Journal of Cancer Therapy, 2013, 4, 28-33
Published Online November 2013 (
Open Access JCT
Pancreatic Neuroendocrine Tumors Are Characterized by
Loss of Noxa Expression that Can be Recovered by the
Proteasome Inhibitor Bortezomib
E. P. Slater, J. Waldmann, C. López, E. Matthäi, V. Fendrich, D. K. Bartsch
Department of Surgery, Philipps University, Marburg, Germany.
Received August 2nd, 2013; revised September 2nd, 2013; accepted September 10th, 2013
Copyright © 2013 E. P. Slater et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Prognosis in well-differentiated neuroendocrine carcinomas varies considerably and therapeutic targets for metastatic
diseas e are urgently need ed. cDNA microar ray studies in our laboratory revealed a significantly lower expression level
of the Noxa-gene in human pancreatic neuroendocrine neoplasms (PNENs) as compared to normal islet cells. To deter-
mine the validity of the downregulation of Noxa in PNENs, benign and malignant tumors from both sporadic and
MEN1 patients were selected for expression analysis. To further verify the findings, neuroendocrine BON1 and QGP
cell lines were tested for Noxa expression and its recovery by treatment with the proteasome inhibitor bortezomib. The
expression of Noxa was significantly downregulated in 20 PNENs (p = 0.0036). There was no significant difference
between MEN1 and sp oradic tumors. However, the malignant tumors showed a more significant decrease as compared
to benign tumors (p = 0.0385 ) and the decrease in expression in tumor s greater than 20 mm was very highly sign ificant
(p < 0.0001). Neither the BON1 nor QGP1 cell lines displayed expression of Noxa protein in the absence of the protea-
some inhibitor, Bortezo mib. After stimulation with the drug for 16 h, the expression was induced in both cell lines that
are correlated with an increase in the level of c-MYC expression, cleaved caspase 3 and cell death. The low expression
level of Noxa in PNENs contributes to the inability o f these tumor entities to undergo apoptos is. The recovery of Noxa
expression following treatment with the proteasome inhibitor, bortezomib, leading to caspase activation and cell death
supports the use of such drugs for the treatment of these tumor entities.
Keywords: Pancreatic Neuro end ocrine Neoplasms; Noxa, Bortezomib
1. Introduction
Pancreatic neuroendocrine neoplasms (PNENs) represent
5% - 10% of pancreatic tumors and include gastrinoma,
insulinoma and non-functioning endocrine tumors. They
occur sporadically or in the autosomal dominant inher-
ited syndromes such as multiple endocrine neoplasia type
1 (MEN1) and von-Hippel-Lindau. Although plasma
chromogranin A and 24-h urine for 5-hydroxyindole ace-
tic acid are known prognostic markers for survival in
patients with such tumors, the role of other tumor mark-
ers is not well described [1,2]. Altho ugh these tumors are
surgically manageable, metastatic disease is present in
~50% of patients at the time of diagnosis, and the overall
5-year surviv al for patien ts with d istant metastasis is on ly
22%. Somatostatin analogues, IFN-α or hepatic artery
chemoembolization prov ide good palliation of the symp-
toms of carcinoid syndrome associated with such tumors.
However, few systemic therapies have been shown to
consistently elicit tumor responses and prolong survival.
Recently, the mTor-inhibitor, everolimus, and the multi-
targeting sunitinib have been shown to increase overall
survival in randomized controlled trials [3,4]. The inef-
fectiveness of most systemic chemotherapy in metastatic
neuroendocrine tumors may relate to their unique bio-
logical features such as slow growth pattern or hyper-
vascularity [5].
The p53 tumor-suppressor protein functions as a tran-
scriptional activator and several p53-indu cible genes that
play a role in the induction of apoptosis in response to
p53 have been described [6]. The pro-apoptotic gene,
Noxa encodes a Bcl-2 homology 3 (BH3)-only member
of the Bcl-2 family of proteins that inactivates the pro-
Pancreatic Neuroendocrine Tumors Are Characterized by Loss of Noxa Expression that
Can be Recovered by the Proteasome Inhibitor Bortezomib 29
tective function of the pro-surv ival members of the Bcl-2
family and unleashes the mitochondrial-based apoptotic
cascade by activating the Bax/Bak-like pro-apoptotic
family members. In fact treatment of metastatic mela-
noma or multiple myeloma with proteasome inhibitors
has been reported to result in selective tumor cell killing
by triggering the induction of Noxa expression [7,8].
A cDNA microarray analysis with 11,500 genes was
performed using RNA from 10 gastrinoma tumor sam-
ples, both sporadic and MEN1, and compared to RNA
from normal islet cells obtained from healthy donors as
reference. The comparative analysis of gene expression
patterns revealed overexpression of 15 and underexpres-
sion of 123 genes greater than 3-fold [9]. Included in this
list was the gene Noxa, a proapoptotic member of the
Bcl-2 family [10]. We hypothesized that this loss could
contribute to tumor progression and proceeded to analyze
the expression in a wider range of PNENs to verify these
results and correlate them with clinical findings.
2. Materials and Methods
2.1. Patients
We randomly selected 10 patients with benign and 10
patients with malignant neuroendocrine pancreatic tu-
mors from the tumor bank at our institute. Five of each
group were genetically confirmed MEN1 patients. Clini-
cal data were prospectively recorded and analyzed with
special regard to symptoms. The diagnosis of a func-
tional tumor was established if hormone excess was pre-
sent, excluding an isolated elevation of pancreatic poly-
peptide (PP). Zollinger-Ellison Syndrome (ZES) was
diagnosed in the presence of clinical symptoms, an ele-
vated fasting serum gastrin level (ref < 125 pg/ml), a
positive secretin stimulation test (increase > 200 pg/ml)
and a low pH of the stomach. If symptomatic hypogly-
cemia (<40 mg/ml) was combined with hyperinsulinism
(>20 µU/ml) insulinoma was confirmed by a supervised
fasting test. A Vipoma was defined in case of watery
diarrhea (>6l/day) and elevated VIP serum levels (>130
pg/ml). Pancreatic tumors were considered non-func-
tional if neither hormone levels were elevated nor symp-
toms associated with hormone excess were evident ex-
cept PP. Tumors were classified as malignant if they
demonstrated infiltrating growth, angio invasion or lymph
node or distant metastases. The pathological classifica-
tion was performed according to Rindi and Klöppel [11].
The twenty fresh frozen tissue samples were obtained
from the tumor bank of Philipps-University of Marburg.
Clinical follow-up was conducted by the patients’ per-
sonal physicians or at outpatient attendance. Survival
was calculated as the time from surgical resection to ei-
ther death or last follow up. Informed consent was ob-
tained from all patients. The Ethics Committee of the
University approved this study and all patients partici-
pating in the study consented to sampling.
Before proceeding with RNA isolation from these 20
tumor samples, an initial histochemical staining was
performed to confirm a neoplastic cellularity of 85%.
Again, the reference contained pooled RNA from normal
human islet cells.
2.2. RNA Isolation and Quantitative Real-Time
Frozen control and tumor tissue samples were homoge-
nized in the presence of guanidinium thiocyante-phe-
nol-chloroform (Trizol Reagent; Invitrogen, Paisley,
Scotland, UK) following the manufacturer’s protocol.
Total RNA was then further purified by digestion with
DNaseI and recovery of RNA using an RNeasy kit
(Qiagen, Hilden, Germany) according to the supplier’s
protocol. The relative expression of Noxa mRNA in tu-
mors and normal islet cells was determined by Real Time
PCR on a Roche Light Cycler using the following prim-
con 298 bp; Accession number NM 21127; 6) and
previously described [12]. Negative controls contained
no cDNA. Expression levels were normalized to GAPDH.
2.3. Cell Culture and Biological Reagents
The BON-1 cell line was derived from a peripancreatic
lymph node metastasis of a serotonin-producing PNEN
[13]. The cells were cultured in a 1:1 mixture of Hams
F12 Nutrient Mixture and 1x Dulbecco’s Modification of
Eagle’s Medum (DMEM) (Life Technologies). The
QGP-1 cell line was derived from a somatostatinoma and
was grown in RPMI 1640. Both cell lines were supple-
mented with 10% fetal calf serum, 1% L-glutamine
(Biochrom KG, Berlin, Germany) and 1% penicillin-
streptomycin (Life technologies) and grown in an incu-
bator maintaining an atmosphere of 95% humidity and
5% CO2 at 37˚C.
Each cell line was plated on day minus 1 and incu-
bated on day 1 with 150 nM Bortezomib (LC Laborato-
ries, Boston) or not for 16 h. The control cells received
vehicle, DMSO. Both floating and adherent cells were
harvested and whole cell extracts were obtained using
RIPA buffer.
2.4. Western Blot
For the Western blot, 30 µg of total protein from cell
Open Access JCT
Pancreatic Neuroendocrine Tumors Are Characterized by Loss of Noxa Expression that
Can be Recovered by the Proteasome Inhibitor Bortezomib
Open Access JCT
lysates were analyzed by 17% SDS-PAGE and blotted
onto nitrocellulose membrane (Schleicher & Schuell,
Wh atman GmbH, Dassel, Germany). Filters were blocked
with 5% nonfat dry milk in PBS and incubated overnight
at 4˚C with specific antibodies (Noxa: Imgenex, San
Diego, California, 1:100; Cleaved Caspase 3, Cell Sig-
naling Technology, 1:1000; Actin, Sigma-Aldrich, 1:
2500; c-Myc, Santa Cruz sc-42, 1:200). After washing,
blots were incubated with horseradish peroxidase-con-
jugated secondary antibodies (Vectastain) and then re-
vealed by enhanced chemiluminescence (Amersham Bio-
2.5. Statistics
Data were analyzed using SPSS, version 14.0 for Micro-
soft Windows. Means were compared using paired t-tests.
A p-value < 0.05 was considered significant.
3. Results
Based on previous results from our cDNA microarray
analysis of 10 gastrinomas in comparison to pooled RNA
from normal islet cells we proceeded to verify the finding
of a downregulation of the Noxa gene in PNENs. For this
purpose we selected 20 additional PNEN tumor samples.
3.1. Patients
The median age of patients (12 male and 8 female) was
44 years (29 - 61) and these were followed for a median
period of 61 months (13 - 193). The median tumor di-
ameter was 15 mm (5 - 2 50). A summary appear s in Ta-
ble 1.
3.2. PNENs vs. Normal Islet Cells
Real time PCR analysis of 20 samples, 10 malignant and
Table 1. Summary of clinical data.
ID Spordic/MEN
1 Tumor Stage
WHO Age at
Dx (yr) Tumor
Size (mm)LNM Liver
Met Ki 67Follow up
(mos) Other therapies Ct Ct-Ref
1 MEN1 M GAS III 46 6 y N 1% NED (136)none 33.017.58
2 MEN1 M GAS III 49 5 n Y ND DOC (84) none 32.97.47
3 MEN1 M GAS III 35 8 y N 2-3%DOD (136)DOTATOC 31.095.66
4 MEN1 M VIP III 32 250 y N ND DOD (240)somatostatin,
Interferon 34.38.87
5 MEN1 NECA III 33 10 n N 6% AWD (120)none 32.817.38
6 sporadic M GAS III 58 15 n Y ND DOD (78) none 30.515.08
7 sporadic M GAS III 29 15 y N 2% NED (146)none 35.129.69
8 sporadic M GAS III 50 20 n N ND NED (235)none 34.819.38
9 sporadic NECA III 45 25 y Y 30% AWD (25)somatosttin-LAR 35.8110.38
10 sporadic NECA III 44 15 y N 2% AWD (18)none 33.17.67
11 MEN1 INS II 48 40 n N ND NED (107)none 31.986.55
12 MEN1 INS I 32 20 n N 3% NED (79) none 31.245.81
13 MEN1 NFPET I 30 10 n N 1% AWD (72)none 29.794.36
14 MEN1 NFPET II 54 50 n N 2% AWD (108)none 35.6510.22
15 MEN1 NFPET II 37 40 n N 1% NED (60) none 34.969.53
16 sporadic GAS II 61 15 n N ND NED (125)none 30.735.3
17 sporadic GAS I 45 9 n N ND DOD (124)none 31.25.77
18 sporadic INS I 61 10 n N 1% NED (13) none 28.573.14
19 sporadic INS II 32 13 n N 5% NED (15) none 33.598.16
20 sporadic INS I 32 8 n N 2% NED (52) none 27.442.01
LNM, lym ph node m etasta sis; Me t, meta stasis; M , m aligna nt; Gas, gastri nom a; VIP, vipom a; NE CA, ne uroend ocrine carc inom a; IN S, i nsulin oma ; NFPE T, non-
functional pancreatic endocrine tumor; NED, no evidence of disease; DOC, dead of unrelated causes; DOD, dead of disease; AWD, alive with disease; ND, not
determin ed; y, yes; n, no; Ref, reference.
Pancreatic Neuroendocrine Tumors Are Characterized by Loss of Noxa Expression that
Can be Recovered by the Proteasome Inhibitor Bortezomib 31
10 benign, was performed to determine the expression of
Noxa using normal human islet cells pooled from three
patients as a reference. All values for Noxa were normal-
ized according to the results with GAPDH. All samples
tested demonstrated a decrease in the level of expression
of Noxa. Figure 1 shows the results of this analysis
where the average threshold cycle (Ct) is presented for
the group of 20 PNENs (Ct = 32.1) compared to that of
normal islet cells (Ct = 25.3). A paired t test of these re-
sults suggests that this result is very statistically signifi-
cant (p = 0.0036).
Of the 20 PNENs, 10 were from MEN1 patients: 5
malignant and 5 benign. Interestingly, there was no sig-
nificant difference in the downregulation of Noxa be-
tween these two groups: MEN1 and non-MEN1 (data not
3.3. Malignant vs. Benign
The results of the real time PCR analysis were graphed
according to the designation malignant (Ct = 33.05) or
benign (Ct = 31.21). Interestingly, h ere there is a statisti-
cally significant difference between the two groups as
determined by a two tailed test (p = 0.0385). The loss of
Noxa expression is greater in malignant tumors as com-
pared to benign tumors (Figure 1).
3.4. PNENs Larger Than 20 mm
A further analysis compared the tumors >20 mm in size
to the expression in normal islet cells. Here the decrease
in expression of the tumor suppressor, Noxa is most evi-
dent with an average Ct of 33.5. The statistical analysis
results in a two-tailed p value of less than 0.0001 which
is extremely statistically significant (Figure 1).
Thus, the results of the initial microarray analysis were
confirmed by real time PCR on 20 additio nal tumor sam-
3.5. Noxa Expression in Tumor Cell Lines,
QGP1 and BON1
The neuroendocrine tumor cell lines, BON1 (serotonin-
producing PNEN) and QGP1 (somatostatinoma) were
analyzed for their expression of Noxa. For this purpose
the cells were grown in culture and total protein extracts
were prepared and applied to a 17% SDS PAGE. The
results of this Western blot analysis are presented in
Figure 2. Lanes 1 and 3 contain the extracts from BON1
and QGP1 cells, respectively. Although the presence of
Actin (42 kDa) verifies the presence of protein in the
lane, there is no band at 11 kDa where Noxa protein
would be expected to run. This lack of or very low level
of expres- sion mimics the real ti me PCR results with the
PNEN patient samples.
Figure 1. Results of the real time PCR Analyses for Noxa
expression in PNENs.
3.6. Expression of Noxa in Cell Lines Inducible
by Bortezomib
Both cell lines were treated with or without 150 nM bo r-
tezomib for 16 h. The cell cultures were analyzed micro
scopically to confirm cell death in the treated cultures.
Both floating and adherent cells were harvested, pelleted
and RIPA buffer was used to isolate cellular protein.
Following quantitation, 30 µg were applied to the SDS
PAGE, blotted and the membrane was hybridized with
antibodies. As shown in Figure 2 Actin as a loading
control was not changed by treatment with the drug.
However, the level of expression of Noxa, which was not
visible in untreated cells (lanes 1 and 3) was clearly in-
duced following treatment with bortezomib (lanes 2 and
Cleavage of Caspase 3 was observed following the
treatment with the drug as evidence that the cell killing is
apoptosis. The known function of Noxa as a pro-apop-
totic member of the Bcl-2 family would suggest that the
increased expression of this protein leads to the apoptotic
death of the cells.
The level of expression of c-MYC was visible before
treatment with the drug and was induced by treatment of
the cells with bortezomib. As MYC is known to tran-
scriptionally activate the expression of Noxa [14,15], th is
would be the proposed mechanism for the increased ex-
Thus, treatment with Bortezomib stabilizes MYC that
re-induces Noxa expression in these cells, leading to
apoptotic killing.
4. Discussion
In this study we have validated the down regulation of
the pro-apoptotic Noxa gene in PNENs. These tumors
express Noxa at a lower level than that of normal islet
cells. Malignant tumors display a more significant de-
crease than benign and larger tumors (>20 mm) show the
greatest decrease. This low level of expression is mir-
rored in the tumor cell lines, BON1 and QGP1.
Open Access JCT
Pancreatic Neuroendocrine Tumors Are Characterized by Loss of Noxa Expression that
Can be Recovered by the Proteasome Inhibitor Bortezomib
Figure 2. Western Blot Analysis of BON1 and QGP1 cells
treated with and without bortezomib.
Bortezomib, also known as Velcade and previously
known as PS-341, is a dipeptidyl borinic acid that is a
specific, potent and reversible inhibitor of the 26S pro-
teosome. Many regulatory proteins governing the cell
cycle, transcription factor activation, apoptosis and cell
trafficking are the substrates for proteosome-mediated
degradation. This proteosome inhibitor has shown anti-
tumor activity in a wide range of malignancies [7,8].
It has been previously shown that bortezomib pro-
motes a proapoptoitc shift in the levels of proteins in-
volved in mitochondrial outer-membrane permeabiliza-
tion and is a potent activator of the mitochondrial path-
way of apoptosis [7,8,16]. It induces the proapoptotic
BH3-only family member Noxa in a p53 independent
fashion that is a key element in the triggering of a cas-
pase cascade culminating in apoptosis in melanoma and
myeloma cells. The proteasome modulates not only the
half-life of Noxa protein but also its mRNA levels [7,8]
Noxa was identified as the first proapoptotic factor in-
duced by bortezomib preferentially in cancer cells [7,8,
14]. Tumor cell-restricted induction of Noxa is a unique
impact of bortezomib on the apopotoic machinery of
tumor cells.
The protooncogene, c-MYC, itself is a proteasomal
target whose levels of function are invariably upregulated
during tumor progression. c-MYC is known to be over-
expressed in PNENS [17,18]. The identification of con-
served MYC binding sites in the Noxa promoter ex-
plains the mechanism of induction of Noxa expression
following stabilization of MYC protein following pro-
teasomal inhibition [14]. This regulation of the apoptotic
machinery by c-MYC underscores a strategy to exploit
the altered genetic background of tumor cells for a spe-
cific induction of cell death. The function of c-MYC
could be the long sought after oncogenic event confer-
ring sensitivity of cancer cells to proteaso me inh ibition in
contrast to healthy cells [14].
In the present study treatment with the proteosome in-
hibitor, bortezomib induces the protein level of c-MYC
(Figure 2, lanes 2 and 4). C-MYC acts as an inducer of
the expression of Noxa (Figure 2). The increased level of
Noxa in neuroendocrine tumor cells leads to apoptosis as
evidenced by the cleavage of Caspase-3 [19] (Figure 2).
Although validation in animal studies should still be
performed, these results support the use of proteasome
inhibitors for the treatment of neuroendocrine tumors.
A common feature of PNENs is their slow-growing
nature and a long standing question has been as to why
these tumor entities, although slow growing, do not die.
The present study describes a lesion in the apoptotic
pathway that may be partially explained by the lack of
Noxa expression. Although the mechanism leading to
lower Noxa expression levels in these tumors relative
islet cells is not yet known, this finding can be exploited
in the treatment of these tumor entities. Proteasome in-
hibitors such as bortezomib can be used to re-induce this
expression and lead to cell killing. In addition explora-
tion into other drugs that exploit this pathway would be
warranted to further the treatment of neuroendocrine tu-
mor patients.
5. Acknowledgements
We express our appreciation to all patients who partici-
pated in the study. This research did not receive any spe-
cific grant from any funding agency in the public, com-
mercial or not-for-profit sector.
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