Signaling Pathways as Specific Pharmacologic Targets for Neuroendocrine Tumor Therapy: RET, PI3K, MEK, Growth Factors, and Notch.

Abstract

Neuroendocrine tumors are rare tumors with a common progenitor - the neural crest cell. Included in this category are pulmonary and gastrointestinal tract carcinoid tumors and medullary thyroid cancer. The majority of these tumors are sporadic in nature, however they can be hereditary. Medullary thyroid cancers can present sporadically, with other endocrine tumors, as in the complex of multiple endocrine neoplasias 1, 2A, or 2B, or as familial medullary thyroid cancer. These tumors can become evident at later stages, with metastases already present at the time of diagnosis. Despite the small size and rare incidence of gastrointestinal neuroendocrine (carcinoid) tumors, they can be debilitating when present. Their natural history presents as early lymph node and distant metastases, as well as symptoms of the carcinoid syndrome, which result from the overproduction and secretion of serotonin and somatostatin. As a consequence of their metastases, surgical resection is non-curative and hence there is a need for novel treatment strategies to address tumor burden and symptom control. There are multiple intracellular pathways which can be targeted, either individually or in combination, to address these tumors. Here, we review some of the intracellular pathways, and identify some specific targets, which are vital to the generation and propagation of neuroendocrine tumorigenesis, and thus, can be the foci of novel drug therapies. We also elaborate on present pharmacological strategies and clinical trials involving these intracellular pathways.

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Neuroendocrinology
DOI: 10.1159/000335136
Signaling Pathways as Specific Pharmacologic
Targets for Neuroendocrine Tumor Therapy: RET,
PI3K, MEK, Growth Factors, and Notch
Yvette Carter Renata Jaskula-Sztul Herbert Chen Haggi Mazeh
Section of Endocrine Surgery, Department of Surgery, University of Wisconsin, Madison, Wisc. , USA

ple intracellular pathways which can be targeted, either in-
dividually or in combination, to address these tumors. Here,
we review some of the intracellular pathways, and identify
some specific targets, which are vital to the generation and
propagation of neuroendocrine tumorigenesis, and thus,
can be the foci of novel drug therapies. We also elaborate on
present pharmacological strategies and clinical trials involv-
ing these intracellular pathways.
Copyright © 2012 S. Karger AG, Basel
Introduction
Neuroendocrine tumors (NETs), though rare in inci-
dence (2–5 per 100,000), are of clinical significance due
to their presentation and symptoms [1] . These tumors are
often well differentiated, with indolent behaviors; how-
ever, some patients present late, with widespread meta-
static disease. As a result of their common embryologic
derivation from neural crest cells, these tumors can se-
crete a variety of substances, including: chromogranin A
(CgA), serotonin or 5-hydroxytryptamine, synaptophy-
sin, somatostatin, and neuron-specific enolase. Accord-
ingly, patients may present with different isolated symp-
toms or in carcinoid syndrome resulting from overpro-
duction and secretion of these bioactive products (right
Key Words
Neuroendocrine tumors ? Carcinoid ? Medullary thyroid
cancer ? Signaling pathways ? RET ? PI3K ? MEK ? Growth
factors ? Notch
Abstract
Neuroendocrine tumors are rare tumors with a common
progenitor – the neural crest cell. Included in this category
are pulmonary and gastrointestinal tract carcinoid tumors
and medullary thyroid cancer. The majority of these tumors
are sporadic in nature, however they can be hereditary. Med-
ullary thyroid cancers can present sporadically, with other
endocrine tumors, as in the complex of multiple endocrine
neoplasias 1, 2A, or 2B, or as familial medullary thyroid can-
cer. These tumors can become evident at later stages, with
metastases already present at the time of diagnosis. Despite
the small size and rare incidence of gastrointestinal neuro-
endocrine (carcinoid) tumors, they can be debilitating when
present. Their natural history presents as early lymph node
and distant metastases, as well as symptoms of the carcinoid
syndrome, which result from the overproduction and secre-
tion of serotonin and somatostatin. As a consequence of
their metastases, surgical resection is non-curative and
hence there is a need for novel treatment strategies to ad-
dress tumor burden and symptom control. There are multi-
Received: June 19, 2011
Accepted after revision: November 11, 2011
Published online: February 14, 2012
Haggi Mazeh, MD
Section of Endocrine Surgery, Department of Surgery
University of Wisconsin, K4/739 Clinical Science Center
600 Highland Avenue, Madison, WI 53792 (USA)
Tel. +1 608 263 1387, E-Mail mazeh   @   surgery.wisc.edu
© 2012 S. Karger AG, Basel
0028–3835/12/0000–0000$38.00/0
Accessible online at:
www.karger.com/nen

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2
heart valvular disease, congestive heart failure, flushing,
and diarrhea) [2, 3] . Gastrointestinal NETs frequently
metastasize to the liver, and these are usually evident at
the time of presentation, making curative resection less
feasible. When surgical resection cannot offer cure, treat-
ment with long-acting somatostatin analogs may de-
crease the progression and symptoms of the NETs as ap-
proximately 90% have somatostatin receptors [4, 5] .
Long-acting octreotide may increase the time to progres-
sion of both active and inactive metastatic midgut NETs,
however this is a costly treatment with documented side
effects and does not provide a therapeutic option to many
patients [5, 6] . The resistance of NETs to conventional
chemotherapy and radiation therapy leads to the investi-
gation of novel therapies targeting intracellular signaling
pathways, as pertinent to the treatment of these debilitat-
ing tumors.
The intracellular pathway initiator, which plays a role
in all neuroendocrine-derived tumors, is the rearranged
during transfection (RET) oncogene. This well-studied
oncogene can be a general, non-specific therapeutic tar-
get, as it activates three intracellular pathways: Ras/Raf/
mitogen-activated protein kinase kinase (MEK)/extra-
cellular signal-related kinase (ERK), c-Jun N-terminal
kinases (JNK), and phosphotidylinositol 3 ? -kinase (PI3K-
Akt) [7] . In addition, its inactivation results in caspase-
dependent apoptotic neuronal cell death [8] . Due to con-
cerns that inhibition at this level would result in an over-
all negative effect of multiple intracellular pathways,
more specific therapies, focused on specific downstream
targets, may be less toxic and more beneficial.
Stimulation of the PI3-Akt pathway is well document-
ed in ovarian, breast and colon cancer [9, 10] . Limiting
Akt activation suppresses gastrointestinal carcinoid and
small cell lung cancer growth [10, 11] . The Ras/Raf/MEK/
ERK mitogen-activating protein (MAP) kinase pathway
plays a similar dual role, in melanoma and colon and lung
cancers, acting as an oncogene, and as a tumor suppres-
sor in NETs [12–15] . More recently, the role of insulin-like
growth factor-1 (IGF-1) and its receptor (IGF-1R) in the
development of NETs is being elucidated [16] . It is cur-
rently known that NETs express vascular endothelial
growth factor (VEGF), epithelial growth factor (EGF)
and their receptors, and IGF-1 and IGF-1R, which have
all been noted to have an effect on tumor growth, inva-
sion and motility via MEK and other intracellular path-
ways [17] .
Another promising signaling pathway is Notch. Notch
is a transmembrane protein that has been studied exten-
sively in multiple malignancies. The isoform Notch-1 has
been shown to act both as an oncogene and as a tumor
suppressor. It has a role as an oncogene in pancreatic, co-
lon and non-small cell lung cancers, and a tumor sup-
pressor role in NETs, including pancreatic carcinoid tu-
mors, medullary thyroid cancer (MTC), and small cell
lung cancer [8, 18–23] . The role of other Notch isoforms
in cancer progression is still being investigated. Table 1
summarizes specific pharmacological agents that have an
effect in each pathway.
The aim of this study is to review the intracellular
pathways that are studied in NETs while focusing on spe-
cific targets that may serve as potential pharmacologic
therapies. Current clinical trials with the use of drugs for
the different pathways are discussed in the following text
and summarized in table 1 .
RET Proto-Oncogene
The RET proto-oncogene is a major focus of the intra-
cellular pathways that determine the morphologic out-
come of neuroendocrine tissues. It is the common de-
nominator that transmits extracellular signals and thus
affects multiple intracellular pathways [24] . RET has been
thoroughly studied and its role is best known in MTC [25,
26] . However, its exact role in other NETs has yet to be
completely elucidated.
Point mutations which lead to a gain of function in the
RET tyrosine kinase have been noted in cases of multiple
endocrine neoplasia (MEN) 2A and 2B and familial
MTC; however, more information regarding the intracel-
lular mechanism(s), is still needed [25, 26] . This gene with
29 exons is located on chromosome 10q11.2 and encodes
for a tyrosine kinase [26] . Alternate splicing results in the
two intracellular tyrosine kinase domains stimulating
several intracellular signal transduction pathways via
three isoforms (RET 9, 43, and 51) known as the short,
medium, and long domains ( fig. 1 , 2 ) [24] . Present on
these isoforms are binding regions for glial cell-line-
derived neurotrophic factor, neurtorin, artemin, and
persephin, which are located on one of four receptors
(growth factor receptor ? 1, 2, 3, or 4) [27–32] .
The knowledge that accumulated on the role of RET
in endocrine tumors has led to a change in clinical prac-
tice. Genetic testing for RET oncogene mutations has
proven key in the advancement of the treatment of
MEN2A gene carriers presenting with MTC. As a result
of their haplotype analysis, prophylactic thyroidectomy
proved curative and provided insight into the nodal sta-
tus in relation to the timing of intervention [33] .

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Tyrosine kinase inhibitors target this NET common
denominator to give a general non-specific inhibition of
multiple downstream end products. XL184 and ZD6474
(vandetanib) target RET, resulting in suppression of cell
proliferation and phosphorylation of RET and ERK, and
inhibition of EGF and VEGF receptor (VEGFR) kinases,
in vitro and in vivo [34–36] . Other tyrosine kinase in-
hibitors, including ST1571 (Gleevac) and AMG706, have
also been shown to inhibit MTC cell growth in vitro and
have undergone limited phase II clinical trials for hered-
itary MTC, with almost immediate reductions in calcito-
nin levels [37, 38] ; however their role as a systemic thera-
py for decreasing tumor burden has yet to be demonstrat-
ed. Other tyrosine kinase inhibitors, which target the
RET oncogene, work via the VEGF, EGF, or Raf-1/MEK/
ERK pathways [39–42] . Some success has been seen in
disease stabilization, with vandetanib in stage II clinical
trials for MTC; however as a whole, the results have not
proven promising for metastatic disease [43] . Gefitinib,
an EGFR tyrosine kinase inhibitor, blocks the anti-apo-
ptotic Ras signal transduction cascade, and is showing
some success in phase II clinical trials of patients with
advanced thyroid cancers, including papillary, anaplastic
and medullary variants [44] .
Additional investigation into the role of the down-
stream targets of RET in familial and sporadic MTC, as
well as carcinoid tumors, may further elucidate more spe-
cific therapeutic targets.
PI3-Akt Pathway
The PI3-Akt pathway is important in cell motility,
proliferation and survival [9, 10] . The focal points of these
lipid kinases are the p85 and p110 subunits, which cata-
lyze the conversion of phosphotidylinositol 4,5-bisphos-
phate to phosphotidylinositol 3,4,5-triphospate ( fig. 1 ).
Phosphotidylinositol 3,4,5-triphosphate plays a key role
in activating Akt, a serine/threonine kinase. Akt, in turn,
affects multiple downstream targets, including glycogen
synthase kinase-3 ? (GSK3 ? ), nuclear transcription fac-
tor ? B (NF ? B), and mammalian target of rapamycin
(mTOR) [45] . These genes have been shown to be in-
volved in the progression of various cancers. The Akt1
isoform has been noted in multiple cancers, including hu-
man pancreatic carcinoid tumors, and works through
mTOR, GSK3 ? and NF ? B activation [9, 10, 46] . Muta-
tions in the p85 or p110 subunits lead to loss of function
in the phosphatase and tensin homolog (PTEN) leads to
Table 1. Pharmacologic therapies, the intracellular pathway affected, their specific targets, and clinical trial
status
PathwayDrugMechanism Clinical trial
RET Vandetanib (ZD6474)
Gleevac (ST1571)
AMG706
Afinitor (everolimus)
Tyrosine kinase, VEGR, EGFR inhibitor
Tyrosine kinase inhibitor
Tyrosine kinase inhibitor
mTOR inhibitor
Phase III
Phase II
Phase II
RADIANT-3
Phase III
N/A
N/A
N/A
N/A
N/A
PI3-Akt
BEZ-235
Lithium
RAF265
Teriflunomide
ZM336372
Torc 1&2 inhibitor
GSK3 inhibitor
Raf-1 inhibitor
Ras/Raf-1 inhibitor
Raf activator
GSK3? inhibitor
Tyrosine kinase, PDGFR, VEGFR inhibitor
Ras/Raf-1
Growth factorsSutent (SU11248) SUN1111
Phase III
Phase II
Multi-center
Phase III
N/A
Pilot phase II
Gefitinib
Bevacuzimab
EGFR
VEGF-A antibody
Notch Suberoyl bis-hydroxamic acid Histone deacetylase inhibitor
Valproic acidNotch-1 activator; HDAC inhibitor

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unregulated activation of Akt [47, 48] . The specific role of
Akt1 and the other isoforms, in the generation and pro-
liferation of NETs, needs further elucidation. Due to its
diverse activators, effectors and downstream targets, this
pathway is of interest as a treatment for NETs.
Phosphotidylinositol kinase (PI3K) can be activated
by various integrins, tyrosine kinases, and B- and T-cell
receptors. It subsequently acts as a docking protein for
Akt. Akt then directly acts on p21 and p27, and indirect-
ly on cyclin D1 and p53, via mTOR, to effect cell growth
RAS
Gene transcription
Cell cycle progression
RET EGFR VEGFR
GTP
AMG706, gefitinib, Gleevac, XL184
Afinitor, BEZ-235
Teflunomide, ZM336372
BEZ-235
GSK3?
ZM336372, lithium
PI3K
ERK1/2
RAF
AKT
Extracellular
Intracellular
Cell membrane
Sutent, vandetanib
RAF265
Fig. 1. Illustration of the RET, PI3K/Akt,
Ras/Raf-1, and growth factors, intracellu-
lar pathways and pharmacological agents
involved in NETs.
NH2—
Target genes – Hes, Hey
RET Notch
NICD
SBHA, valproic acid
AMG706, gefitinib,
Gleevac, Sutent,
vandetanib, XL184
Nucleus
Extracellular
Intracellular
Cell membrane
Tyrosine kinase domain
Fig. 2. Illustration of the RET and Notch
intracellular pathways and pharmacologi-
cal agents involved in NETs.

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5
and pro-apoptotic signals [48, 49] . Phosphotidylinositol
is activated by RET tyrosine kinase 1062, which has a
binding site for phospholipase C ? (PLC ? ), as well as for
multiple docking proteins (including for activation of
ERK, p38 and JNK MAP kinases). The tyrosine kinase
1062-Akt pathway is important in NF ? B activation [47]
and pancreatic and pulmonary carcinoid cell survival
[50, 51] .
The multiple isoforms of Akt also have various activa-
tion pathways. Akt activation can be independent of, or
dependent on PI3. Irrespective of its stimulation method,
it plays critical event in nerve tissue generation; hence this
may be yet another therapeutic target [48, 50] . Investiga-
tions of this pathway have elucidated how Akt signaling
in both gastrointestinal and pulmonary carcinoid tu-
mors suppresses both cell proliferation and tumor mark-
er expression, and induces apoptosis [18, 50, 52, 53] . In
addition, Akt over activation plays a critical role in the
pathogenesis of MTC and MEN2A and MEN2B RET on-
cogene expression [52, 53] .
Several effective agents in this pathway have already
been used, and show some promise as therapeutic targets
in NETs, including lithium and ZM336372 which altered
some of the downstream products, including GSK3 ? ,
achaete-scute complex homolog-1 (ASCL1), and CgA
[54–56] . Everolimus, an mTOR inhibitor, received Fed-
eral Drug Administration (FDA) approval in February
2011, after undergoing a successful phase III clinical trial,
in which it attenuated disease progression and increased
progression free survival in patients presenting with ad-
vanced pancreatic NETs [57] . Two recent studies have
noted some success in the treatment of advanced MTC,
utilizing dual therapy with medications that cross-talk
amongst the intracellular pathways. A clinical trial com-
paring ZD6474, a RET, VEGFR and EGFR inhibitor, to
placebo, after two successful phase II trials was just re-
cently completed, and noted a significant improvement
in progression-free survival in those patients with MTC
[58] . Preclinical studies of RAF265 and BEZ-235, which
inhibit the Raf and PI3K and target of rapamycin com-
plex 1 (Torc1) and target of rapamycin complex 2 (Torc2)
pathways, respectively, noted a synergistic effect, via
blockage of both the ERK and PI3K signaling pathways,
resulting in an attenuation of MTC growth [59] .
Another signaling pathway downstream of Akt is the
GSK3 ? , a serine/threonine protein kinase. This pathway
can be activated by phosphorylation by either the Akt or
MAP kinase pathway. The tuberous sclerosis complex 2
(TSC2) gene is subsequently activated, and in turn, pre-
vents the inhibition of the mTOR complex-1 protein, re-
sulting in attenuation of cell growth, proliferation, angio-
genesis and mitochondrial metabolism. Cell cycle arrest
results via inhibition of cyclin D1 [60] . NF ? B is a dimer-
ic transcription factor which translocates into the nucleus
to induce gene transcription and ultimately leads to cell
survival and proliferation, after being released by phos-
phorylated inhibitor ? B (I ? B) or activated by GSK3 ? .
The activation of I ? B is a result of Akt stimulation of I ? B
kinase. Inhibitors of this downstream target were initial-
ly thought of as potential therapies for diabetes, due to its
role in glucose metabolism. However, further elucidation
of the specific inhibition of its ser-9 position noted regu-
lation of apoptosis and the cell cycle in NETs is needed.
Afinitor (everolimus; Novartis Pharmaceuticals Co., East
Hanover, N.J., USA) is an mTOR inhibitor. The phase III
clinical trial, RAD001 in Advanced NETs (RADIANT)-3,
resulted in a reduction of the risk of cancer progression
and an improvement in progression-free survival in pa-
tients with progressive pancreatic NETs [57] . These more
recent studies suggest a focus towards multiple down-
stream targets. The concerns to date are with tumor sta-
bilization, side effects, and the therapeutic modality
(neoadjuvant therapy, adjuvant therapy with surgery, or
with a somatostatin analog).
Ras/Raf-1/MEK/ERK1/2 Pathway
Upon activation, Ras binds Raf-1 at both the Ras-bind-
ing domain and the cysteine-rich domain, resulting in
activation at four inducible sites (Ser 338, Tyr 341, Thr
491, and Ser 494). Phosphorylation of these activation
sites results in activation of the MAPK/ERK1 kinase1 and
2 (MEK1 and MEK2), via phosphorylation of Ser 217 and
Ser 221. ERK1 and ERK2, which are 44 and 42 kDa re-
spectively, can then be phosphorylated and become ac-
tive. Phosphorylation of all sites (Thr 202/Tyr 204 for
ERK1 and Thr 185/Tyr 187 for ERK2) is required for full
activation of ERK. Complete elucidation of all of the
docking proteins involved and their activation of the
MAP kinases is still being investigated [7, 13] .
This pathway can be targeted upstream by inducing
Ras mutations, which impair guanine triphosphatase ac-
tivity, by stabilizing guanine triphosphate in the bound
state, or more downstream, where ERK1 and ERK2 affect
gene transcription ( fig. 1 ). It is known that ERK regulates
growth factor-responsive targets in the cytosol and regu-
lates gene expression after translocating into the nucleus.
It also indirectly phosphorylates protein S6 kinase, which
regulates mRNA transcription. Nuclear translocation is

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6
critical for gene expression and DNA replication, making
this pathway a therapeutic target for cancer therapy.
Activation of the Raf-1/MEK/ERK pathway may be a
therapeutic target for neuroendocrine cancers. Sippel et
al. [12] demonstrated that activation of this pathway is
associated with a reduction in neuroendocrine hormone
production. Ectopic raf-1 expression leads to MTC
growth suppression both in vitro and in vivo [55, 61] .
Others have also demonstrated the role of this pathway
in MTC. Ning et al. [61] noted its regulation of cell-cell
contact molecules and its association with a metastatic
phenotype in MTC. Increased Raf-1 activity, in gastroin-
testinal and pulmonary carcinoid tumor cell lines, at the
transcriptional level, resulted in increased ERK1/2 phos-
phorylation and activation, with a decrease in NE hor-
mone production, after treatment with ZM336372, a spe-
cific Raf activator [55, 56] . Raf-1 overexpression after
MTC treatment with ZM336372 resulted in growth sup-
pression and morphologic differentiation of the cells,
which more closely resemble normal C cells [62, 63] . This
treatment is of interest as it has been shown to decrease
bioactive hormone levels and expression of the transcrip-
tion factor ASCL1 via upregulation and phosphorylation
of Raf/MEK/ERK1/2 and suppress cell proliferation and
induce cell cycle inhibitors p21 and p18. On the horizon
are further, more specific therapies as a result of the de-
creased MTC cell viability noted [64, 65] .
Pancreatic carcinoid cells (BON) have also been noted
to have morphologic changes, including sharper boarders
and a flatter shape, mimicking cellular differentiation,
and representing the non-carcinogenic phenotype, with
the activation of raf-1 and its downstream target, ERK1/2
[64] . As a result of this pathway’s role in gastrointestinal
and pulmonary carcinoid tumors, as well as MTC, it is a
reasonable therapeutic target. Teriflunomide, a novel
Raf-1 pathway activator, has recently been proven to in-
hibit gastrointestinal carcinoid cell proliferation and de-
crease production of the tumor marker ASCL1 via inhibi-
tion of the MAPK kinase, activation of Raf-1/MEK and
ERK1/2 and induction of G2-M arrest [65] .
Growth Factors
Further downstream targets of the RET pathway,
which could be more specific targets, are growth factors.
NETs are highly vascularized and depend on growth fac-
tors which affect tumor cells, as well as endothelial cells,
for survival. Several pro-angiogenic factors are overex-
pressed in NETs. VEGF, its receptors, and related signal-
ing pathway components, including IGF-1, IGF-1R, EGF
and hepatocyte growth factor (HGF), may also play role
in vascular invasion, NET growth and metastases. The
role of these growth factors is still being elucidated in
NET growth and invasion. IGF-1 is a 70-amino-acid hor-
mone, with a receptor (IGF-1R) that is a member of the
tyrosine kinase super-family, with 70% homology to the
insulin receptor [66] . This hormone has a role in differ-
entiation, transformation and the prevention of apoptosis
[67, 68] . Two cascades have been identified in which this
hormone utilizes, to effect tumor growth, VEGF expres-
sion and tumor invasion [68, 69] . Cell survival is medi-
ated via the PI3K, protein kinase B (PKB), GSK3 ? , ? -
katenin and Myc-TCT 4 protein cascade; while the Ras-
Raf-MAPK pathway regulates cell proliferation ( fig. 1 ).
The expression of IGF-1 and/or IGF-1R, as well as HGF,
in NETs has been associated with advanced stage, in-
creased size, poor prognosis/survival, and recurrence or
metastases [16, 17, 70–72] .
This pathway is a downstream target of at least three
other intracellular pathways; hence it allows the option of
a more specific therapeutic target. IGF-1-mediated neu-
roendocrine product regulation can be attenuated by
stimulation of the raf-1/MEK1 pathway, inhibition of
its autocrine loop, or as a result of stimulation of the
Ras/PI3K/AKT system. Manipulation of this pathway, via
stimulation of the PI3/AKT pathway, effects human BON
NET cell survival and ultimate neuropeptide production,
via alteration of cyclin D1 expression and increases CgA
secretion.
EGF and HGF play key roles in tumor growth, aggres-
siveness, recurrence, motility and invasion [73–76] . These
are important as overexpression of their receptors has
been associated with increased tumor size, lymph node
metastases, and poor prognosis/survival [77–84] . Nilsson
et al. [84] demonstrated the role of EGFR in pheochromo-
cytoma and MTC, as blockade with a monoclonal anti-
body, decreased tumor growth. Sutent (sunitinib; Pfizer,
New York, N.Y., USA) is the first anti-VEGFR tyrosine
kinase inhibitor approved. The SUN 111 phase III study
resulted in a significant improvement in progression-free
survival in patients with progressive, well-differentiated
pancreatic NETs [85] . The results stem from the simulta-
neous inhibition of receptors for platelet-derived growth
factor and VEGF, which reduced tumor vascularization
and increased cancer cell death and tumor shrinkage.
Another drug which may prove more promising in com-
bination with other drugs is bevacizumab. This monoclo-
nal VEGF-A antibody is currently in phase III clinical
trials after demonstrating improved progression-free

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7
survival and attenuated tumor perfusion and progression
in a phase II clinical trial of patients with advanced car-
cinoid tumors [86] .
NOTCH-1/Achaete-Scute Complex-Like 1 (ASCL1)
This highly conserved pathway plays an important
role in embryonic development. It maintains stem cells,
influences the final fate of cells, and generates terminal
differentiation processes [20, 87, 88] . Notch proteins are
comprised of four 300-kDa transmembrane receptors
and five ligands. After stimulation via cell-to-cell contact
and ligand binding (DLL 1, 3, 4 or Jagged 1, 2), a sequence
of proteolytic cleavages occurs, with the subsequent ac-
tivation of the Notch intracellular domain ( fig. 2 ) [87, 88] .
This domain translocates into the nucleus and interacts
with CBF-1/RBPjk resulting in the activation of various
genes, including the suppressor of hairless and Lag-1
(HES-1). Notch-1 has been identified as both an onco-
gene and a tumor suppressor. It was first noted to stimu-
late cell proliferation and attenuate apoptosis in T-cell
acute lymphocytic leukemia, breast cancer, melanoma,
non-small cell lung cancer, and the renal epithelial cell
[20, 87, 88] . Its role as a tumor suppressor was noted in
keratinocytes and astrocytomas, with a loss of function
of Notch-1 resulting in a negative regulation of tumori-
genesis [46] . More recent research has noted a fine bal-
ance which must be adhered to with regard to Notch ex-
pression. Excessive up- or downregulation can be detri-
mental [87, 88] .
The role of the Notch pathway in NETs appears to be
opposite of that in epithelial-derived cancers. Overex-
pression of Notch inhibits cell proliferation and induces
apoptosis, rather than promoting the growth of these tu-
mors [22, 23, 89] . VEGF induces Notch-1 and ? -like-
4 expression via activation of the PI3K-Akt pathway,
and elevated Jagged 1 expression, in tumors, increases
Notch-1 activation in endothelial cells, resulting in vas-
cular network formation and hence vascular supply to
tumors. Notch activation suppresses MTC and carcinoid
tumor growth and hormone production [89–93] . This
pathway is conserved in gastrointestinal and pulmonary
carcinoid tumors. Additional studies of this pathway
have noted attenuation of ASCL1 production and modu-
lation of the neuroendocrine phenotype in carcinoid tu-
mors, associated with Notch expression [22] ; hence it is
a novel therapeutic target to exploit. Further studies have
shown that downregulation of ASCL1 transcription and
growth suppression of NETs by Notch is HES-1-depen-
dent [22, 89] . In vitro studies with valproic acid and his-
tone deacetylase (HDAC) inhibitors proved beneficial in
inducing apoptosis and decreasing cell growth in papil-
lary, follicular, papillary and MTCs, as well as carcinoid
tumors, via alter ations in Notch expression [89–91] . A
recent phase II pilot study utilizing valproic acid, to in-
duce Notch-1 expression, noted its efficacy in the treat-
ment of low-grade neuroendocrine carcinoma [92] . This
supported a previous in vivo study in which the expres-
sion of active Notch-1 correlated with a decrease in MTC
growth [51] .
This multifunctional transmembrane receptor also
plays a role in cell differentiation, proliferation and sur-
vival [20, 22, 87] . Noted to be absent or have minimal ex-
pression in NETs, Notch-1 activation and expression has
been shown to be a tumor suppressor in MTC and carci-
noid tumors, resulting in a reduction of both in vivo and
in vitro tumor growth, as well as attenuation of the pro-
duction of neuropeptides CgA, serotonin neuron-specif-
ic enolase and synaptophysin, and the tumor marker
ASCL1 [90, 93] . Due to its multiple binding sites, more
than one pathway is probably involved in the generation
of NETs, especially MTC, as some mutations are evident
with more aggressive phenotypes. Hence, the Notch
pathway is a prime target for new, unconventional phar-
macologic therapeutic agents. Valproic acid and suberoyl
bis-hydroxamic acid, HDAC inhibitors, have been dem-
onstrated both in vitro and in vivo, to upregulate Notch-1
signaling and suppress growth in papillary thyroid can-
cer, follicular thyroid cancer, MTC, and carcinoid cells
[45, 93] . In addition, ASCL1 expression and cyclin D1
were suppressed, and p21 production was increased. Re-
cently reported phase II clinical trial results from patients
treated with valproic acid are promising. Patients with
low-grade carcinoid or pancreatic NETs had upregula-
tion of Notch-1 signaling, and demonstrated either a par-
tial tumor response or stable disease [92] . With more
knowledge of the downstream targets, both the individ-
ual isoforms and downstream effectors can be targeted
for more specific pathway inhibition. Another area be-
ginning to be focused on is combination therapy. Adler
et al. [94] recently concluded an in vitro study of carci-
noid tumors treated with lithium and HDAC inhibitors,
demonstrating tumor growth suppression. With more
targets and combination therapy, specific downstream
effectors can be targeted, and toxicity and side effects can
be minimized.

Page 8

Carter/Jaskula-Sztul/Chen/Mazeh
Neuroendocrinology
8
Conclusion
There are several pathways which regulate the prolif-
eration of neuroendocrine cancers. However, further elu-
cidation of these pathways may hold the key to not only
improvement and resolution of symptoms, but inhibition
of tumor growth. The downstream targets in these path-
ways are providing more therapeutic options, as well as
more information regarding the pathogenesis of these tu-
mors. By having more specific targets, treatments can be
tailored to appropriate tumors in order to enhance effec-
tiveness and minimize toxicity and side effects. Some
current ongoing clinical trials attempting to target these
specific pathways show encouraging results and offer
hope to the patients who otherwise have limited other
therapies available.
Acknowledgements
We thank Nicholas Yeutter for his contribution to the data
search for this review. This study was funded by NIH/NCI Sup-
plemental Grant RO1CA121115-S1.

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