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Epigenetic Regulation in
Gastroenteropancreatic
Neuroendocrine Tumors
Judy S. Crabtree*
Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, United States
Gastroenteropancreatic neuroendocrine neoplasms are a rare, diverse group of
neuroendocrine tumors that form in the pancreatic and gastrointestinal tract, and often
present with side effects due to hormone hypersecretion. The pathogenesis of these
tumors is known to be linked to several genetic disorders, but sporadic tumors occur due
to dysregulation of additional genes that regulate proliferation and metastasis, but also the
epigenome. Epigenetic regulation in these tumors includes DNA methylation, chromatin
remodeling and regulation by noncoding RNAs. Several large studies demonstrate the
identification of epigenetic signatures that may serve as biomarkers, and others identify
innovative, epigenetics-based targets that utilize both pharmacological and theranostic
approaches towards the development of new treatment approaches.
Keywords: DNA methylation, neuroendocrine, histone modification, miRNA, pancreatic
1 INTRODUCTION
Gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) have steadily increased in
prevalence and incidence, and this number is expected to continue rising primarily due to
improvements in diagnostic imaging and physician awareness (1–3). Patients with GEP-NENs
often present with advanced disease at diagnosis and surgery alone is rarely curative (4). Despite
association of GEP-NENs with several genetically linked syndromes, the molecular mechanisms of
GEP-NENs are not well understood. Recent studies have provided evidence for the importance of
particular cellular processes such as angiogenesis, or pathways such as G-protein coupled receptor
(GPCR) activation in the pathogenesis of GEP-NENs, but there remains significant work to be done.
Hanahan and Weinberg outlined the “Hallmarks of Cancer”first in 2000 (5), then again in 2011 (6)
that included the cancer-driving categories of tumor-promoting inflammation, genome instability
and mutation, enabling replicative immortality, resisting cell death, activating invasion and
metastasis, inducing angiogenesis, evading growth suppressors, sustained proliferative signaling,
deregulating cellular energetics, and avoiding immune destruction. Studies on GEP-NENs suggest
that the generalized Hallmarks of Cancer also apply to this tumor type [Figure 1 and reviewed in
(7)] and many of these categories will be discussed below as they related to epigenetic regulation of
GEP-NENs. The goal of this review is to briefly touch on the current therapeutic options for patients
with GEP-NENs [these have been extensively reviewed in (8–11)], then explore details of the
emerging epigenetic approaches in terms of utility as diagnostic or prognostic biomarkers, or
therapeutics for this rare tumor type.
Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 9014351
Edited by:
Emilio Cusanelli,
University of Trento, Italy
Reviewed by:
Giovanni Vitale,
University of Milan, Italy
Massimiliano Mazza,
Scientific Institute of Romagna for the
Study and Treatment of Tumors
(IRCCS), Italy
Jonathan Strosberg,
Moffitt Cancer Center, United States
*Correspondence:
Judy S. Crabtree
jcrabt@lsuhsc.edu
Specialty section:
This article was submitted to
Cancer Genetics,
a section of the journal
Frontiers in Oncology
Received: 21 March 2022
Accepted: 09 May 2022
Published: 07 June 2022
Citation:
Crabtree JS (2022) Epigenetic
Regulation in Gastroenteropancreatic
Neuroendocrine Tumors.
Front. Oncol. 12:901435.
doi: 10.3389/fonc.2022.901435
REVIEW
published: 07 June 2022
doi: 10.3389/fonc.2022.901435
2 CURRENT CLINICAL APPROACHES
Neuroendocrine neoplasms (NENs) in general are clinically and
biologically diverse tumors that arise in a variety of tissues
including pituitary, parathyroid, lung, skin, pancreas and
gastrointestinal tract. The diversity of these tumors presents
challenges when establishing tumor classification categories
and patient management guidelines, and the nomenclature has
changed several times over the last several decades. Recently, the
guidance for these tumors has begun to stabilize as a result of
significant efforts by the World Health Organization and other
regulatory bodies, allowing pathologists and clinicians a more
consistent taxonomy. Currently, NENs are divided into three
major classes including 1) neuroendocrine tumors (NET) which
included well differentiated tumors, 2) neuroendocrine
carcinoma (NEC) which are poorly differentiated tumors, and
3) mixed adenocarcinoma (MANECs or MiNEN; mixed
neuroendocrine-non-neuroendocrine neoplasm) (12).
A subset of NENs are the gastroenteropancreatic neoplasms
(GEP-NENs) that are within themselves a very heterogeneous
group of tumors arising from the neuroendocrine cells of the
pancreas and gastrointestinal tract. Well differentiated GEP-
NETs are classified as either Grade 1 if they demonstrate <3%
Ki67 index and mitoses <2 per 10 high-power visual field, or
Grade 2 if they have 3-20% Ki67 index and mitoses 2-20 per 10
high-power visual field. All poorly differentiated GEP-NECs are
classified as Grade 3, meaning >20% Ki67 index and >20 per 10
high-power visual field. MANECs are defined as having >30% of
each component in the tumor and were defined as NEC Grade 3
containing non-neuroendocrine components (typically
adenocarcinoma) (13).
In terms of treatment, surgical resection is often the first line
therapy for either curative (for localized, non-metastastic cancer)
or palliative (for advanced metastatic disease) care, followed by
pathway-specific chemotherapeutic approaches that are
administered systemically. For patients with advanced disease
and functional tumors, controlling symptoms as a result of
hormone hypersecretion becomes paramount. Somatostatin
analogs (SSA) such as octreotide, lanreotide and the second
generation compound pasireotide are typically the first line
therapy for symptom control. These drugs work by binding
tightly to one of several somatostatin receptors (SSTR) which
have variable expression both across different tumor types and
also within the same tumor type. In general, 70-90% of GEP-
FIGURE 1 |Hallmarks of Cancer and Epigenetic Regulation of GEP-NETs. The original Hallmarks of Cancer as published in 2000 by Hanahan and Weinberg
(gray), were updated in 2011 to include additional enabling factors (purple) and emerging hallmarks of cancer (teal). GEP-NETs have been studied in several of
the defined hallmarks of cancer, and listed here are the epigenetic mechanisms and therapeutics associated with each. ARID2, AT-rich interactive domain 2;
ATRX, alpha-thalassemia/mental retardation, X-linked; CDK4/6, cyclin dependent kinase 4/6; CDKN2A, cyclin dependent kinase inhibitor 2A gene; CR,
chromatin remodeling; DAXX, death domain-associated protein; DNMT, DNA methyltransferase; HAc, histone acetylation; HDAC, histone deacetylase;HMe,
histone methyltation; HMT, histone methyltransferase; MEN1, multiple endocrine neoplasia type 1; MGMT, O-6-methylguanine DNA methyltransferase gene;
MLH1, mutL homolog 1; MLL, mixed lineage leukemia lysine methyltransferase 2A; mTOR, mammalian target of rapamycin; PMe, promoter methylation;
RASSF1A, ras associated domain family 1 gene; SETD2, set domain-containing protein 2; SMARCA4, SWI/SNF-related matrix associated actin dependent
regulator of chromatin subfamily a, member 4; SSTR, somatostatin receptor; TIMP3, tissue inhibitor of metalloproteinase 3 gene; TKI, tyrosine kinase inhibitor;
VEGF, vascular epithelial growth factor. Adapted from “Hallmarks of Cancer”by BioRender.com (2022), Retrieved from https://app.biorender.com/biorender-
templates.
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NENs express SSTR2, followed by SSTR5 (14,15). The efficacy of
SSA treatment is dictated by the amount and subtype of SSTR
expressed by the tumor. SSAs have been rigorously tested in
clinical trials, including the PROMID (16) and CLARINET (17)
trials, which demonstrated improved symptom control, tumor
growth control and progression free survival (PFS) compared
to placebo.
Peptide receptor radionuclide therapy (PRRT) is the systemic
delivery of cytotoxic radionuclides that specifically target cells
expressing high levels of SSTR. The most commonly used
approach is (177)Lu-DOTATATE that was studied in the
NETTER-1, phase III trial (18,19). In this trial of midgut
neuroendocrine tumors, PRRT treatment provided superior
PFS, and improved response rates, symptom control and
quality of life. Additional trials that included pancreatic NETs,
also demonstrated increased PFS and overall survival (OS), along
with improved symptom control and quality of life (20,21).
The mammalian target of rapamycin (mTOR) pathway has
also been a target of systemic therapies for GEP-NENs. The
mTOR inhibitor everolimus was used in the RADIANT-3 (22)
and RADIANT-4 (23) trials and demonstrated increased PFS
compared to placebo, and similarly, sunitinib, a broad spectrum
tyrosine kinase inhibitor showed superiority in PFS compared to
placebo in a phase III trial (24). There is no consensus on the use
of cytotoxic chemotherapy such as 5-fluorouracil, platinum
drugs, or alkylating agents, but some combinations have
yielded durable response rates in GEP-NENs, and additional
trials such as the SENECA trial (NCT03387592) are ongoing to
address these systemic therapies. Comprehensive reviews on
drug therapy for GEP-NENs have been published recently
(8–11).
3 GENETIC BASIS OF GEP-NENS
GEP-NENs most often arise as sporadic tumors, but roughly 10%
of pancreatic NETs (pNETs) occur in hereditary syndromes like
multiple endocrine neoplasia, type I (MEN1) (25), Von Hippel-
Lindau (VHL) (26), neurofibromatosis type I (NF1) (27)and
tuberous sclerosis complex (TSC) (28). MEN1 is associated with
two or more tumors of the parathyroid, pituitary, and GEP-NENs
and is associated with germline mutations in the MEN1 gene on
chromosome 11q13 (25). While more than 1300 germline
mutations have been identified to date, sporadic mutations can
also occur. MEN1 has an important role in cellular proliferation
pathways and is reported to impact Wnt/b-catenin (29)and
hedgehog (30) signaling, NF-kB transactivation (31), MAPK-
ERK signaling (32) and PI3K-mTOR-Akt signaling (33). VHL is
another tumor syndrome that phenotypically includes pNETs
along with pheochromocytoma, renal cell carcinoma and
hemangioblastoma and results from mutation in the VHL gene
on chromosome 3p25 (26). It is thought that mutated VHL
facilitates degradation of hypoxia inducible factor 1, which
induces production of growth factors, angiogenesis and tumor
growth (34). TSC arises from mutations in the TSC1 gene on
chromosome 9q34 or TSC2 gene on chromosome 16p13.3 that
lead to increased proliferation by activation of the mTOR signaling
pathway and includes phenotypic skin abnormalities, renal system
angiomyolipomas, hamartomas and neurological defects.
Pancreatic neuroendocrine carcinoma are a rare part of this
genetic syndrome (35). Finally, NF1 arises from mutations in
the NF1 gene located on chromosome17q11.2 that result in
activation of the RAS-MAPK and PI3K-mTOR pathways.
Patients with these mutations develop a spectrum of cancers
including myeloid leukemia, pheochromocytoma,
rhabdomyosarcoma, central nervous system tumors and GEP-
NENs, including pNETs (36).
Recent large scale studies have been performed to define
pNET genetic abnormalities (37–40). Using whole exome
sequencing of 10 nonfunctional, sporadic pNETs followed by
directed sequencing of an additional 58 pNETs, Jiao, et al.
confirmed the frequency of MEN1 mutations in 40% of cases,
inactivation of TSC1/2 in 6% of cases and dysregulation of the
P13K/mTOR signaling pathway by identifying PTEN
inactivating mutations in 5% of cases. Scarpa, et al. performed
a study on 102 primary pNETs and reported new germline
mutations in DNA repair genes MUTYH,CHEK2 and BRCA2,
along with additional somatic mutations involved in chromatin
remodeling, mTOR signaling (new EWSR1 gene fusions), and
hypoxia (38).
A study completed in 2022 by Simon et al. performed in-
depth genomic analysis of 57 pNENs (43 pNET, 14 pNEC) using
DNA sequencing to identify recurrent genetic mutations. From
this analysis, the authors identified two groups: Group A
included mutations that are often associated with pNETs
including MEN1,DAXX,ATRX,VHL,PTEN and TSC2, while
Group B contained no mutations in DAXX,ATRX or MEN1 but
contained KRAS,SMAD4 and TP53 mutations (39). Allen, et al.
investigated the clinical significance of BRAF alterations in a
series of 80 well-differentiated, metastatic pNETs. BRAF
mutations were identified in 6 samples (7.5%): two harbored
V600E, one tumor each for K601E, T599K and T310I mutations,
and the final tumor carried both G596D and E451K mutations
(41). In vitro studies suggested that these mutations may serve as
biomarkers for therapy response to RAF and MEK inhibition in
this small subset of pNET tumors (41). NTRK fusions have also
been investigated in NENs and in a study of 2417 tumors, NTRK
alterations were identified in six cases (0.3%; 2 pancreas, one
uterus, one lung and 2 unknown origin) (42). Fusion partners
included an intergenic region, PIP5K1A,CCDC19,GPATCH4,
ETV6 and SQSTM1.
Gastrointestinal neuroendocrine neoplasms (GI-NENs) have
also been analyzed using large scale DNA sequencing to uncover
genomic and epigenomic alterations. Yachida, et al. studied 115
cases of GI-NENs using whole genome/exome sequencing. The
collection of 60 GI-NECs analyzed included pancreatic,
colorectal, gastric, biliary, ampullary and esophageal tumors,
while the remaining 55 GI-NET samples included pancreatic,
colorectal and duodenal. Analysis of mutations allowed GI-NECs
to be distinguished from GI-NETs, with GI-NECs containing
mutations in TP53,KRAS,RB1,CCNE1,CDKN2A and
MYC (43).
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4 DNA METHYLATION
DNA promoter methylation is one of the most well-studied
epigenetic modifications, and the methylation of cytosine
residues to 5-methylcytosine is a cornerstone of gene regulation
and genomic homeostasis (Figure 2). DNA methylation alters the
availability of transcription activation binding sites, inhibiting
transcription. Moreover, additional inhibitory proteins, such as
methyl CpG binding domain (MBD) and zinc finger proteins,
bind to the methylated DNA further reinforcing transcriptional
repression (44). There is also significant crosstalk with histone
modifications (discussed below) where activating histone
modifications prohibit binding of DNA methyltransferases
(DNMTs) resulting in activation of transcription. The reverse is
also true when repressive MBD proteins bound to methylated
DNA prohibit histone modifications, resulting in transcriptional
repression (44). In cancer, dysfunction of DNA methylation often
results in genome-wide hypomethylation and increased
chromosomal instability (CIN) alongside localized regions of
DNA hypermethylation, specifically at CpG islands upstream of
tumor suppressor genes which facilitate cancer growth
and metastasis.
4.1 Role of DNMTs and Alterations in
Global Methylation
In both healthy and cancer cells, DNA methylation is performed
by the DNMT family of enzymes that transfer a methyl group
from S-adenosyl-L-methionine to cytosine (45). Many cancers
have overexpression or increased activity of DNMT1, DNMT3a
and/or DNMT3b (46). One study examined the expression levels
of DNMT1, DNMT3a and DNMT3b in a series of 63 GEP-NETs
using immunohistochemistry (47). The authors found that
DNMT1, DNMT3a and DNMT3b expression was detected in
87%, 81% and 75% of tumors, respectively. Further, the
expression of DNMT3b was significantly elevated in poorly
differentiated GEP-NECs when compared to well-differentiated
GEP-NETs and NECs, and elevated in foregut tumors compared
to mid- or hindgut NETs (47). The Simon et al. study performed
DNA methylation profiling of the 57 pNENs (43 pNET, 14
pNEC) and was able to delineate pNET tumors from NEC based
on t-distributed stochastic neighbor embedding (tSNE) analysis
of methylation profiles (39).
Several early studies also examined global methylation
patterns in small intestinal NETs (SI-NETs). A study of 20
primary SI-NETs and their metastases revealed decreased
global methylation in metastases when compared to their
primary tumors (48). Long interspersed nucleotide element 1
(LINE1) methylation, which can be used as a surrogate for global
methylation patterns, was measured in 58 GEP-NETs (14 gastric,
15 pancreatic, 17 small intestinal, 8 appendiceal, and 4
colorectal). LINE1 hypomethylation was detected in 50% of
gastric, 100% of pancreatic and colorectal, 82% small intestinal,
and 87.5% of appendiceal tumors. Again, matched metastases
exhibited decreased global methylation compared to their
FIGURE 2 |Mechanisms of epigenetic regulation in GEP-NETs. Regulation of gene expression in GEP-NETs is controlled in part by DNA methylation, histone
methylation and histone acetylation. Addition of methyl groups to cytosines in CpG islands of genes, along with hypermethylation and hypoacetylation of histones
results in gene silencing. DNMT, DNA methyltransferases; HATs, histone acetyltransferases; HDAC, histone deacetylases; KDM, histone lysine demethylases;
HMT, histone methyltransferases. Adapted from “Epigenetic Levels”by BioRender.com (2022), Retrieved from https://app.biorender.com/biorender-templates.
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primary tumor counterparts (49). These smaller studies formed
the foundation for a larger study performed by Karpathakis, et al.
wherein the global landscape of 97 SI-NETs was analyzed using
an integrated genomic approach. Based on this data, the authors
categorized SI-NET tumors into three groups based on PFS after
primary tumor resection. Twenty-one genes were identified with
changes in methylation in as many as 85% of tumors (50). The
more recent study by Yachida, et al. also evaluated methylation
profiles and using unsupervised hierarchical cluster analysis of
methylation status, divided their samples into three groups.
pNETs were grouped into one group, non-pNETs in another,
and the remaining group featured a CpG island methylator
phenotype (CIMP) due to alterations in genes related to
DNA methylation and/or due to MLH1 promoter
hypermethylation (43).
4.2 Altered Promoter Methylation of Genes
in GEP-NENs
Specific genes impacted by promoter methylation have been
identified in GEP-NENs and include RASSF1 (51–57), CDKN2A
(51,52,55,58,59), TIMP3,MGMT (51,52,55,60), MLH1 (61),
and IGF2 (62) genes, among others. Methylation of these genes
correlates with downregulated expression in GEP-NENs –often
with molecular differences based on tumor tissue of origin.
Downregulated expression of genes can also be the result of
silencing by gene loss as noted by Capurso, et al. Many of the
same genes silenced by hypermethylation, can also be genetically
lost in GEP-NENs (63), suggesting a selective advantage for the
tumor by inhibiting key proliferative processes within the cell.
4.2.1 RASSF1
Ras-association domain gene family 1 (RASSF1) functions as a
tumor suppressor gene that works through the cell cycle to arrest
cells in G1 (64). Downregulation of RASSF1 is a result of
epigenetic silencing by promoter methylation or allelic loss of
3p21.3, and results in cell cycle activation due to an accumulation
of cyclinD1. The RASSF1 gene includes a control region with two
promoters, termed A and C, which control the production of
eight RASSF1 isoforms. Selective methylation leads to only two
of the isoforms being produced, RASSF1A and RASSF1C.
RASSF1A not only regulates cellular proliferation, but also
influences apoptosis and stabilization of microtubules, and
downregulation has been reported in many cancers, including
GEP NETs and other cancers of neuroendocrine origin (51–55,
64). Overexpression of RASSF1C occurs primarily in pNETs and
is thought to inhibit beta-catenin within the Wnt signaling
pathway, suggesting a role in development of pNETs (65).
In a collection of 48 pNETs, 87% of tumors were positive for
aberrant methylation in at least one of 11 queried genes, with
RASSF1A being hypermethylated in 75% of tumors when
compared to non-tumor surrounding tissue. Furthermore,
tumor size and aggressiveness correlated with methylation
status, with tumors >5cm and those associated with lymph
node or liver metastasis demonstrating a higher frequency of
hypermethylation (55). Similarly, Malpeli, et al. noted an inverse
correlation between RASSF1A expression and gene methylation
when studying a group of 20 primary pNETs. In this study,
the expression of RASSF1A was 6.8 times lower than normal
tissue (57).
A study of 118 well differentiated fore- and midgut GEP-
NETs (including 46 pancreatic, and 72 extrapancreatic) analyzed
11 genes and found that 71.3% of tumors had hypermethylation
of RASSF1A (51). Liu, et al. studied a different set of 47 GEP-
NETs including pancreatic (n=16), nonilieal (including lung,
gastric, duodenum, appendix, colon and rectum; n=15), and
ilieal (n=16) tumors. Hypermethylation of RASSF1A was found
in 57% of tumors with no significant correlation to site of origin,
and was also associated with lymph node metastasis (52).
Another study examined 62 gastrointestinal NETs and found
aberrant methylation of RASSF1A in 32% of cases (all of foregut
origin), cyclin D1 hyperexpression in 53% of cases, and deletion
of 3p21.3 in 26% of tumors (53). Zhang, et al. analyzed 33 small
bowel and matched metastatic tumors by methylation-specific
PCR (56). RASSF1A was methylated in 60% of primary tumors
and 84.8% of metastatic sites (56). Collectively, these studies
suggest a significant role for methylation of RASSF1A in
progression and metastasis of GEP-NENs. In vitro work from
Pizzi, et al. shows that after treatment with 5-aza-2’-
deoxycytidine or decitabine, RASSF1A mRNA was reexpressed
(53), suggesting a treatment mechanism that may have promise
for GEP-NENs.
4.2.2 CDKN2A
The cyclin dependent kinase inhibitor 2A (CDKN2A)geneis
located on chromosome 9p21.3 and generates several different
transcripts through alternative splicing of the first exon. P16ink4a
functions as an inhibitor of CDK4, and p14ARF functions as a
stabilizer of p53 –both acting as tumor suppressors towards the
common goal of cell cycle G1 checkpoint control. CDKN2A loss
by promoter hypermethylation has been demonstrated in a
number of cancers (66,67), including GEP-NENs. A study from
Liu, et al. indicated methylation present upstream of the p14ARF
gene in 49% and upstream of p16ink4a gene 25% of the 47 well-
differentiated NEN tumors analyzed (52). Bartsch, et al. identified
only 2/17 (11.7%) pNET insulinomas with aberrant methylation
of the p16ink4a promoter region (59). In a study of 48 well-
differentiated pancreatic NETs, p16ink4a was hypermethylated in
40% of the cases, yet none of the pNETs in this study exhibited
hypermethylation of p14ARF (55). A contradicting study from
2004 indicates that in a study of 29 GEP-NENs, no
hypermethylation of p16ink4a was observed (58) consistent with
an additional study of 118 fore- and midgut NETs that found no
evidence of p16ink4a methylation (51). In aggregate, these data
suggest that hypermethylation of the CDKN2A promoter may
contribute to tumorigenesis in only a subset of GEP-NENs.
4.2.3 MGMT
O-6-methylguanine-DNA methyltransferase (MGMT) is a DNA
repair protein that catalyzes the transfer of methyl groups from O-
6-alkylguanine and to repair toxic lesions resulting from DNA
alkylation. MGMT promoter methylation is linked to several
cancer types, including GEP-NENs (68), and is predictive of
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tumor response to alkylating agents such as temozolomide.
Indeed, MGMT was found to be hypermethylated in 40% of the
tumors in a study of 48 pNETs (55). Another study of 118 well
differentiated fore- and midgut GEP-NETs from 71 patients
evaluated the promoter methylation of MGMT, and found that
16.1% of tumors had hypermethylation (51). Interestingly, within
this group of tumors, gastrinomas had a significantly reduced
MGMT promoter methylation when compared to insulinoma
(p=0.023). MGMT methylation occurred in 13% of GEP-NENs
(n=47), but not in those tumors of ileal neuroendocrine origin
(52). Further, Kulke, et al. found MGMT deficiency in 19/37 (51%)
pNETs by immunohistochemistry, while no MGMT deficiency
was identified in GI-NETs. In this study, patients with pNETs had
a much better response to temozolomide, and this association with
clinical response may be due to MGMT deficiency (69). The
ongoing MGMT-NET trial (NCT03217097) is studying whether
MGMT methylation status is a predictive factor for response to
alkylating agents (70).
4.2.4 TIMP3
Other gene loci have been identified as having decreased gene
expression as a result of promoter methylation in GEP-NENs.
Wild, et al. identified methylation of tissue inhibitor of
metalloproteinase-3 (TIMP3)in8/18pNETsamples,along
with corresponding loss or reduction in protein expression (71,
72). TIMP-3 could play a role in metastasis as evidenced by
detection of methylation changes in 79% metastatic versus 14%
of nonmetastatic pNETs. Similarly, in a study of 56 sporadic G1
and G2 pNETs, TIMP3 methylation correlated with stage IV
aggressive cancers with poor prognosis (73). Whereas TIMP3
expression has been noted in gastric cancer methylation of this
gene in NENs of gastric/small intestinal origin has not
been identified.
4.2.5 Other Genes –IGF2, MLH1, APC
Insulin like growth factor 2 (IGF2) is an imprinted locus on
chromosome 11p15.5 and overexpression has been linked to
hypermethylation of the CpG islands in the differentially
methylated region 2. Development of insulinoma has been
linked to the loss of imprinting and overexpression of IGF2
(74,75).
Other gene loci including RAR,MLH1,E-cadherin,APC and
p73 exhibited hypermethylated in 25%, 23%, 23%, 21% and 17%
of tumors, respectively, in a study of 48 pNET (55). Mei, et al.
observed MLH1 methylation in 15/48 (31%) of insulinomas (76).
Another study of 29 GEP-NENs found only one tumor
hypermethylated at the hMLH1 promoter but APC was
hypermethylated in 65% (58).
4.3 MEN1 Status in DNA Methylation
In pNETs, the presence of mutations in MEN1 also contributes
to methylation status (77,78). In a study of 29 pNETs, CpG
hypermethylation correlated with MEN1 loss when compared to
sporadic or VHL-associated pNETs (77). In a followup study,
Tirosh, et al. examined 96 GEP-NEN samples by genome-wide
methylation assays (querying a total of 835,424 CpGs). The
methylome signature was compared between MEN1-related,
VHL-related and sporadic pNET tumors. Unsupervised
hierarchical clustering analysis identified two groups, with
VHL-related tumors exhibiting marked DNA hypomethylation,
and MEN1-related and sporadic tumors clustering together,
although each group with a distinct methylation profile (79).
Further, the authors identified elevated APC promoter
hypermethylation in MEN1-related tumors (79)–anew
finding that supported previous identification of APC
mutations in 7/46 NET samples (80)
Coneman, et al. specifically analyzed MEN1-related pNETs
for promoter methylation using methylation-specific multiplex
ligation dependent probe amplification in 61 MEN1-related
pNETs versus 34 sporadic pNETs (78). The cumulative
methylation index (CMI; the sum of methylation percentages
of all genes analyzed) showed no significant difference between
the groups, but within the MEN1-related pNET group the CMI
was significantly higher in larger pNETs. CASP8 was one of only
a few genes that demonstrated elevated promoter methylation in
the MEN1-related pNET group compared to the sporadic
pNETs (78).
Chan and colleagues found that 58% of pNETs (n=64),
contained combined mutations in ATRX,DAXX, and MEN1
(ADM mutant), a signature that also correlated with poorer
clinical outcomes (81). Methylation analysis revealed a profile of
hypermethylation in 59 genes in the ADM mutant tumors, with
13 of these genes demonstrating reduced expression. Seven genes
were hypomethylated and overexpressed, including APOH,
CCL15,EMID2,PDZK1,HAO1,BAIAP2L2 and NPC1L1 (81).
Of note, the PDX1 promoter in the ADM mutant tumors was
hypermethylated at all 4 CpG islands and exhibited decreased
expression compared to pNETs without the ADM mutations.
The authors of the study suggest trans-differentiation of the
tumor cells during development, a finding further supported by
Cejas, et al. for nonfunctional pNETs (82).
4.4 DNA-Methylation Based Therapeutics
The cellular enzymes controlling the addition of DNA
methylation marks are DNA methyl transferases (DNMTs)
(83). The function, or more accurately the dysfunction,of
these enzymes has an enormous impact on gene regulation in
cancers, including GEP-NETs. To combat cancers driven by
this mechanism, DNMT inhibitors have been developed,
including azacitidine, decitabine, and guadecitabine (a
dinucleotide prodrug of decitabine+guanine). All are
approved by the FDA and function as nucleoside analogs to
inducedoublestrandedDNAbreaks and subsequent apoptosis
in actively dividing cells (84). These compounds are systemic in
action and do not target a particular methylated CpG promoter
region, but have been shown to promote re-expression of tumor
suppressor genes. Efficacy of DNMT inhibitors in vitro shows
promise: azacitidine caused a dose-dependent reduction in
NET cell lines BON1, H727 and CNDT2.5 (85)and
decitabine decreased proliferation of QGP1 cells (86).
However, in other cancers the therapeutic potential of
decitabine is decreased due to compound instability and
dose-limiting hematological toxicities (87), suggesting the
need for new compounds without these liabilities.
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5 CHROMATIN REMODELING
DNA wraps around histone proteins to form nucleosomes which
then compact to form chromatin. Histone proteins can be
modified by the addition of acetyl, methyl, phosphoryl or
ubiquitin groups to specific residues, often present in the N-
terminal histone tails. Modification of histones in this way
changes the ability of transcription factors to bind chromatin,
altering gene expression. The histone complex normally includes
two copies of H2A, H2B, H3 and H4 along with one copy of H1.
Histone H1 is a family of linker histones that plays a role in
chromatin stability, regulating gene expression, and participating
in chromatin-based DNA repair. Histones can also have variants,
resulting in modified histones, that have been identified in a
variety of cancers, including GEP-NETs. A study of 13 primary
NETs, showed the histone H1 family member H1x was
overexpressed as measured by immunohistochemistry in tumor
compared to surrounding normal tissue (88).
The novel identification of mutations in ATRX and DAXX in
17% and 25% of cases respectively, suggested alternative
lengthening of telomeres (ALT) (89) as a contributing
mechanism to pNET development. ATRX and DAXX are
chromatin remodeling complex proteins that are required to
incorporate histone variant H3.3 into chromosomal telomeres.
In a study performed by Heaphy, et al, there was perfect
correlation between the loss of ATRX/DAXX nuclear
expression and the presence of ALT as measured by telomere-
specificfluorescence in situ hybridization (89). Scarpa, et al.
confirmed the mutation of ATRX/DAXX and the presence of
ALT in 33/98 pNETs (38). Interestingly, the combination of
mutations in MEN1,ATRX and DAXX,orMEN1 with either
ATRX or DAXX was associated with better outcomes compared
to pNETs lacking these mutation combinations (37).
A study of structural rearrangements in pNETs identified
chromosomal rearrangements in several genes involved in
chromatin remodeling including SETD2 (a histone lysine
methyltransferase), ARID2 (involved in chromatin structure
modification), KMT2C/MLL3 (histone lysine methyltransferase)
and SMARCA4 (part of the SWI/SNF chromatin remodeling
complex) (38). SETD2 and ARID2 were identified as having
mutations in 18 and 13% of advanced well differentiated pNETs,
respectively (90).
5.1 Histone Acetylation
Histone remodeling by addition/removal of acetylation is
facilitated by two enzymes (Figure 2). Histone deacetylases
(HDACs) catalyze the removal of acetyl groups from specific
lysine residues in histone proteins allowing for closing of
chromatin and gene silencing. Histone acetyltransferases
(HATs) do the opposite and mediate acetylation of histones
resulting in a reduction in the positive charge on the surface of
histones to loosen the attraction between DNA and the histones
resulting in more open chromatin and gene accessibility for
transcriptional activation (65).
Expression of all classes of HDACs (I, IIA, IIB, III and IV) was
measured in a study of 57 pNETs on tissue microarrays (91).
Significant upregulation (1.5 to >7 fold) of all HDACs was
identified in pNET versus control, with the greatest increases
in expression noted in the G3 tumors. The association of elevated
HDAC expression with tumor grade, markers of proliferation
and patient survival elevated inhibitors of HDACs as a potential
therapy for pNETs and other neuroendocrine tumors (91). In
addition to HDAC expression levels, functional histone
acetylation has been studied in MEN1-related pNETs. After
acetylation marks are written by HATs, these marks are read
by the bromodomain and extraterminal (BET) protein. JQ1, a
small molecule inhibitor of BET protein-protein interaction,
decreased proliferation and apoptosis in an MEN1-dependent
model of insulinoma (92).
The first histone deacetylase inhibitor (HDACi) to receive
FDA approval was vorinostat in 2006 for the treatment of
cutaneous T-cell lymphoma, and the history of HDACi
development has been recently reviewed (93,94). There is
significant preclinical evidence in NET cell lines suggesting
efficacy in GEP-NENs and in vitro studies performed with
HDACi like trichostatin A, sodium butyrate, valproic acid and
entinostat on pNET cell lines demonstrate dose dependent
inhibition on cancer-related endpoints of cellular proliferation,
apoptosis and cell cycle arrest (94–97). Preliminary clinical
evidence evaluating panobinostat in GEP-NETs demonstrated
stable disease in treated patients, but showed no effect on tumor
volume (98). Additional clinical trials evaluating HDACi in
GEP-NENs alone and in combination with mTOR inhibitors
(everolimus/emsirolimus) or anti-PD-L1 immunotherapy
(sintilimab) are in progress.
5.2 Histone Methylation
Methylation of core histones, particularly on H3 and H4, is a
well-known phenomenon in cancer. Histone methylation can
either be repressive or activating, depending on the histone, the
location of the amino acid (lysine or arginine) to be modified,
and the extent of methylation (i.e. di-, or trimethylation; me2 or
me3, respectively). H3K9me2/3, H3K27me2/3 and H4K20me3
are known inhibitory methylation marks, while H3K4me2/3,
H2K36me3 and K3K79me3 are known activating marks. Histone
methylation is performed in the cell by histone methyl
transferases (HMTs) and histone demethylases (HDMs), often
in complexes with many cofactors (Figure 2).
An early study in NENs used immunohistochemistry to
evaluate the presence of LSD1, a histone demethylating
enzyme, and the methylating complex Ash2, using a tissue
microarray that included 16 primary intestinal NECs. Strong
immunostaining was present in 100% of NEC for Ash2 and 87%
for LSD1. 93% of the tumors showed demethylation at H3K4,
with only weak staining in surrounding normal tissue (99).
Similarly, immunostaining for histone lysine methyltransferase
enhancer of zeste homolog 2 (EZH2) indicated high differential
expression of EZH2 in small intestinal NETs (100). EZH2
catalyzes H3K27me3 marks to function as a master regulator
in a variety of cellular processes, including cancer, and its role in
cancer is linked with high proliferation rates, metastasis and poor
overall survival. In vitro, EZH2 inhibitors (CPI-1205/
Lirametostat and GSK126) decreased cell viability, proliferation
and migration capacity while increasing apoptosis in GI-NET
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Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 9014357
cell lines (100). Histone methyltransferase inhibitors (HMTis)
still in active development include EZH2 inhibitors CPI-1205/
Lirametostat, Tazemetostat, and SHR2554, along with
Pinometostat (DOT1L inhibitor), and should be considered the
next frontier for pNET therapy (101).
Much of the work to understand histone methylation
has been performed using MEN1/menin-related pNETs
and model systems (102,103). Menin plays a key role in
epigenetic regulation by interacting with the histone lysine
methyltransferases (MLL1/KMT2A and MLL2/KMT2B) (104)
to influence writing of H3K4me3 methylation marks. Indeed,
genome-wide studies of H3K4 methylation in pancreatic islets
(105) indicate that loss of Men1 alters the epigenetic landscape of
its target genes such as insulin like growth factor binding protein
2(Igf2bp2), p18ink4c (CDKN2C) and p27kip1 (CDKN1B) (106).
In mouse models, decreased expression of Igfbp2 is accompanied
by changes in H3K4 and H3K27 histone methylation at the
promoter, changes that can be partially abrogated by deletion of
the histone demethylase retinoblastoma binding protein 2
(Rbp2). This suggests that Rbp2 can reverse changes induced
by the Menin/MLL complexes (105).
The histone lysine demethylase RBP2/KDM5A/JARID1A
may also be involved in regulation of GEP-NEN tumorigenesis.
RBP2 was overexpressed in a 20/25 human NEN primary tumors
and metastases, with elevated expression most evident in the
metastatic sites (107). Further in vitro study in NET cell lines
demonstrated that overexpression of RBP2 significantly
increased proliferation, migration, invasion and colony
formation, whereas knockdown of RBP2 decreased the same
parameters in a demethylase-dependent manner supporting the
hypothesis that aberrant RBP2 expression and altered histone
demethylation, is a frequent contributing factor to NET
pathogenesis (108).
Histone modification does not always function independently
and there is a unique interplay between DNMTs and histone
methyltransferases in the context of malignancy (83). In general,
active chromatin regions are characterized by acetylated histones
and unmethylated DNA, while inactive regions of the genome
are associated with methylated histones and methylated DNA
(109). These two epigenetic mechanisms are intertwined such
that DNA methylation guides histone modifications, and vice
versa. Although not performed in GEP-NENs, two studies have
demonstrated that EZH2-written H3K27 methylation marks
directly control DNA methylation (110,111), suggesting that
abnormal methylation in tumor cells can be intimately linked
with histone modification.
Protein arginine methyltransferase (PRMT5) functions with
cofactors to mediate methylation of histones H2A and H4 at
arginine 3, and H3 at arginine 8 (112). In vitro work in
neuroendocrine cells demonstrates a direct binding between
PRMT5 and menin at Gas1 and Gli1 promoters, resulting in
repressive H4R3me2s methylation marks, decreased Gas1 and
Gli1 expression, and decreased cell proliferation that is
independent of classical Hedgehog signaling (30,113). pNETs
are known to have elevated GLI1 levels in the absence of menin
(113), implying that inhibition of GLI1 could suppress formation
of MEN1-related neuroendocrine tumors. Although not yet
tested in GEP-NENs, loss of the enzyme methylthioadenosine
phosphorylase (MTAP) due to genetic deletion of the nearby
CDKN2A tumor suppressor may confer dependence on PRMT5
arginine methyltransferase activity as happens in other cancers
(114). Inhibition of PRMT5 showed decreased cell viability in
preclinical testing and as a result, several PRMT5 inhibitors
(GSK3326595, AMG193, JNJ64619178/Onametostat,
PF06939999, TNG908, PRT811, PRT543, and MRTX1719)
have been developed and are in early phase clinical
development for metastatic solid tumors and lymphoma
demonstrating MTAP deletion. More work is needed in GEP-
NENs to elucidate the role of PRMT5 and potentially other
arginine methyltransferases in disease progression.
6 MIRNA REGULATION
miRNAs are small (18-23 nucleotide), noncoding RNA
modulators of gene expression that are implicated in the
control of critical processes involved in tumor development
and metastasis by inducing inhibition and/or degradation of
target mRNAs. miRNAs can be easily detected in biological fluids
such as blood, saliva, urine, et cetera and also detected in
exosomes (115). MiRNAs have potential utility as prognostic
or diagnostic biomarkers as well as therapeutic targets, and
recent data also suggests miRNAs may also play a role in
therapy sensitivity and/or resistance (116).
Several miRNA profiling studies have been performed on
NENs towards the goal of identifying miRNAs that hold
promiseasbiomarkerstodistinguish between the different
tissues of origin within GEP-NENs as well as pancreatic
acinar cell carcinoma (9,117,118).Onecasereportanalyzed
the miRNA signature of normal pancreas, gastrinoma and
pNET from a single MEN1 patient and highlighted sets of
miRNAs that fall into two gene regulatory networks with
distinct functional features (119). Klieser identified a
“proliferative”miRNA signature that correlated with
expression of HDAC as well as staging, grade and hormone
activity in pNETs (120). Taken together, these data confirm
much of the previous work focused on selected miRNAs such as
miR-24 (121,122), miR-21 (123), and others as reviewed
recently (9) and is a critical step towards understanding the
prognostic and diagnostic role of miRNAs.
Therapeutically, there are two current approaches in the
preclinical stages of development that show promise for
pNETs. The first uses stabilized miR-targeted locked nucleic
acids that bind tightly to their target gene to block oncogenic
miRNAs from interacting (124) and the second strategy uses
antagomiRs, or miRNA mimetics (125) to restore function of
tumor suppressing miRNAs. The field of nucleic acid-based
therapeutics is growing exponentially based on past
experiences in gene therapy, but challenges such as delivery
modes and stability of RNAs remain. As biomarkers, miRNAs
can be monitored as a measure of treatment response. For
example, miRNA profiles correlated with pNET treatment
response to metformin-induced decreased proliferation in cell
lines (126).
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Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 9014358
7 REGULATION OF SSTR
SSTR2 and SSTR5 are the most frequently expressed SSTRs in
GEP-NENs and are the target of somatostatin analog therapy.
Recently, a natural antisense transcript, SSTR5-AS1, was
identified that regulates the production and availability of
SSTR5. Examination of expression levels in 15 pNET and
adjacent normal tissue revealed that the expression level of
SSTR5-AS1 was significantly higher in tumor samples, while
SSTR5 expression was similar in both regions. Further studies in
BON1 and QGP1 cells suggest that the expression may be linked
as silencing SSTR5 with siRNA decreased expression of SSTR5-
AS1. Methylation of CpG islands upstream of SSTR5 may be
associated with upregulated expression of SSTR5 and SSTR5-
AS1. SSTR5-AS1 promotes aggressiveness in pNET cells and
may be involved in the limited response of these cells to
pasireotide (127).
SSTR2 is also regulated by DNA methylation and histone
modifications. BON1 and QGP1 cell lines both express SSTR2 at
a relatively low level, despite mRNA levels greater than that seen
in other cancer types and expression of SSTR2 is inversely related
to the level of CpG island methylation (128). Further, treatment
of BON1 cells with the DNMT inhibitor 5-aza-2’-deoxycytidine
revealed stimulation of SSTR2 (129). Histone acetylation is also
likely to be involved in regulation of SSTR2 levels in GEP-NETs
and several in vitro studies in NET cell lines have investigated
HDACi (romidepsin/FK228, vorinostat/SAHA, and AB3 along
with valproic acid) with varying results (130,131). Overall, these
studies suggest the involvement of epigenetic mechanisms in the
regulation of SSTR2 which may be capitalized upon to
upregulate SSTR2 in patients with low SSTR levels prior to
treatment with PRRT.
8 DISCUSSION AND FUTURE
PERSPECTIVES
GEP-NENs are a complex, very diverse family of tumors with
many germline and somatic genetic abnormalities identified. In
recent years, studies are revealing more about the epigenetic
regulation that overlays the identified genetic mutations. Great
strides have been made towards understanding the regulation of
gene expression by DNA methylation, histone modifications and
miRNAs in this group of tumors.
As with all new knowledge, our understanding of the
epigenetics of GEP-NENs provokes addition questions about
the etiology of these tumors, and the best approaches to take in
patient management. There are newly revealed avenues of
investigation that should be pursued to study the impact of
therapies like PRMT5 inhibitors, EZH2 inhibitors, BET
inhibitors like JQ1 (although chemical modification may be
necessary to circumvent dose limiting adverse events), and
combination strategies that could include HDAC inhibitors
teamed with mTOR tyrosine kinase inhibitors, as has been
done with other cancer types (132). More study is needed to
understand the roles of different HDAC family members in NET
development as proposed by Gagliano (94), as well as the efficacy
and hopefully, reduced toxicity of selective HDAC inhibitors. For
example, combination strategies such as EZH inhibitors teamed
with metformin have been pursued in vitro in SI-NETs with
growth arrest and increased apoptosis in GOT1 spheroid models
(100). EZH2 inhibitors combined with dopamine receptor D2
antagonists showed measurable promise in an organoid model of
androgen receptor-insensitive neuroendocrine prostate tumors
(133). Finally, mTOR inhibitors combined with azacitidine also
showed antiproliferative and apoptotic activity in medullary
thyroid carcinoma (134), with the suggestion that this
combination may also have efficacy in neuroendocrine tumors
for which mTOR inhibitors are approved.
The concept of stochastic epigenetic mutations, or a measure
of life-course accumulation of exposure-related, epigenetic
damage as a function of evolution, is also understudied in
cancers in general. Feinberg and Irizarry originally proposed
this model wherein epigenetic variation as a response to
environmental stimuli is a mechanism for inherited,
evolutionary adaptation (135). Recent studies have
demonstrated correlations between SEM and a higher risk of
breast cancer, liver cancer, lung cancer, and mature B-cell
neoplasms (136,137). This has not been studied in GEP-NENs.
Further, there are other completely unexplored territories
including the interplay of signaling pathways. For example, the
role of genes like RASSF1A that are known to have decreased
expression in NETs due to promoter hypermethylation or gene
loss, and how these proteins interact with signaling pathways
such as Notch. A recent publication demonstrates how loss of
RASSF1A expression allows for tumor dedifferentiation and
proliferation as a result of accumulated Hes1, suggesting a role
for RASSF1A/Notch crosstalk in GEP-NENs that has not yet
been explored (138,139). Further, the lysine demethylase protein
KDM5A/RBP2, shown to be overexpressed in GEP-NENs (107),
is a key component of the CSL repressor complex in Notch
signaling and also may play a role in epigenetic regulation of
cancer cell proliferation and stemness. All of this evidence
provides a foothold for epigenetic changes as a key player in
GEP-NEN development, progression, metastasis, and response
to treatment. The large scale omics approaches are beginning to
reveal the mechanisms, both genetic and epigenetic, of
tumorigenesis. Such comprehensive, integrated approaches
teamed with appropriately powered clinical trials based on
specific, molecular-based therapeutics is expected to have a
direct clinical impact on the management of GEP-NENs.
AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and
has approved it for publication.
FUNDING
This work is supported by the Louisiana State University Health
Sciences Center School of Medicine, Department of Genetics.
Crabtree Epigenetics of GEP NETs
Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 9014359
REFERENCES
1. Dasari A, Shen C, Halperin D, Zhao B, Zhou S, Xu Y, et al. Trends in the
Incidence, Prevalence, and Survival Outcomes in Patients With
Neuroendocrine Tumors in the United States. JAMA Oncol (2017) 3
(10):1335–42. doi: 10.1001/jamaoncol.2017.0589
2. Huguet I, Grossman AB, O’Toole D. Changes in the Epidemiology of
Neuroendocrine Tumours. Neuroendocrinology (2017) 104(2):105–11.
doi: 10.1159/000441897
3. Leoncini E, Boffetta P, Shafir M, Aleksovska K, Boccia S, Rindi G. Increased
Incidence Trend of Low-Grade and High-Grade Neuroendocrine
Neoplasms. Endocrine (2017) 58(2):368–79. doi: 10.1007/s12020-017-
1273-x
4. Modlin IM, Moss SF, Chung DC, Jensen RT, Snyderwine E. Priorities for
Improving the Management of Gastroenteropancreatic Neuroendocrine
Tumors. J Natl Cancer Institute (2008) 100(18):1282–9. doi: 10.1093/jnci/
djn275
5. Hanahan D, Weinberg RA. The Hallmarks of Cancer. Cell Jan 7 (2000) 100
(1):57–70. doi: 10.1016/S0092-8674(00)81683-9
6. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell
Mar 4 (2011) 144(5):646–74. doi: 10.1016/j.cell.2011.02.013
7. Walenkamp A, Crespo G, Fierro Maya F, Fossmark R, Igaz P, Rinke A, et al.
Hallmarks of Gastrointestinal Neuroendocrine Tumours: Implications for
Treatment. Endocr Relat Cancer (2014) 21(6):R445–60. doi: 10.1530/ERC-
14-0106
8. Crabtree JS. Clinical and Preclinical Advances in Gastroenteropancreatic
Neuroendocrine Tumor Therapy. Front Endocrinol (Lausanne) (2017)
8:341. doi: 10.3389/fendo.2017.00341
9. Havasi A, Sur D, Cainap SS, Lungulescu CV, Gavrilas LI, Cainap C, et al.
Current and New Challenges in the Management of Pancreatic
Neuroendocrine Tumors: The Role of miRNA-Based Approaches as New
Reliable Biomarkers. Int J Mol Sci (2022) 23(3):1109–29. doi: 10.3390/
ijms23031109
10. Rinke A, Auernhammer CJ, Bodei L, Kidd M, Krug S, Lawlor R, et al.
Treatment of Advanced Gastroenteropancreatic Neuroendocrine Neoplasia,
are We on the Way to Personalised Medicine? Gut Sep (2021) 70(9):1768–
81. doi: 10.1136/gutjnl-2020-321300
11. Bocchini M, Nicolini F, Severi S, Bongiovanni A, Ibrahim T, Simonetti G,
et al. Biomarkers for Pancreatic Neuroendocrine Neoplasms (PanNENs)
Management-An Updated Review. Front Oncol (2020) 10:831. doi: 10.3389/
fonc.2020.00831
12. Rindi G, Klimstra DS, Abedi-Ardekani B, Asa SL, Bosman FT, Brambilla E,
et al. A Common Classification Framework for Neuroendocrine Neoplasms:
An International Agency for Research on Cancer (IARC) and World Health
Organization (WHO) Expert Consensus Proposal. Mod Pathol (2018) 31
(12):1770–86. doi: 10.1038/s41379-018-0110-y
13. Bosman F, Carneiro F, Hruban R, Theise N. WHO Classification of
Tumours of the Digestive System. In: WHO Classification of Tumours,4th
ed. Lyon, France: IARC Press (2010).
14. de Herder WW, Hofland LJ, van der Lely AJ, Lamberts SW. Somatostatin
Receptors in Gastroentero-Pancreatic Neuroendocrine Tumours. Endocr
Relat Cancer (2003) 10(4):451–8. doi: 10.1677/erc.0.0100451
15. Frilling A, Akerstrom G, Falconi M, Pavel M, Ramos J, Kidd M, et al.
Neuroendocrine Tumor Disease: An Evolving Landscape. Endocr Relat
Cancer (2012) 19(5):R163–85. doi: 10.1530/ERC-12-0024
16. Rinke A, Muller HH, Schade-Brittinger C, Klose KJ, Barth P, Wied M, et al.
Placebo-Controlled, Double-Blind, Prospective, Randomized Study on the Effect
of Octreotide LAR in the Control of Tumor Growth in Patients With Metastatic
Neuroendocrine Midgut Tumors: A Report From the PROMID Study Group. J
Clin Oncol (2009) 27(28):4656–63. doi: 10.1200/JCO.2009.22.8510
17. Caplin ME, Pavel M, Cwikla JB, Phan AT, Raderer M, Sedlackova E, et al.
Lanreotide in Metastatic Enteropancreatic Neuroendocrine Tumors. N Engl
J Med (2014) 371(3):224–33. doi: 10.1056/NEJMoa1316158
18. Strosberg J, El-Haddad G, Wolin E, Hendifar A, Yao J, Chasen B, et al. Phase
3 Trial of (177)Lu-Dotatate for Midgut Neuroendocrine Tumors. N Engl J
Med (2017) 376(2):125–35. doi: 10.1056/NEJMoa1607427
19. Strosberg J, Wolin E, Chasen B, Kulke M, Bushnell D, Caplin M, et al.
Health-Related Quality of Life in Patients With Progressive Midgut
Neuroendocrine Tumors Treated With (177)Lu-Dotatate in the Phase III
NETTER-1 Trial. JClinOncol(2018) 36(25):2578–84. doi: 10.1200/
JCO.2018.78.5865
20. Starr JS, Sonbol MB, Hobday TJ, Sharma A, Kendi AT, Halfdanarson TR.
Peptide Receptor Radionuclide Therapy for the Treatment of Pancreatic
Neuroendocrine Tumors: Recent Insights. Onco Targets Ther (2020)
13:3545–55. doi: 10.2147/OTT.S202867
21. Zandee WT, Brabander T, Blazevic A, Kam BLR, Teunissen JJM, Feelders
RA, et al. Symptomatic and Radiological Response to 177Lu-DOTATATE
for the Treatment of Functioning Pancreatic Neuroendocrine Tumors. J Clin
Endocrinol Metab Apr 1 (2019) 104(4):1336–44. doi: 10.1210/jc.2018-01991
22. Yao JC, Shah MH, Ito T, Bohas CL, Wolin EM, Van Cutsem E, et al.
Everolimus for Advanced Pancreatic Neuroendocrine Tumors. N Engl J Med
(2011) 364(6):514–23. doi: 10.1056/NEJMoa1009290
23. Yao JC, Pavel M, Lombard-Bohas C, Van Cutsem E, Voi M, Brandt U, et al.
Everolimus for the Treatment of Advanced Pancreatic Neuroendocrine
Tumors: Overall Survival and Circulating Biomarkers From the
Randomized, Phase III RADIANT-3 Study. JClinOncol(2016) 34
(32):3906–13. doi: 10.1200/JCO.2016.68.0702
24. Raymond E, Dahan L, Raoul JL, Bang YJ, Borbath I, Lombard-Bohas C, et al.
Sunitinib Malate for the Treatment of Pancreatic Neuroendocrine Tumors.
N Engl J Med (2011) 364(6):501–13. doi: 10.1056/NEJMoa1003825
25. Chandrasekharappa SC, Guru SC, Manickam P, Olufemi SE, Collins FS,
Emmert-Buck MR, et al. Positional Cloning of the Gene for Multiple
EndocrineNeoplasia-Type1.Science (1997) 276(5311):404–7.
doi: 10.1126/science.276.5311.404
26. Hosoe S, Brauch H, Latif F, Glenn G, Daniel L, Bale S, et al. Localization of
the Von Hippel-Lindau Disease Gene to a Small Region of Chromosome 3.
Genomics (1990) 8(4):634–40. doi: 10.1016/0888-7543(90)90249-t
27. Wallace MR, Marchuk DA, Andersen LB, Letcher R, Odeh HM, Saulino AM,
et al. Type 1 Neurofibromatosis Gene: Identification of a Large Transcript
Disrupted in Three NF1 Patients. Science (1990) 249(4965):181–6.
doi: 10.1126/science.2134734
28. Young J, Povey S. The Genetic Basis of Tuberous Sclerosis. Mol Med Today
(1998) 4(7):313–9. doi: 10.1016/s1357-4310(98)01245-3
29. Cao Y, Liu R, Jiang X, Lu J, Jiang J, Zhang C, et al. Nuclear-Cytoplasmic
Shuttling of Menin Regulates Nuclear Translocation of {Beta}-Catenin. Mol
Cell Biol (2009) 29(20):5477–87. doi: 10.1128/mcb.00335-09
30. Gurung B, Feng Z, Iwamoto DV, Thiel A, Jin G, Fan C-M, et al. Menin
Epigenetically Represses Hedgehog Signaling in MEN1 Tumor Syndrome.
Cancer Res (2013) 73(8):2650–8. doi: 10.1158/0008-5472.Can-12-3158
31. Heppner C, Bilimoria KY, Agarwal SK, Kester MB, Whitty LJ, Guru SC, et al.
The Tumor Suppressor Protein Menin Interacts With NF-kB Proteins and
Inhibits NF-kB-Mediated Transactivation. Onco 2001/08/01 (2001) 20
(36):4917–25. doi: 10.1038/sj.onc.1204529
32. Chamberlain CE, Scheel DW, McGlynn K, Kim H, Miyatsuka T, Wang J,
et al. Menin Determines K-RAS Proliferative Outputs in Endocrine Cells. J
Clin Invest (2014) 124(9):4093–101. doi: 10.1172/JCI69004
33. Wang Y, Ozawa A, Zaman S, Prasad NB, Chandrasekharappa SC, Agarwal
SK, et al. The Tumor Suppressor Protein Menin Inhibits AKT Activation by
Regulating Its Cellular Localization. Cancer Res (2011) 71(2):371–82.
doi: 10.1158/0008-5472.Can-10-3221
34. Blansfield JA, Choyke L, Morita SY, Choyke PL, Pingpank JF, Alexander HR,
et al. Clinical, Genetic and Radiographic Analysis of 108 Patients With Von
Hippel-Lindau Disease (VHL) Manifested by Pancreatic Neuroendocrine
Neoplasms (PNETs). Surg Dec (2007) 142(6):814–8. doi: 10.1016/
j.surg.2007.09.012
35. Arva NC, Pappas JG, Bhatla T, Raetz EA, Macari M, Ginsburg HB, et al.
Well-Differentiated Pancreatic Neuroendocrine Carcinoma in Tuberous
Sclerosis–Case Report and Review of the Literature. Am J Surg Pathol Jan
(2012) 36(1):149–53. doi: 10.1097/PAS.0b013e31823d0560
36. Mafficini A, Scarpa A. Genetics and Epigenetics of Gastroenteropancreatic
Neuroendocrine Neoplasms. Endocr Rev (2019) 40(2):506–36. doi: 10.1210/
er.2018-00160
37. Jiao Y, Shi C, Edil BH, de Wilde RF, Klimstra DS, Maitra A, et al. DAXX/
ATRX, MEN1, and mTOR Pathway Genes Are Frequently Altered in
Pancreatic Neuroendocrine Tumors. Science (2011) 331(6021):1199–203.
doi: 10.1126/science.1200609
Crabtree Epigenetics of GEP NETs
Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 90143510
38. Scarpa A, Chang DK, Nones K, Corbo V, Patch AM, Bailey P, et al. Whole-
Genome Landscape of Pancreatic Neuroendocrine Tumours. Nature (2017)
543(7643):65–71. doi: 10.1038/nature21063
39. Simon T, Riemer P, Jarosch A, Detjen K, Di Domenico A, Bormann F, et al.
DNA Methylation Reveals Distinct Cells of Origin for Pancreatic
Neuroendocrine Carcinomas and Pancreatic Neuroendocrine Tumors.
Genome Med (2022) 14(1):24. doi: 10.1186/s13073-022-01018-w
40. Ishida H, Lam A-y. Pancreatic Neuroendocrine Neoplasms: Updates on
Genomic Changes in Inherited Tumour Syndromes and Sporadic Tumours
Based on WHO Classification. Crit Rev Oncology/Hematol (2022)
172:103648. doi: 10.1016/j.critrevonc.2022.103648
41. Allen A, Qin ACR, Raj N, Wang J, Uddin S, Yao Z, et al. Rare BRAF
Mutations in Pancreatic Neuroendocrine Tumors may Predict Response to
RAF and MEK Inhibition. PloS One (2019) 14(6):e0217399. doi: 10.1371/
journal.pone.0217399
42. Sigal DS, Bhangoo MS, Hermel JA, Pavlick DC, Frampton G, Miller VA,
et al. Comprehensive Genomic Profiling Identifies Novel NTRK Fusions in
Neuroendocrine Tumors. Oncotarget (2018) 9(88):35809–12. doi: 10.18632/
oncotarget.26260
43. Yachida S, Totoki Y, Noë M, Nakatani Y, Horie M, Kawasaki K, et al.
Comprehensive Genomic Profiling of Neuroendocrine Carcinomas of the
Gastrointestinal System. Cancer Discovery (2022) 12(3):692–711.
doi: 10.1158/2159-8290.Cd-21-0669
44. Moore LD, Le T, Fan G. DNA Methylation and its Basic Function.
Neuropsychopharmacology (2013) 38(1):23–38. doi: 10.1038/npp.2012.112
45. Schubeler D. Function and Information Content of DNA Methylation.
Nature (2015) 517(7534):321–6. doi: 10.1038/nature14192
46. Cheng Y, He C, Wang M, Ma X, Mo F, Yang S, Han J, et al. Targeting
Epigenetic Regulators for Cancer Therapy: Mechanisms and Advances in
Clinical Trials. Signal Trans Target Ther (2019) 4:62. doi: 10.1038/s41392-
019-0095-0
47. Rahman MM, Qian ZR, Wang EL, Yoshimoto K, Nakasono M, Sultana R,
et al. DNA Methyltransferases 1, 3a, and 3b Overexpression and Clinical
Significance in Gastroenteropancreatic Neuroendocrine Tumors. Hum
Pathol (2010) 41(8):1069–78. doi: 10.1016/j.humpath.2010.01.011
48. Verdugo AD, Crona J, Starker L, Stalberg P, Akerstrom G, Westin G, et al.
Global DNA Methylation Patterns Through an Array-Based Approach in
Small Intestinal Neuroendocrine Tumors. Endocr Relat Cancer (2014) 21(1):
L5–7. doi: 10.1530/ERC-13-0481
49. Stricker I, Tzivras D, Nambiar S, Wulf J, Liffers ST, Vogt M, et al. Site- and
Grade-Specific Diversity of LINE1 Methylation Pattern in
Gastroenteropancreatic Neuroendocrine Tumours. Anticancer Res (2012)
32(9):3699–706. PMID: 22993308
50. Karpathakis A, Dibra H, Pipinikas C, Feber A, Morris T, Francis J, et al.
Prognostic Impact of Novel Molecular Subtypes of Small Intestinal
Neuroendocrine Tumor. Clin Cancer Res (2016) 22(1):250–8.
doi: 10.1158/1078-0432.CCR-15-0373
51. Arnold CN, Sosnowski A, Schmitt-Graff A, Arnold R, Blum HE. Analysis of
Molecular Pathways in Sporadic Neuroendocrine Tumors of the Gastro-
Entero-Pancreatic System. Int J Cancer (2007) 120(10):2157–64.
doi: 10.1002/ijc.22569
52. Liu L, Broaddus RR, Yao JC, Xie S, White JA, Wu TT, et al. Epigenetic
Alterations in Neuroendocrine Tumors: Methylation of RAS-Association
Domain Family 1, Isoform A and P16 Genes Are Associated With
Metastasis. Mod Pathol (2005) 18(12):1632–40. doi: 10.1038/
modpathol.3800490
53. Pizzi S, Azzoni C, Bottarelli L, Campanini N, D'Adda T, Pasquali C, et al.
RASSF1A Promoter Methylation and 3p21.3 Loss of Heterozygosity are
Features of Foregut, But Not Midgut and Hindgut, Malignant Endocrine
Tumours. J Pathol (2005) 206(4):409–16. doi: 10.1002/path.1784
54. Dammann R, Schagdarsurengin U, Strunnikova M, Rastetter M, Seidel C,
Liu L, et al. Epigenetic Inactivation of the Ras-Association Domain Family 1
(RASSF1A) Gene and its Function in Human Carcinogenesis. Histol
Histopathol (2003) 18(2):665–77. doi: 10.14670/HH-18.665
55. House MG, Herman JG, Guo MZ, Hooker CM, Schulick RD, Lillemoe KD,
et al. Aberrant Hypermethylation of Tumor Suppressor Genes in Pancreatic
Endocrine Neoplasms. Ann Surg (2003) 238(3):423–31.doi:10.1097/
01.sla.0000086659.49569.9e
56. Zhang HY, Rumilla KM, Jin L, Nakamura N, Stilling GA, Ruebel KH, et al.
Association of DNA Methylation and Epigenetic Inactivation of RASSF1A
and Beta-Catenin With Metastasis in Small Bowel Carcinoid Tumors.
Endocrine (2006) 30(3):299–306. doi: 10.1007/s12020-006-0008-1
57. Malpeli G, Amato E, Dandrea M, Fumagalli C, Debattisti V, Boninsegna L,
et al. Methylation-Associated Down-Regulation of RASSF1A and Up-
Regulation of RASSF1C in Pancreatic Endocrine Tumors. BMC Cancer
Aug 12 (2011) 11:351. doi: 10.1186/1471-2407-11-351
58. Arnold CN, Sosnowski A, Blum HE. Analysis of Molecular Pathways in
Neuroendocrine Cancers of the Gastroenteropancreatic System. Ann N Y
Acad Sci Apr (2004) 1014:218–9. doi: 10.1196/annals.1294.023
59. Bartsch DK, Kersting M, Wild A, Ramaswamy A, Gerdes B, Schuermann M,
et al. Low Frequency of P16(INK4a) Alterations in Insulinomas. Digestion
(2000) 62(2-3):171–7. doi: 10.1159/000007810
60. Ekeblad S, Sundin A, Janson ET, Welin S, Granberg D, Kindmark H, et al.
Temozolomide as Monotherapy Is Effective in Treatment of Advanced
Malignant Neuroendocrine Tumors. Clin Cancer Res (2007) 13(10):2986–
91. doi: 10.1158/1078-0432.CCR-06-2053
61. How-Kit A, Dejeux E, Dousset B, Renault V, Baudry M, Terris B, et al. DNA
Methylation Profiles Distinguish Different Subtypes of
Gastroenteropancreatic Neuroendocrine Tumors. Epigenomics (2015) 7
(8):1245–58. doi: 10.2217/epi.15.85
62. Cao Y, Gao Z, Li L, Jiang X, Shan A, Cai J, et al. Whole Exome Sequencing of
Insulinoma Reveals Recurrent T372R Mutations in YY1. Nat Commun
(2013) 4:2810. doi: 10.1038/ncomms3810
63. Capurso G, Festa S, Valente R, Piciucchi M, Panzuto F, Jensen RT, et al.
Molecular Pathology and Genetics of Pancreatic Endocrine Tumours. J Mol
Endocrinol Aug (2012) 49(1):R37–50. doi: 10.1530/jme-12-0069
64. Shivakumar L, Minna J, Sakamaki T, Pestell R, White MA. The RASSF1A
Tumor Suppressor Blocks Cell Cycle Progression and Inhibits Cyclin D1
Accumulation. Mol Cell Biol (2002) 22(12):4309–18. doi: 10.1128/
mcb.22.12.4309-4318.2002
65. Mapelli P, Aboagye EO, Stebbing J, Sharma R. Epigenetic Changes in
Gastroenteropancreatic Neuroendocrine Tumours. Oncogene (2015) 34
(34):4439–47. doi: 10.1038/onc.2014.379
66. Di Fiore R, D’Anneo A, Tesoriere G, Vento R. RB1 in Cancer: Different
Mechanisms of RB1 Inactivation and Alterations of pRb Pathway in
Tumorigenesis. J Cell Physiol (2013) 228(8):1676–87. doi: 10.1002/jcp.24329
67. Serrano J, Goebel SU, Peghini PL, Lubensky IA, Gibril F, Jensen RT.
Alterations in the P16ink4a/CDKN2A Tumor Suppressor Gene in
Gastrinomas. J Clin Endocrinol Metab (2000) 85(11):4146–56.
doi: 10.1210/jcem.85.11.6970
68. Choi IS, Estecio MR, Nagano Y, Kim DH, White JA, Yao JC, et al.
Hypomethylation of LINE-1 and Alu in Well-Differentiated
Neuroendocrine Tumors (Pancreatic Endocrine Tumors and Carcinoid
Tumors). Mod Pathol (2007) 20(7):802–10. doi: 10.1038/
modpathol.3800825
69. Kulke MH, Hornick JL, Frauenhoffer C, Hooshmand S, Ryan DP, Enzinger
PC, et al. O6-Methylguanine DNA Methyltransferase Deficiency and
Response to Temozolomide-Based Therapy in Patients With
Neuroendocrine Tumors. Clin Cancer Res (2009) 15(1):338–45.
doi: 10.1158/1078-0432.CCR-08-1476
70. Lemelin A, Barritault M, Hervieu V, Payen L, Peron J, Couvelard A, et al.
O6-Methylguanine-DNA Methyltransferase (MGMT) Status in
Neuroendocrine Tumors: A Randomized Phase II Study (MGMT-NET).
Dig Liver Dis (2019) 51(4):595–9. doi: 10.1016/j.dld.2019.02.001
71. Wild A, Ramaswamy A, Langer P, Celik I, Fendrich V, Chaloupka B, et al.
Frequent Methylation-Associated Silencing of the Tissue Inhibitor of
Metalloproteinase-3 Gene in Pancreatic Endocrine Tumors. JClin
Endocrinol Metab (2003) 88(3):1367–73. doi: 10.1210/jc.2002-021027
72. Li Z, Jing Q, Wu L, Chen J, Huang M, Qin Y, et al. The Prognostic and
Diagnostic Value of Tissue Inhibitor of Metalloproteinases Gene Family and
Potential Function in Gastric Cancer. J Cancer (2021) 12(13):4086–98.
doi: 10.7150/jca.57808
73. Stefanoli M, La Rosa S, Sahnane N, Romualdi C, Pastorino R, Marando A,
et al. Prognostic Relevance of Aberrant DNA Methylation in G1 and G2
Pancreatic Neuroendocrine Tumors. Neuroendocrinology (2014) 100(1):26–
34. doi: 10.1159/000365449
Crabtree Epigenetics of GEP NETs
Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 90143511
74. Larsson C. Epigenetic Aspects on Therapy Development for
Gastroenteropancreatic Neuroendocrine Tumors. Neuroendocrinology
(2013) 97(1):19–25. doi: 10.1159/000336087
75. Dejeux E, Olaso R, Dousset B, Audebourg A, Gut IG, Terris B, et al.
Hypermethylation of the IGF2 Differentially Methylated Region 2 is a
Specific Event in Insulinomas Leading to Loss-of-Imprinting and
Overexpression. Endocr Relat Cancer (2009) 16(3):939–52. doi: 10.1677/
ERC-08-0331
76. Mei M, Deng D, Liu TH, Sang XT, Lu X, Xiang HD, et al. Clinical
Implications of Microsatellite Instability and MLH1 Gene Inactivation in
Sporadic Insulinomas. J Clin Endocrinol Metab (2009) 94(9):3448–57.
doi: 10.1210/jc.2009-0173
77. Tirosh A, Mukherjee S, Lack J, Gara SK, Wang S, Quezado MM, et al.
Distinct Genome-Wide Methylation Patterns in Sporadic and Hereditary
Nonfunctioning Pancreatic Neuroendocrine Tumors. Cancer (2019) 125
(8):1247–57. doi: 10.1002/cncr.31930
78. Conemans EB, Lodewijk L, Moelans CB, Offerhaus GJA, Pieterman CRC,
Morsink FH, et al. DNA Methylation Profiling in MEN1-Related
Pancreatic Neuroendocrine Tumors Reveals a Potential Epigenetic
Target for Treatment. Eur J Endocrinol (2018) 179(3):153–60.
doi: 10.1530/eje-18-0195
79. Tirosh A, Killian JK, Petersen D, Zhu YJ, Walker RL, Blau JE, et al. Distinct
DNA Methylation Signatures in Neuroendocrine Tumors Specificfor
Primary Site and Inherited Predisposition. J Clin Endocrinol Metab (2020)
105(10):3285–94. doi: 10.1210/clinem/dgaa477
80. Zehir A, Benayed R, Shah RH, Syed A, Middha S, Kim HR, et al. Mutational
Landscape of Metastatic Cancer Revealed From Prospective Clinical
Sequencing of 10,000 Patients. Nat Med Jun (2017) 23(6):703–13.
doi: 10.1038/nm.4333
81. Chan CS, Laddha SV, Lewis PW, Koletsky MS, Robzyk K, Da Silva E, et al.
ATRX, DAXX or MEN1 Mutant Pancreatic Neuroendocrine Tumors are a
Distinct Alpha-Cell Signature Subgroup. Nat Commun (2018) 9(1):4158.
doi: 10.1038/s41467-018-06498-2
82. Cejas P, Drier Y, Dreijerink KMA, Brosens LAA, Deshpande V, Epstein CB,
et al. Enhancer Signatures Stratify and Predict Outcomes of non-Functional
Pancreatic Neuroendocrine Tumors. Nat Med Aug (2019) 25(8):1260–5.
doi: 10.1038/s41591-019-0493-4
83. Zhang W, Xu J. DNA Methyltransferases and Their Roles in Tumorigenesis.
biomark Res (2017) 5(1):1. doi: 10.1186/s40364-017-0081-z
84. Nunes SP, Henrique R, Jeronimo C, Paramio JM. DNA Methylation as a
Therapeutic Target for Bladder Cancer. Cells (2020) 9(8):1850–79.
doi: 10.3390/cells9081850
85. Alexander VM, Roy M, Steffens KA, Kunnimalaiyaan M, Chen H.
Azacytidine Induces Cell Cycle Arrest and Suppression of
Neuroendocrine Markers in Carcinoids. Int J Clin Exp Med (2010) 3
(2):95–102. doi: 10.1016/j.jss.2009.11.053
86. Habbe N, Bert T, Simon B. Identification of Methylation-Associated Gene
Expression in Neuroendocrine Pancreatic Tumor Cells. Pancreatology
(2007) 7(4):352–9. doi: 10.1159/000107270
87. Samlowski WE, Leachman SA, Wade M, Cassidy P, Porter-Gill P, Busby L,
et al. Evaluation of a 7-Day Continuous Intravenous Infusion of Decitabine:
Inhibition of Promoter-Specific and Global Genomic DNA Methylation.
J Clin Oncol (2005) 23(17):3897–905. doi: 10.1200/JCO.2005.06.118
88. Warneboldt J, Haller F, Horstmann O, Danner BC, Fuzesi L, Doenecke D,
et al. Histone H1x Is Highly Expressed in Human Neuroendocrine Cells and
Tumours. BMC Cancer (2008) 8:388. doi: 10.1186/1471-2407-8-388
89. Heaphy CM, de Wilde RF, Jiao Y, Klein AP, Edil BH, Shi C, et al. Altered
Telomeres in Tumors With ATRX and DAXX Mutations. Science (2011) 333
(6041):425. doi: 10.1126/science.1207313
90. Raj NP, Soumerai T, Valentino E, Hechtman JF, Berger MF, Reidy DL.
Next-Generation Sequencing (NGS) in Advanced Well Differentiated
Pancreatic Neuroendocrine Tumors (WD pNETs): A Study Using MSK-
IMPACT. JClinOncol(2016) 34(4_suppl):246–6. doi: 10.1200/
jco.2016.34.4_suppl.246
91. Klieser E, Urbas R, Stättner S, Primavesi F, Jäger T, Dinnewitzer A, et al.
Comprehensive Immunohistochemical Analysis of Histone Deacetylases in
Pancreatic Neuroendocrine Tumors: HDAC5 as a Predictor of Poor Clinical
Outcome. Hum Pathol (2017) 65:41–52. doi: 10.1016/j.humpath.2017.02.009
92. Lines KE, Stevenson M, Filippakopoulos P, Muller S, Lockstone HE, Wright
B, et al. Epigenetic Pathway Inhibitors Represent Potential Drugs for
Treating Pancreatic and Bronchial Neuroendocrine Tumors. Oncog May
15 (2017) 6(5):e332. doi: 10.1038/oncsis.2017.30
93. Ho TCS, Chan AHY, Ganesan A. Thirty Years of HDAC Inhibitors: 2020
Insight and Hindsight. J Med Chem (2020) 63(21):12460–84. doi: 10.1021/
acs.jmedchem.0c00830
94. Gagliano T, Brancolini C. Targeting Histone Deacetylases for Combination
Therapies in Neuroendocrine Tumors. Cancer Gene Ther (2021) 28(6):547–
50. doi: 10.1038/s41417-020-00260-x
95. Cives M, Simone V, Rizzo FM, Silvestris F. NETs: Organ-Related Epigenetic
Derangements and Potential Clinical Applications. Oncotarget (2016) 7
(35):57414–29. doi: 10.18632/oncotarget.10598
96. Klieser E, Swierczynski S, Mayr C, Schmidt J, Neureiter D, Kiesslich T, et al.
Role of Histone Deacetylases in Pancreas: Implications for Pathogenesis and
Therapy. World J Gastrointest Oncol (2015) 7(12):473–83. doi: 10.4251/
wjgo.v7.i12.473
97. Marini F, Giusti F, Brandi ML. Epigenetic-Based Targeted Therapies for
Well-Differentiated Pancreatic Neuroendocrine Tumors: Recent Advances
and Future Perspectives. Expert Rev Endocrinol Metab (2021) 16(6):295–307.
doi: 10.1080/17446651.2021.1982382
98. Jin N, Lubner SJ, Mulkerin DL, Rajguru S, Carmichael L, Chen H, et al. A
Phase II Trial of a Histone Deacetylase Inhibitor Panobinostat in Patients
With Low-Grade Neuroendocrine Tumors. Oncologist (2016) 21(7):785–6.
doi: 10.1634/theoncologist.2016-0060
99. Magerl C, Ellinger J, Braunschweig T, Kremmer E, Koch LK, Holler T, et al.
H3K4 Dimethylation in Hepatocellular Carcinoma is Rare Compared With
Other Hepatobiliary and Gastrointestinal Carcinomas and Correlates With
Expression of the Methylase Ash2 and the Demethylase LSD1. Hum Pathol
(2010) 41(2):181–9. doi: 10.1016/j.humpath.2009.08.007
100. Barazeghi E, Hellman P, Norlen O, Westin G, Stålberg P. EZH2 Presents a
Therapeutic Target for Neuroendocrine Tumors of the Small Intestine. Sci
Rep (2021) 11(1):22733. doi: 10.1038/s41598-021-02181-7
101. April-Monn SL, Andreasi V, Schiavo Lena M, Sadowski MC, Kim-Fuchs C,
Buri MC, et al. EZH2 Inhibition as New Epigenetic Treatment Option for
Pancreatic Neuroendocrine Neoplasms (PanNENs). Cancers (Basel) (2021)
13(19):5014–32. doi: 10.3390/cancers13195014
102. Feng Z, Ma J, Hua X. Epigenetic Regulation by the Menin Pathway. Endocr
Relat Cancer (2017) 24(10):T147–59. doi: 10.1530/ERC-17-0298
103. Brandi ML, Agarwal SK, Perrier ND, Lines KE, Valk GD, Thakker RV.
Multiple Endocrine Neoplasia Type 1: Latest Insights. Endocr Rev (2021) 42
(2):133–70. doi: 10.1210/endrev/bnaa031
104. Hughes CM, Rozenblatt-Rosen O, Milne TA, Copeland TD, Levine SS, Lee
JC, et al. Menin Associates With a Trithorax Family Histone
Methyltransferase Complex and With the Hoxc8 Locus. Mol Cell (2004)
13(4):587–97. doi: 10.1016/s1097-2765(04)00081-4
105. Lin W, Watanabe H, Peng S, Francis JM, Kaplan N, Pedamallu CS, et al.
Dynamic Epigenetic Regulation by Menin During Pancreatic Islet Tumor
Formation. Mol Cancer Res (2015) 13(4):689–98. doi: 10.1158/1541-
7786.Mcr-14-0457
106. Milne TA, Martin ME, Brock HW, Slany RK, Hess JL. Leukemogenic MLL
Fusion Proteins Bind Across a Broad Region of the Hox A9 Locus,
Promoting Transcription and Multiple Histone Modifications. Cancer Res
(2005) 65(24):11367–74. doi: 10.1158/0008-5472.CAN-05-1041
107. Maggi EC, Trillo-Tinoco J, Struckhoff AP, Vijayaraghavan J, Del Valle L,
Crabtree JS. Retinoblastoma-Binding Protein 2 (RBP2) is Frequently
Expressed in Neuroendocrine Tumors and Promotes the Neoplastic
Phenotype. Oncogenesis (2016) 5(8):e257. doi: 10.1038/oncsis.2016.58
108. Maggi EC, Crabtree JS. Novel Targets in the Treatment of Neuroendocrine
Tumors: RBP2. Int J Endo Oncol (2017) 4(1):31–41. doi: 10.2217/ije-2016-
0022
109. Du J, Johnson LM, Jacobsen SE, Patel DJ. DNA Methylation Pathways and
Their Crosstalk With Histone Methylation. Nat Rev Mol Cell Biol (2015) 16
(9):519–32. doi: 10.1038/nrm4043
110. Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J,
et al. Polycomb-Mediated Methylation on Lys27 of Histone H3 Pre-Marks
Genes for De Novo Methylation in Cancer. Nat Genet (2007) 39(2):232–6.
doi: 10.1038/ng1950
Crabtree Epigenetics of GEP NETs
Frontiers in Oncology | www.frontiersin.org June 2022 | Volume 12 | Article 90143512
111. Vire E, Brenner C, Deplus R, Blanchon L, Fraga M, Didelot C, et al. The
Polycomb Group Protein EZH2 Directly Controls DNA Methylation. Nature
(2006) 439(7078):871–4. doi: 10.1038/nature04431
112. Karkhanis V, Hu YJ, Baiocchi RA, Imbalzano AN, Sif S. Versatility of
PRMT5-Induced Methylation in Growth Control and Development. Trends
Biochem Sci (2011) 36(12):633–41. doi: 10.1016/j.tibs.2011.09.001
113. Gurung B, Feng Z, Hua X. Menin Directly Represses Gli1 Expression
Independent of Canonical Hedgehog Signaling. Mol Cancer Res (2013) 11
(10):1215–22. doi: 10.1158/1541-7786.MCR-13-0170
114. Kryukov GV, Wilson FH, Ruth JR, Paulk J, Tsherniak A, Marlow SE, et al.
MTAP Deletion Confers Enhanced Dependency on the PRMT5 Arginine
Methyltransferase in Cancer Cells. Science (2016) 351(6278):1214–8.
doi: 10.1126/science.aad5214
115. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-
Mediated Transfer of mRNAs and microRNAs Is a Novel Mechanism of
Genetic Exchange Between Cells. Nat Cell Biol (2007) 9(6):654–9.
doi: 10.1038/ncb1596
116. Smolarz B, Durczynski A, Romanowicz H, Szyłło K, Hogendorf P. miRNAs
in Cancer (Review of Literature). Int J Mol Sci (2022) 23(5):2805–23.
doi: 10.3390/ijms23052805
117.MalczewskaA,KiddM,MatarS,Kos-KudlaB,ModlinIM.A
Comprehensive Assessment of the Role of miRNAs as Biomarkers in
Gastroenteropancreatic Neuroendocrine Tumors. Neuroendocrinology
(2018) 107(1):73–90. doi: 10.1159/000487326
118. Thorns C, Schurmann C, Gebauer N, Wallaschofski H, Kumpers C, Bernard
V, et al. Global microRNA Profiling of Pancreatic Neuroendocrine
Neoplasias. Anticancer Res May (2014) 34(5):2249–54. PMID: 24778027
119. Luzi E, Pandolfini L, CiuffiS, Marini F, Cremisi F, Nesi G, et al. MicroRNAs
Regulatory Networks Governing the Epigenetic Landscape of MEN1 Gastro-
Entero-Pancreatic Neuroendocrine Tumor: A Case Report. Clin Transl Med
Apr (2021) 11(4):e351. doi: 10.1002/ctm2.351
120. Klieser E, Urbas R, Swierczynski S, Stattner S, Primavesi F, Jager T, et al.
HDAC-Linked “Proliferative”miRNA Expression Pattern in Pancreatic
Neuroendocrine Tumors. Int J Mol Sci (2018) 19(9):2781–94. doi: 10.3390/
ijms19092781
121. Luzi E, Marini F, Giusti F, Galli G, Cavalli L, Brandi ML. The Negative
Feedback-Loop Between the Oncomir Mir-24-1 and Menin Modulates the
Men1 Tumorigenesis by Mimicking the “Knudson’s Second Hit”.PloS One
(2012) 7(6):e39767. doi: 10.1371/journal.pone.0039767
122. Vijayaraghavan J, Maggi EC, Crabtree JS. miR-24 Regulates Menin in the
Endocrine Pancreas. Am J Physiol Endocrinol Metab (2014) 307(1):E84–92.
doi: 10.1152/ajpendo.00542.2013
123. Dettori D, Orso F, Penna E, Baruffaldi D, Brundu S, Maione F, et al.
Therapeutic Silencing of miR-214 Inhibits Tumor Progression in Multiple
Mouse Models. Mol Ther (2018) 26(8):2008–18. doi: 10.1016/
j.ymthe.2018.05.020
124. Zhang T, Choi S, Zhang T, Chen Z, Chi Y, Huang S, et al. miR-431 Promotes
Metastasis of Pancreatic Neuroendocrine Tumors by Targeting DAB2
Interacting Protein, a Ras GTPase Activating Protein Tumor Suppressor.
Am J Pathol (2020) 190(3):689–701. doi: 10.1016/j.ajpath.2019.11.007
125. Bai J, Na H, Hua X, Wei Y, Ye T, Zhang Y, et al. A Retrospective Study of
NENs and miR-224 Promotes Apoptosis of BON-1 Cells by Targeting
PCSK9 Inhibition. Oncotarget (2017) 8(4):6929–39. doi: 10.18632/
oncotarget.14322
126. Yamana H, Kato K, Kobara H, Fujihara S, Fujita K, Namima D, et al.
Metformin Inhibits Proliferation and Tumor Growth of QGP-1 Pancreatic
Neuroendocrine Tumor Cells by Inducing Cell Cycle Arrest and Apoptosis.
Anticancer Res (2020) 40(1):121–32. doi: 10.21873/anticanres.13933
127. Pedraza-Arevalo S, Ibañez-Costa A, Blazquez-Encinas R, Branco MR,
Vazquez-Borrego MC, Herrera-Martínez AD, et al. Epigenetic and Post-
Transcriptional Regulation of Somatostatin Receptor Subtype 5 (SST(5)) in
Pituitary and Pancreatic Neuroendocrine Tumors. Mol Oncol (2022) 16
(3):764–79. doi: 10.1002/1878-0261.13107
128. Torrisani J, Hanoun N, Laurell H, Lopez F, Maoret JJ, Souque A, et al.
Identification of an Upstream Promoter of the Human Somatostatin
Receptor, Hsstr2, Which is Controlled by Epigenetic Modifications.
Endocrinology (2008) 149(6):3137–47. doi: 10.1210/en.2007-1525
129. Taelman VF, Radojewski P, Marincek N, Ben-Shlomo A, Grotzky A, Olariu
CI, et al. Upregulation of Key Molecules for Targeted Imaging and Therapy.
J Nucl Med (2016) 57(11):1805–10. doi: 10.2967/jnumed.115.165092
130. Veenstra MJ, van Koetsveld PM, Dogan F, Ben-Shlomo A, Grotzky A, Olariu
CI, et al. Epidrug-Induced Upregulation of Functional Somatostatin Type 2
Receptors in Human Pancreatic Neuroendocrine Tumor Cells. Oncotarget
(2018) 9(19):14791–802. doi: 10.18632/oncotarget.9462
131. Klomp MJ, Dalm SU, de Jong M, Feelders RA, Hofland J, Hofland LJ.
Epigenetic Regulation of Somatostatin and Somatostatin Receptors in
Neuroendocrine Tumors and Other Types of Cancer. Rev Endocr Metab
Disord (2021) 22(3):495–510. doi: 10.1007/s11154-020-09607-z
132. Malone CF, Emerson C, Ingraham R, Barbosa W, Guerra S, Yoon H, et al.
mTOR and HDAC Inhibitors Converge on the TXNIP/Thioredoxin Pathway
to Cause Catastrophic Oxidative Stress and Regression of RAS-Driven Tumors.
Cancer Discovery (2017) 7(12):1450–63. doi: 10.1158/2159-8290.CD-17-0177
133. Mosquera MJ, Kim S, Bareja R, Fang Z, Cai S, Pan H, et al. Extracellular
Matrix in Synthetic Hydrogel-Based Prostate Cancer Organoids Regulate
Therapeutic Response to EZH2 and DRD2 Inhibitors. Adv Mater (2022) 34
(2):e2100096. doi: 10.1002/adma.202100096
134. Vitale G, Dicitore A, Pepe D, Gentilini D, Grassi ES, Borghi MO, et al.
Synergistic Activity of Everolimus and 5-Aza-2’-Deoxycytidine in Medullary
Thyroid Carcinoma Cell Lines. Mol Oncol (2017) 11(8):1007–22.
doi: 10.1002/1878-0261.12070
135. Feinberg AP, Irizarry RA. Evolution in Health and Medicine Sackler
Colloquium: Stochastic Epigenetic Variation as a Driving Force of
Development, Evolutionary Adaptation, and Disease. Proc Natl Acad Sci U
SA(2010) 107 Suppl 1:1757–64. doi: 10.1073/pnas.0906183107
136. Gagliardi A, Dugue PA, Nost TH, Southey MC, Buchanan DD, Schmidt DF,
et al. Stochastic Epigenetic Mutations Are Associated With Risk of Breast
Cancer, Lung Cancer, and Mature B-Cell Neoplasms. Cancer Epidemiol
Biomarkers Prev (2020) 29(10):2026–37. doi: 10.1158/1055-9965.EPI-20-
0451
137. Gentilini D, Scala S, Gaudenzi G, Garagnani P, Capri M, Cescon M, et al.
Epigenome-Wide Association Study in Hepatocellular Carcinoma:
Identification of Stochastic Epigenetic Mutations Through an Innovative
Statistical Approach. Oncotarget (2017) 8(26):41890–902. doi: 10.18632/
oncotarget.17462
138. Crabtree JS, Singleton CS, Miele L. Notch Signaling in Neuroendocrine
Tumors. Front Oncol (2016) 6:94. doi: 10.3389/fonc.2016.00094
139. Papaspyropoulos A, Angelopoulou A, Mourkioti I, Polyzou A, Pankova D,
Toskas K, et al. RASSF1A Disrupts the NOTCH Signaling Axis via SNURF/
RNF4-Mediated Ubiquitination of HES1. EMBO Rep (2022) 23(2):e51287.
doi: 10.15252/embr.202051287
Conflict of Interest: The author is a member of the Amgen Neuroendocrine
Tumor Expert Advisory Board and receives an honorarium for this service.
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Crabtree Epigenetics of GEP NETs
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