Features
[203]
Primary tumor fragments from patients’
lymph node metastases used for s.c.
transplantion into NOD scid mouse (NSG)
Successful engraftment required MET proto-oncogene activation by
HGF or its analogue.
Critical comments: validity, utility unclear, no studies
utilizing the model.
[102] pNET liver metastases pNETs were well-differentiated, pNET gene signatures were retained,
tumor growth inhibition observed with everolimus and sapanisertib.
Chamberlain et al., developed the first validated PDX model in 2018 (Table 13) [
102
].
Tumor sections removed during resection of pNET liver metastases were implanted subcu-
taneously into female athymic nude mice to establish the xenograft passage 0 (P0), which
were subsequently passaged to obtain P1 and P2 xenografts. These PDX-pNETs were
well-differentiated and retained the common pNET gene signatures, including MEN1 loss
and mTOR activation. Two different mTOR inhibitors, everolimus (mTORC1 inhibitor)
and sapanisertib (a dual mTORC1 and mTORC2 inhibitor), displayed excellent antitumor
activity in the PDXs although resistance to everolimus emerged in some of the tumors.
Sapanisertib treatment was remarkably able to cause tumor regression in most of the
everolimus resistant lesions, supporting the clinical potential of this investigational drug.
Several groups have generated PDXs from neuroendocrine neoplasm of other organs.
Tanaka et al., obtained PDXs from metastatic inguinal lymph node of renal neuroendocrine
carcinoma [
352
], whereas another group derived a ‘GA0087’ PDX model from a human
gastric neuroendocrine carcinoma [
353
]. More comprehensive PDX generation attempts
were made by Yang et al., from 106 NETs comprising 58 pancreatic, 1 gallbladder, 38 small
bowel, 3 renal, and 6 liver metastases of unknown primary site [
354
]. Although seven of
these tumors (three pancreatic, three intestinal, and one gall bladder) were successfully
Cancers 2021,13, 5117 33 of 51
engrafted and gave rise to first generation xenografts, only the gallbladder PDX was sus-
tained when re-engrafted into a new set of animals. The gallbladder PDX was propagated
for eight passages without losing key molecular signatures of the original primary tumor.
Interestingly, PDXs from other NET types (e.g., pituitary) have been successfully
generated in zebrafish [
355
,
356
]. In such models, stained primary cells or tumorspheres
generated from post-surgical patient NETs are injected into the sub-peridermal cavity of
zebrafish embryos [
357
,
358
]. This facilitates rapid evaluation of individual tumor character-
istics including angiogenesis, invasion, and metastasis as well as development of precision
medicine [
356
,
358
]. Some advantages of zebrafish over murine PDX models include
high transplantation efficiency, limited sample size requirement, simplicity, and low cost.
Moreover, the zebrafish adult pancreas shares anatomic and metabolic similarities with
mammalian pancreas. However, the wide application of zebrafish PDXs could be limited
by the fact that it is a non-mammalian system that requires unique expertise
and facilities.
3.4. Genetically Engineered Mouse Models
In cancer research, genetically engineered mouse models (GEMMs) are used to under-
stand the role of specific gene mutations in tumor development and progression
in vivo
.
These also enable scientists to evaluate the role of tumor vasculature, tumor microenviron-
ment, and immune system which are overlooked in
in vitro
systems. Moreover, GEMMS
are essential tools for preclinical assessment of drug efficacy and safety, and for diagnostic
or prognostic marker discovery [12].
The RIP-Tag2 (RT2) mouse is the first developed pNET GEMM (Table 2) [
57
]. In this
pNET model, overexpression of transgenic simian virus (SV) 40 large T-antigen by rat in-
sulin gene promoter transforms pancreatic
β
-cells forming hyperplastic islets by
3–5 weeks,
which can further grow into angiogenic islets by 5–9 weeks and tumors by
9–12 weeks [359].
Histopathological analysis revealed that the majority of tumors in RIP-Tag2 mice are well
differentiated insulinomas while a small percentage are poorly differentiated invasive
carcinomas with high mitotic index resembling poorly differentiated human pNECs [
360
].
The latter extreme pNET phenotype is unsurprising because tumorigenesis in the RT2
model is driven by inactivation of p53 and Rb, whose genetic inactivation is more specific
to high-grade, poorly differentiated pNECs in humans. Because the genetic background
of this mouse model resembles that of rare pancreatic neuroendocrine carcinomas, and
additionally, tumor formation is exceptionally rapid, critics of this mouse model advise
its use with caution. Nevertheless, RT2 is a validated model for pNET drug screening
as it was originally used in the preclinical assessment of everolimus and sunitinib. Most
impressively, their results successfully predicted the outcomes of clinical trials [
95
,
361
],
leading to the use of those agents in patient therapies today.
The genetic background of RIP-Tag2 mice influences the type of pNET they develop.
Under the original C57BL6 background, RIP-Tag2 mice form insulinomas. By comparison,
RIP-Tag2; AB6 F1 hybrid mice generated by breeding RIP-Tag2 (C57BL6) males with wild
type A/J strain females switch to developing non-functional pNETs in a high percentage
of animals [
58
]. This is the first model of non-functional tumors, the most common pNET
in patients, making it a relevant and powerful model for future investigations. Genetic
studies reveal RT2 AB6F1 mice inherently express reduced levels of Insm1, a
β
-cell specific
transcription factor required for insulin synthesis compared to their C57BL6 counterparts.
RIP-Tag2;