Chromogranin A and Its Fragments as Regulators of
Small Intestinal Neuroendocrine Neoplasm Proliferation
Francesco Giovinazzo1,2☯, Simon Schimmack1,3☯, Bernhard Svejda1,4, Daniele Alaimo1, Roswitha
Pfragner4, Irvin Modlin1, Mark Kidd1*
1 Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, United States of America, 2 Laboratory of Translational Surgery-LURM,
University of Verona, Verona, Italy, 3 University Hospital of General, Visceral- and Transplantation-Surgery of Heidelberg, Heidelberg, Germany, 4 Department
of Pathophysiology and Immunology, University of Graz, Graz, Austria
Abstract
Introduction: Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN dedifferentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis
and played a role in the regulation of NEN proliferation.
Methods: Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human
mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability
(WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot)
in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT
antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines.
Results: Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs
compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands
including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in
metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by
inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05)
and 5-HT (p<0.05) as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated
(50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by
RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05).
Conclusion: Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell
lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway
indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.
Citation: Giovinazzo F, Schimmack S, Svejda B, Alaimo D, Pfragner R, et al. (2013) Chromogranin A and Its Fragments as Regulators of Small Intestinal
Neuroendocrine Neoplasm Proliferation . PLoS ONE 8(11): e81111. doi:10.1371/journal.pone.0081111
Editor: Salvatore Papa, Institute of Hepatology - Birkbeck, University of London, United Kingdom
Received March 19, 2013; Accepted October 17, 2013; Published November 19, 2013
Copyright: © 2013 Giovinazzo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: mark.kidd@yale.edu
☯ These authors contributed equally to this work.
Introduction
of the mechanism of processing, its peptides have been
proposed to regulate a range of physiological processes [6], an
example being colonic motility [7]. Transcripts [8,9] and plasma
levels of the protein are specifically elevated in patients with
different endocrine tumors such as pheochromocytomas and
medullary thyroid carcinoma, as well as in bronchopulmonary
and gastroenteropancreatic neuroendocrine neoplasms (GEPNENs) [1,2].
Western blot identifies a range of CgA fragment sizes from
~9-85 kDa depending on processing, neuroendocrine cell type
and antibody used [10], but the parent CgA molecule is
generally considered to have a molecular mass of ~70-85 kDa
Chromogranin A (CgA), a heat stable, hydrophilic acidic
protein of ~460 amino acids, is a member of the granin family
of secretory proteins that are ubiquitous to the nervous,
endocrine and immune system [1,2]. It forms the principal
component of the soluble core of dense-core secretory
granules in neuroendocrine cells and is secreted from these
cells in a physiologically regulated manner [3]. The
biosynthesis of CgA can be influenced both at a transcriptional
level and post-translationally [4]. CgA is elevated in a number
of pathological conditions e.g. renal failure [5] and, irrespective
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Figure 1. CgA coding regions, putative functional domains and targets. The primary transcript, on chromosome 14, is derived
from 8 exons and includes exon 1 which is untranslated but contains a signal peptide region for protein processing. In this study,
PCR was performed using intron spanning primers to examine exons I-VI. Mature CgA mRNA includes 439 coding base pairs which
are translated into a primary peptide of 431 amino acids. Processing of CgA following cleavage at dibasic and monobasic residues
e.g. by PC1/3 and CPE results in production of a range of intermediate peptides as well biologically active peptides [36].
doi: 10.1371/journal.pone.0081111.g001
protein expression in normal mucosa and tumor tissue
samples, and then measured proliferation in four tumor cell
lines, two primary tumor-derived lines, KRJ-I and P-STS
[20,24], and two metastases, L-STS and H-STS [24]. As
proliferation of tumors is related to AKT/mTOR activation and
signaling and can be inhibited by rapamycin-derivatives [20],
we specifically evaluated the effects of candidate peptides on
this pathway as well as on tumor cell proliferation. To
characterize the role of post-translational effectors, we
evaluated mRNA and protein expression of the CgA processing
enzyme prohormone convertase in both tumor tissue samples
as well as cell lines and evaluated the effect of proliferation on
CgA and this processing enzyme in vitro. We also examined
the effect of CgA silencing and pharmacologic inhibition of
prohormone convertase on tumor cell proliferation, secretion
and post-transcriptional changes in CgA fragment expression.
The results identified a role for CgA peptides in the regulation
of NEN proliferation at the level of AKT signaling. Targeting
AKT or prohormone convertases specifically decreased
proliferation, especially in metastases.
[11]. Smaller fragments reflects post-translational processing
and the production of a series of smaller biologically active
peptides such as vasostatin I and II (corresponding to CgA
residues 1-76 and 1-113, respectively), chromostatin (CgA
173-194), pancreastatin (CgA 250-301), WE14 (CgA 324-337)
and catestatin (CgA 344-372) [1,2,12] (Figure 1). Posttranscription (translational) modifications are regulated by a
number of enzymes. These include the serine protease
prohormone convertase 1-3 (PC1-3) [13], which is associated
with production of pancreastatin [14], the cysteine protease
cathepsin L [15], associated with production of the middle and
C-terminal fragments e.g. catestatin, or by the fibrinolytic
enzyme, plasmin [16].
Although CgA is a well-characterized product of NENs, very
few studies have investigated the role of either CgA or its
cleavage fragments as regulators of small intestinal NEN (SINEN) proliferation, the commonest tumor in this class [17,18].
Given the differences in transcription and processing of CgA in
different neuroendocrine tissues and their neoplasia [1,2], e.g.
SI-NENs with liver metastases [19], we hypothesized that CgA
transcripts were differentially expressed during NEN
metastasis, that this translated into differences in CgA fragment
expression and that these specific fragments may regulate
NEN proliferation.
We specifically focused on SI-NENs since these are
common and there are a number of well-characterized cell
lines available [20-24]. Initially, we examined mRNA and
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Chromogranin A, Proliferation and GEP-NENs
corresponding amino acids 1-251 and >85% of the coding
region; see Figure 1) and prohormone convertase (PCSK1)
expression were quantified using Assays-on-DemandTM
products and the ABI 7900 Sequence Detection System (both
Applied Biosystems) according to the manufacturers’
instructions. PCR data were normalized to the housekeeping
gene, ALG9 (asparagine linked glycosylation 9) [28] using the
CT method [29].
Table 1. Peptide fragments used for the in vitro proliferation
studies.
CgA fragment peptides
Position
Position
Size (kDa)
Vasostatin I
N-terminal
1-76
8.5 kDa
Source
Phoenix
Vasostatin II
N-terminal
1-133
12.8 kDa
Phoenix
Pancreastatin
Middle
250-301
4.2 kDa
Phoenix
Chromostatin
C-terminal
173-194
2.0 kDa
Phoenix
Fragment 1
C-terminal
286-301
1.7 kDa
Phoenix
Fragment 2
C-terminal
342-355
1.4 kDa
Phoenix
WE14
C-terminal
324-337
1.6 kDa
N E Peptides
Catestatin
C-terminal
352-372
2.2 kDa
N E Peptides
Protein isolation, ELISA and Western blot analysis
Small pieces (~20mg) of tissue or cell line lysates (from
1x106 cells) were processed as described [27] including
manual homogenization with RIPA lysis buffer (Millipore,
Temecula, CA) with addition of complete protease inhibitor
(Roche, Indianapolis, IN), phosphatase inhibitor sets 1 and 2
(Calbiochem,
La
Jolla,
CA),
100
mM
phenylmethylsulfonylfluoride (Roche), 200 mM Na3VO4 (Acros
Organics,Geel, Belgium), and 12.5 mg/ml sodium dodecyl
sulfate (American Bioanalytical, Natick, MA) and protein
quantification undertaken using the Pierce BCA protein assay
(Thermo Scientific, Rockford, IL).
CgA protein levels were firstly quantified via ELISA using a
commercially available kit (DAKO, K0025, Glostrup, Denmark)
as described in the manufacturer’s instructions. Final results
were normalized to protein levels and expressed as Units
CgA/µg protein for samples and cell lines. For western blot
analysis, total protein lysates (15µg) were denatured in SDS
sample buffer (Invitrogen), separated on an SDS-PAGE gel
(10%, Invitrogen) and transferred to a PVDF membrane with a
pore size of 0.45 mm (Bio-Rad, Hercules, CA). After blocking
(5% bovine serum albumin [BSA]) for 60 min at room
temperature (RT), the membrane was incubated with either
anti-CgA (complete protein, DAK-A3, 1:200) or antiprohormone convertase 1-3 (Abcam, Boston, MA, 1:1000) and
separately with anti-β-actin (Sigma-Aldrich, St. Louis, MO,
1:10,000) antibodies in 5% BSA/PBS/Tween 20 overnight at
4°C. After washing in PBS/Tween 20, the membranes were
incubated with the horseradish peroxidase-conjugated
secondary antibodies (Cell Signaling, Danvers, MA) for 60 min
at RT. After washing, immunodetection was performed using
the Supersignal West Pico Luminol/ Enhancer solution
(Thermo Scientific). Protein expression in cell lines was
reported relative to β-actin (Sigma-Aldrich).
N E Peptides = New England Peptides
doi: 10.1371/journal.pone.0081111.t001
Materials and Methods
Small intestinal neuroendocrine neoplasm tissue and
cell lines
In total, 10 samples of normal small intestinal mucosa, 5
preparations of normal human enterochromaffin (EC) cells,
obtained from fluorescence-activated cell sorting of normal
mucosa; >98% pure EC cells [25]), 14 primary SI-NENs
(localized – no evidence of metastases), 12 primary SI-NENs
(with evidence of distant metastases) and 9 corresponding liver
metastases were collected for real-time PCR analysis, western
blot and ELISA analysis. All tumors contained >80% pure
neoplastic cells and were tryptophan hydroxylase1-positive and
therefore EC cell-derived [25]). All SI-NENs were classified
pathologically according to the WHO standard 2006 as well
differentiated neuroendocrine tumors (WDNETs, n=26), now
classified as G1 NETs [26]. All samples were collected and
analyzed according to an IRB protocol (Yale University School
of Medicine). The protocol was specifically approved for this
study. Written consent was obtained from all study participants.
To evaluate the biological function of the candidate CgA
fragments (Figure 1 and Table 1), four well-characterized NEN
cell lines (KRJ-I and P-STS: primary tumors, L-STS: lymph
node metastasis, and H-STS: hepatic metastasis) were
cultured in Quantum 263 complete tumor medium (PAA,
Dartmouth, MA) supplemented with 100 IU penicillin/ml and
100 µg streptomycin/ml at 37°C with 5% CO2. During growth
phases, 40-90% of e.g., KRJ-1 cells were Ki67 positive [20-24]
reflecting in vitro growth characteristics. While not
commensurate with in vivo SI-NEN behavior (Ki67<20%), these
provide robust, well-characterized models for assessing
proliferation. All experiments were performed without
antibiotics.
Effect of proliferation on CgA and processing enzymes
H-STS cells were sub-cultured and collected at days 2, 3 and
7, which are time points during logarithmic (~70%) and plateau
(~30%) growth curves [20,21,24]. PCR and western blot were
undertaken on CgA (exons 1-6) and prohormone convertases
1-3, respectively. Transcript and protein results were compared
to day 2 (logarithmic growth) [20,21,24].
RNA isolation and real-time polymerase chain reaction
Effect of CgA, its peptides and processing enzymes on
proliferation and secretion
Messenger RNA was extracted and converted to cDNA from
small pieces (~20mg) of tissue or cell line lysates (1x106 cells)
as described [27] using TRIZOL® (Invitrogen, Carlsbad, CA)
and the High Capacity cDNA Archive Kit (Applied Biosystems,
Carlsbad, CA). Transcript levels of CgA (exons 1-6;
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In order to investigate the role of CgA in tumor cell
proliferation, 2 x 105 H-STS cells/well were seeded in 12-well
plates (Falcon, BD, Franklin Lakes, NJ) and CgA silenced
using the reverse transfection approach with siRNA (Invitrogen,
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Chromogranin A, Proliferation and GEP-NENs
inhibition on vasostatin or chromostatin-regulated proliferation
in each of the cell lines.
40 pmol/well, sense sequence: GCUACAAGGAGAUCCGGAA)
and Lipofectamine 2000 (Invitrogen) in comparison to
scrambled siRNA (Invitrogen) as a control. We confirmed the
knockdown using PCR after 48 and 96 hours of incubation and
performed BrdU (Bromodeoxyuridine) ELISA (Roche
Diagnostics, Indianapolis, IN) according to the manufacturers’
instructions. Briefly, after silencing H-STS cells in 96-well
plates (clear bottom, Costar, Corning, NY), they were labeled
with BrdU uptake solution and incubated for 3 hrs. Cells were
fixed, DNA denatured and anti-BrdU antibody solution added.
After 90 min incubation, the final substrate was added and the
chemiluminescence read on a GLOMAX Luminometer
(Promega, Madison, WI). Luminescence (relative values) in
CgA knocked down cells were compared to scrambled siRNA/
Lipofectamine treated cells and represented as a folddecrease.
A complementary approach to investigate the role of
translational processing on CgA was undertaken by inhibiting
PC activity. H-STS cells were seeded in either 6-well (1 x 106
cells/well) or 96-well plates (2 x 104 cells/well) and treated with
the prohormone convertase inhibitor [30] decanoyl-Arg-ValLys-Arg-chloromethylketone (Millipore, Billercia, MA, 25 and
50µM) for 48 hrs. Western blot was used to evaluate alterations
in CgA processing while effects on proliferation were assessed
by BrdU-ELISA.
To confirm a functional effect of inhibiting CgA itself or its
processing by either silencing CgA or inhibition of CgA
processing enzyme prohormone convertase on cells [31], CgA
and serotonin secretion was evaluated at the termination of the
experiment (48 hrs) in the supernatant (ELISA: Labor
Diagnostika, Nordhorn, Germany) [32].
To examine the effect of processed CgA peptides on
proliferation, 2 x 105 cells/ml were seeded in 96 well plates
(Falcon, BD, Franklin Lakes, NJ) at 100 µl and stimulated after
2 days with different fragments (Phoenix Pharmaceuticals and
New England Peptides, Gardner, MA) (see Table 1,
concentration 10-9M to 10-6M) for 72 hrs in comparison to no
treatment (control) and to pre-incubation with the mTOR
inhibitor everolimus (RAD001, 10-9M) for 30 min prior to
application of the peptide [33]. Cell viability was analyzed using
the WST1 cell proliferation reagent (Roche) according to the
manufacturers’ instructions [34]. Optical density was quantified
spectrophotometrically at 450nm. Results were normalized to
the unstimulated control and effective half-maximal
concentration (EC50 or IC50) was calculated [21,33].
Identification of a candidate “receptor” for CgA
To identify potential CgA receptors, we examined expression
of Cy5-labeled CgA (342-355, Phoenix Pharmaceuticals,
Burlingame, CA) fluorescence in KRJ-I cells and H-STS cells
using confocal microscopy as described [20].
Statistical Evaluation
All statistical analyses were performed using Microsoft Excel
and Prism 5 (GraphPad Software, San Diego, CA). Sigmoidal
dose responses and nonlinear regression analyses were
calculated to identify the EC50 and IC50 concentrations for each
agent. Alterations in signal transduction and transcriptional
activation were assessed using 2-tailed Wilcoxan rank sum
tests for non-parametric data. Multiple group comparisons were
performed using the Kruskal Wallis test, followed by the Dunn’s
post-hoc test where appropriate. A p-value of <0.05 was
designated as significant.
Results
CgA mRNA and protein expression in small intestinal
NENs and cell lines
Messenger RNA levels of all CgA exons that were examined
(exons 1-6) were increased in primary NENs with or without
metastases as well as in metastases compared to normal
mucosa (540-680x, (p<0.0001) and normal EC cell
preparations (270-340x, p<0.001) (Figure 2A). Transcripts
tended to be lower in metastases than in the primaries
suggesting an alteration in transcriptional regulation in these
tumors.
We next examined CgA protein expression in these samples
using ELISA (Figure 2C). The reproducibility of this assay
(inter-assay and intra-assay coefficients of variability) for
mucosal tissue was 7 and 5%, respectively. Normal mucosa
expressed 1.6±0.3 Units CgA/µg protein, normal EC cells
3.7±1.6 Units CgA/µg protein. Levels in neoplasms ranged
from 168.6±53 (localized primaries) to 402.2±132.4 Units/µg
protein in neoplasms with metastases (0.42-fold). CgA protein
levels were significantly increased in SI-NENs compared to
normal mucosa [vs. localized primaries p<0.01 (105-fold), vs.
metastasized primaries p<0.001 (251-fold) and vs. metastases
p<0.05 (86-fold)] as well as compared to EC cell preparations
(p<0.01) (Figure 2C). Protein levels in metastases (138.4±54.9
Units/µg protein) tended to be lower than in the localized
tumors that exhibited metastases (0.33-fold), consistent with
the mRNA results (Figure 2A, 334.8 vs. 1353, 0.25 fold).
Using a western blot approach to examine expression of
different CgA fragments, predominant bands of ~76-80 kDa
were noted for CgA in all NEN samples but not in normal
mucosa or in normal EC cell preparations (Figure 2E),
confirming over-expression of CgA and its fragments in
neuroendocrine neoplastic tissue. Processing of CgA was
evident in all SI-NEN samples. In particular, band sizes
consistent with intermediate fragments including Vasostatin I
AKT/ mTOR pathway activation
H-STS was stimulated with CgA fragments vasostatin I and
P-STS with chromostatin (10-6 M) for 1 hr. One cell line set
were pre-incubated for 30 min with RAD001 (10-9M) [33] or
AKT antisense oligonucleotide (TCCCTCTTGCTCGTGTTCC,
Yale Medical School Keck Oligonucleotide Synthesis Facility)
[35] prior to application of the peptide. AKT signal activity
(pAKT) was determined using SuperArray CASETM ELISA kits
(AKT FE-001, Biomol, Hamburg, Germany) and western blot
using anti-pAKT (Ser473, Cell Signaling) as described [20,33].
BrdU uptake was measured to evaluate the effect of AKT
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Figure 2. Chromogranin A expression in normal mucosa, EC cells, small intestinal NENs (SI-NENs) and primary and
metastatic SI-NEN cell lines. CgA mRNA expression in normal human mucosa (NML), EC cell preparations (EC), localized NENs
(PRIM), primaries with metastasis (MET PRIM) and liver metastases (METS) demonstrated that all NENs expressed higher CgA
levels (Kruskal-Wallis p<0.0001) compared to normal mucosa (*p<0.001) or normal EC cells (#p<0.001) (a). Levels of CgA protein
expression, measured by ELISA, showed a similar pattern (2C, Kruskal-Wallis p<0.0001) and was increased in PRIM (*p<0.01),
MET PRIM (*p<0.001) and METS (*p<0.05) compared to normal mucosa. CgA western blot in normal mucosa, normal EC cells and
SI-NENs identified a mature CgA band of 75-80 kDa in all NENs but not in normal mucosa or EC cells (2E). Fragment sizes
included peptides ranging in size from ~30-60kDa, consistent with CgA processing intermediates [36]. In cell lines, CgA mRNA was
expressed in higher levels in the two primary cell lines in comparison to metastatic cell lines (2B, Kruskal-Wallis p<0.0001),
particularly in P-STS CgA was over-expressed compared to H-STS (* p<0.001) and L-STS cells (#p<0.01). In KRJ-1 CgA was also
elevated in comparison to H-STS cells (*p<0.01, 2B). Protein level (ELISA) followed similar pattern (Kruskal-Wallis p=0.0273,
*p<0.05, 2D). Using western blot, total CgA (75-80 kDa) was identified in all cell lines, highest in the primary cell lines KRJ-1 and PSTS (2F). Band sizes consistent with CgA processing were evident and exhibited different patterns of expression consistent with
alterations in translational modifications (2F). No external receptor was identified for CgA, but Cy5-labeled immunofluorescence (IF)
was identified within KRJ-I and H-STS cells. We interpret this dot-like signal to reflect intracellular uptake of this CgA peptide (2G).
doi: 10.1371/journal.pone.0081111.g002
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Chromogranin A, Proliferation and GEP-NENs
and II [36] (confirmed by a Vasostatin I/II antibody [sc-23556,
Santa Cruz, CA], data not shown) were highly expressed in
metastases. Other processing fragments as well as fragments
that included pancreastatin were also identified in neoplasia;
levels were increased in comparison to normal EC cells (Figure
2E).
Expression of CgA mRNA in the two primary SI-NEN cell
lines (KRJ-1 and P-STS) was also elevated compared to
normal EC cells (3-6000 fold), while the two metastatic cell
lines (L-STS and H-STS) expressed lower levels of CgA mRNA
in comparison to normal EC cells (0.08-0.3 fold). Both primary
cell lines expressed higher levels of CgA mRNA than the
metastatic cell lines (Figure 2B, Kruskal-Wallis p<0.0001 in all
exons). CgA was elevated, particularly in P-STS, compared to
H-STS (p<0.001) and L-STS cells (p<0.01); CgA, likewise, was
higher in KRJ-1 compared to H-STS cells (p<0.01, Figure 2B).
CgA protein levels (measured by ELISA) also differed in the
SI-NEN cell lines (Kruskal-Wallis p=0.027) and were
particularly higher in the primary (P-STS) than in its metastasis
(H-STS, p<0.05) (Figure 2D). The latter observation was mostly
consistent with the mRNA results (Figure 2B).
The four cell lines all expressed detectable parent CgA
bands but expression appeared higher in primary cell lines
(Figure 2F). Since western blot and ELISA use antibodies that
bind to CgA at different amino acids, P-STS was not as highly
over-expressed as measured by ELISA but was still higher
than L-STS. Similar sized bands, consistent with CgA
processing (and identified in clinical samples, Figure 2E) were
also noted in the cell lines (Figure 2F). Interestingly, processing
fragments I/II and vasostatin I/II showed increased expression
in lymph node and liver metastatic cell line compared to the
matched primary (P-STS). Using immunofluorescence,
membrane binding of a CgA fragment could not be detected on
KRJ-I and H-STS cells (Figure 2G). We did, however, identify a
rapid internalization of this peptide with clear cytoplasmic
expression. This is consistent with endocytosis of CgA and
suggests a mechanism by which this protein can enter cells
and potentially affect signaling pathways.
identified an up-regulation of all CgA exons at day 7 (Figure
3B), indicating CgA synthesis may be elevated during slow cell
growth in this cell line. In parallel, expression of the
predominant 75-80 kDa CgA protein was elevated at day 7 in
H-STS compared to day 2 (Figure 3D), consistent with the
mRNA levels. It was further evident that PC1-3 may regulate
CgA processing in those cells since prohormone convertase
levels were decreased at day 7 when total CgA was increased.
We interpret this to demonstrate that alterations occur in CgA
processing enzymes as well as in CgA protein itself as the cells
proliferate, divide and become quiescent.
Effects of CgA and its peptide fragments on cell line
proliferation and AKT phosphorylation
We next evaluated whether CgA and/or its peptide fragments
played a role in regulating proliferation of H-STS cells since
those cells exhibited alterations in CgA expression consistent
with a regulatory role for this protein. To this end, CgA was
successfully silenced (2-fold, data not shown), which resulted
in a significant decrease in proliferation (87%, p<0.05, Figure
4A). To confirm the efficacy of CgA knockdown on inhibition of
cell function, we evaluated CgA and 5-HT secretion. Both were
significantly decreased in CgA knockdown cells (31-52%,
p<0.01, Figure 4B).
Inhibiting the processing enzyme prohormone convertase
using decanoyl-Arg-Val-Lys-Arg- chloromethylketone also
resulted in a decrease in proliferation (down to 62% p<0.05,
Figure 4D) and was also associated with decreases both in
CgA and 5-HT secretion (Figure 4E). CgA secretion was
significantly decreased (down to 69%, p<0.01) and 5-HT (down
to 49%, p<0.05). These physiological alterations, a decrease in
CgA and 5-HT secretion, were associated with alterations in
CgA processing following inhibition of prohormone convertase
(Figure 4C and F).
Thereafter, we performed functional analysis of eight
candidate CgA peptides in the four SI-NEN cell lines. In initial
studies, we have identified Cy5-labeled CgA immunostaining of
KRJ-I and H-STS cells, which was internalized (Figure 2G).
This suggests that a CgA/peptide mediated effect may
represent a process of intracellular activation as opposed to the
more classical, membrane-bound receptor mechanism. In the
current study, C-terminal derived fragments had little effect on
cell line proliferation. Pancreastatin, fragment 1, fragment 2,
catestatin and WE14 had no proliferative effect (data not
shown), while chromostatin had an anti-proliferative effect on
the primary cell line P-STS (Figure 4G). In contrast, N-terminal
and the middle fragments did affect cell proliferation.
Specifically, vasostatin I and II significantly stimulated
proliferation (up to 60%, p<0.02) in both metastatic cell lines (LSTS and H-STS) but had no effects on primary tumor cell lines
(P-STS and KRJ-1) (Figure 4H and I). This effect was
associated with AKT phosphorylation (CASE ELISA: 50%,
p<0.04; western blot: 25%) (Figure 5A, C) and was completely
reversed by pre-incubation with the mTOR inhibitor RAD001
(p<0.01). AKT antisense also was able to reverse vasostatinmediated BrdU uptake (proliferation) (Figure 5E). In contrast,
chromostatin, inhibited localized cell proliferation (CASE
ELISA: ~20%, p=0.03) (Figure 5B) as well as AKT
CgA processing enzyme expression in small intestinal
NENs and cell lines and during proliferation
Given the evidence for different CgA peptide fragments,
mRNA and protein expression of the CgA processing enzyme
prohormone convertase 1 (PCSK1, PC1-3, respectively) was
investigated in normal human mucosa, EC cell preparations,
localized SI-NENs, primaries with metastasis and liver
metastases. Levels were over-expressed in SI-NENs (Figure
3A, Kruskal-Wallis p<0.001) and were increased in metastases
compared to localized tumors (Figure 3A). At a protein level,
PC1-3 were higher in metastases compared to normal mucosa
(Figure 3C, p<0.05) suggesting this enzyme may be a relevant
regulator of CgA processing in SI-NEN metastases.
We next examined the effect of proliferation on CgA and its
processing enzyme prohormone converstase. To undertake
this, we compared expression in H-STS cells, harvested two
and three days after subculture, when cells are in logarithmic
growth and after seven days, when cells are in plateau (growthrestricted) phase [20,21,24]. An analysis of CgA mRNA levels
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Chromogranin A, Proliferation and GEP-NENs
Figure 3. CgA processing enzyme prohormone convertase expression and the effect of tumor growth on CgA and
processing. PCSK1 mRNA expression was increased in SI-NEN metastases (METS) and primaries with metastasis (MET PRIM)
compared to normal mucosa (NML, *p<0.05) and normal EC cells (EC, #p<0.05) (3A, Kruskal-Wallis p=0.0003). Western blot
analysis confirmed that protein levels of prohormone convertase 1-3 were elevated in metastases compared to normal mucosa
(3C,*p<0.05).
CgA mRNA (3B, 6 exons) and protein (3D) were elevated at the plateau growth phase (day 7) compared to logarithmic growth (day
2) in H-STS cells (*p<0.05). PC1-3 proteins were decreased at day 7 (3D), which can be discussed as one reason for the elevation
of total intracellular CgA at this time point.
Mean±SEM. PCSK1: prohormone convertase 1.
doi: 10.1371/journal.pone.0081111.g003
Discussion
phosphorylation (~50%; Figure 5D) in the localized P-STS cell
line, effects that were reversed by RAD001. AKT antisense
reversed chromostatin-mediated BrdU inhibition (Figure 5F).
These growth regulatory effects signaled predominantly via
Ser473 phosphorylation, a known regulator of SI-NEN cell line
proliferation [33].
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Chromogranin A is a pro-hormone that is differentially
processed into peptides that regulate a range of biological
functions including cell proliferation, angiogenesis and
hormonal secretion [36]. The current study identified that CgA
mRNA and proteins were differently expressed in SI-NEN
progression, from normal EC cells to metastatic cancer, and
that advanced disease was associated with gain of specific
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Chromogranin A, Proliferation and GEP-NENs
Figure 4. CgA silencing, processing enzyme inhibition and functional analysis of CgA peptides in SI-NEN cell lines. After
successfully silencing CgA in H-STS cells (data not shown), proliferation was significantly decreased (4A, *p<0.05). Secretion of
CgA (p<0.01) and 5-HT (4B, *p<0.05) was significantly reduced following CgA antisense. Inhibition of the CgA processing enzyme
prohormone convertase using Decanoyl-Arg-Val-Lys-Arg-CMK also decreased proliferation of H-STS cells (25 µM [data not shown]
and 50 µM, 4D, *p<0.05). Additionally, secretion of CgA and 5-HT (4E, *p<0.05) was also significantly reduced. Decreases in CgA
and its fragments (Vasostatin II and Pancreastatin) after treatment with the prohormone convertase inhibitor were confirmed with
western blot (4C and F). Chromostatin (<20 kDa) was too small to appear on this WB. The fragments Vasostatin I and II significantly
stimulated proliferation (up to 60%, *p<0.02) in both metastatic cell lines (L-STS and H-STS, 4H and I, square) but had no effects on
the primary tumor cell lines. Chromostatin inhibited the well-differentiated localized NEN cell line proliferation (P-STS, 4G, square,
~50%, *p<0.05) but not proliferation of the less well-differentiated cell line, KRJ-I. Mean±SEM; n=6, CON: control, KD: knockdown,
SCR: scrambled, P: P-STS, K:KRJ-1, L: L-STS, H: H-STS. 5-HT: Serotonin.
doi: 10.1371/journal.pone.0081111.g004
CgA fragments (largely middle and N-terminal peptides) that
were effectors of growth via AKT/mTOR signal cross-activation.
CgA mRNA and proteins/peptides were, in comparison to
normal mucosa and EC cells, increased in SI-NENs, which
were also characterized by the expression of CgA processing
fragments consistent with N-terminal fragments. The
mechanisms underlying the altered expressions may reflect
either transcriptional or post-translational mechanisms. CgA
transcription is well-known to be regulated via CRE sites in the
PLOS ONE | www.plosone.org
promoter [37]. We have undertaken a microarray screen of SINENs and identified CRE-mediated signaling as a common
pathway in these tumors [38]. It is possible that alterations in
growth signaling pathways that signal through cAMP are
associated with the alterations in CgA transcription identified in
SI-NEN metastases in this study.
While CgA is commonly considered a marker of tumor load
and plasma levels are related to progression free survival in
GEP-NENs [19,39], this most likely reflects tumor mass per se
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Chromogranin A, Proliferation and GEP-NENs
Figure 5. Effect of Vasostatin I and Chromostatin on AKT phosphorylation in metastatic and localized NEN cell
lines. Vasostatin I stimulated AKT phosphorylation in the liver metastasis (H-STS) (CASE ELISA: 50%, *p<0.04, western blot: 25%)
and could be completely reversed by pre-incubation with RAD001 (5A/C, #p<0.01). AKT antisense reversed vasostatin-mediated
proliferation (BrdU uptake) (5E). In contrast, chromostatin, inhibited AKT signaling in the primary cell line (P-STS) (5B/D, ~25%,
*p<0.05). AKT antisense reversed chromostatin-mediated inhibition of proliferation (BrdU uptake) (5F). Mean±SD; AS = antisense,
CON: control, SCR: scrambled, V-I: vasostatin I, R: RAD001, CST: chromostatin.
doi: 10.1371/journal.pone.0081111.g005
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Chromogranin A, Proliferation and GEP-NENs
[40,41]. In the current study, we identified that metastases
expressed less CgA than primary tumors (when normalized to
total protein) and that the two metastatic cell lines we
investigated exhibited lower levels of CgA mRNA and protein
compared to cell lines derived from primary tumors. We
postulate that alterations in CgA expression, particularly at the
level of post-translational processing may be a feature of more
malignant NENs and may play a role in regulating proliferation.
CgA has been identified to play a role in preventing tumor cell
seeding and progression in a mouse model of breast
adenocarcinoma [42], suggesting that elevated CgA levels
(perhaps of specific fragments – this was not assessed in the
study) may have an inhibitory role in neoplastic development.
Our observations suggest that differences occur in the
processing and the production of specific fragments that may
provide an important, under-examined mechanism for these
processes.
One of the CgA fragments that was differentially processed
during SI-NEN metastasis was vasostatin I/II which is
recognized to have vasoconstrictive effects on small and
medium resistance vessels in cardiovascular system [43].
Although considered a candidate factor in cancer gene therapy
[44,45], cell adhesion, spreading and cellular invasion,
vasostatin enhanced malignant behavior in mice implanted with
vasostatin-expressing BON cells through mechanisms that
involved cell cycle regulation (i.e. p27Kip1) [46]. In the current
study, vasostatin I, selectively stimulated proliferation of the
metastatic cell lines, L-STS and H-STS, through AKT/mTOR
activation, a known regulator of p27Kip1 [47]. These vasostatinmediated effects were modulated by phosphorylation at
Ser473, recognized as the phosphorylation site associated with
growth-regulatory signaling in SI-NEN cell lines and neoplasms
[33]. These effects occurred at clinically relevant
concentrations; plasma CgA levels in patients affected with SINEN liver metastases range from 10-4 to 10-7M [19]. The two
localized cell lines, KRJ-I and P-STS, were not affected by
these peptides. Vasostatin-mediated proliferation appeared to
reflect a gain of function consequence of metastasis, an effect
that we consider due to differential CgA processing. These
proliferative effects are most likely due to intracellular activation
of the AKT/mTOR pathway, as we did not identify a membranebound receptor for CgA. Since CgA peptide effects,
particularly, vasostatin, has been demonstrated to occur
through internalization and activation of intracellular proteins in
HUVEC cells [48], we postulate that internalization of peptides
may affect signaling pathways in SI-NENs in a non-membrane
receptor manner.
In contrast to vasostatin, chromostatin inhibited proliferative
activity in P-STS cells through inhibition of AKT
phosphorylation, which is, to the best of our knowledge, the
first identification that this CgA fragment has an antiproliferative effect in NENs.
An emerging area of interest is regulation of pro-hormone
processing enzymes, either spatially or at the level of cellular
expression, that may play an important role in cleavage and
secretion of hormones [49]. The classical prohormone
convertases (PC1-3) selectively process precursors e.g. CgA
to pancreastatin, whose products are stored in secretory
granules [14]. Variation in PC1 and PC2 mRNA expression has
been suggested to play distinct roles in the activation of brain
pro-proteins, particularly CgA, while expression of this enzyme
itself appears regulated at a CRE-level, at least in the
pancreatic NEN cell line BON [50]. In the current study, PC1/2
mRNA was identifiable in normal EC cells, but was overexpressed in SI-NENs while PC1 itself was significantly
elevated in metastases. Inhibiting this enzyme resulted in a
decrease in H-STS proliferation as well as down-regulation of
CgA and 5-HT secretion demonstrating the sensitivity of posttranslational modifications within the neoplastic CgA system.
In conclusion, we identified that CgA was differentially
regulated in primary and metastatic SI-NEN cell lines, which
exhibited elevated (compared to normal EC cells) but different
patterns of CgA fragments with different functions. Specifically,
N-terminal fragments stimulated metastatic NEN cell line
proliferation while middle fragments inhibit localized tumor cell
proliferation via the AKT/mTOR pathway. The presence and
over-expression of prohormone convertases 1-3 in
neuroendocrine neoplasms suggested that CgA was altered at
a post-translational stage via this enzymes. It therefore seems
likely that the CgA fragment pattern may be of value in
evaluating the biologic behavior of NENs and the regionspecific antibodies may be of potential use when seeking to
identify individual malignant tumor phenotypes.
Author Contributions
Conceived and designed the experiments: MK BS FG SS IM
RP. Performed the experiments: MK BS DA SS FG. Analyzed
the data: MK BS SS FG. Contributed reagents/materials/
analysis tools: MK SS RP IM. Wrote the manuscript: MK BS SS
FG.
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