Pancreatic Neuroendocrine Tumors Are Characterized by Loss of <i>Noxa</i> Expression that Can be Recovered by the Proteasome Inhibitor Bortezomib

doi:10.4236/jct.2013.410A004

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Journal of Cancer Therapy, 2013, 4, 28-33

Published Online November 2013 (http://www.scirp.org/journal/jct)

http://dx.doi.org/10.4236/jct.2013.410A004

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.

Email: [email protected]

Received August 2nd, 2013; revised September 2nd, 2013; accepted September 10th, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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

RT-PCR (qRT-PCR)

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-

ers: NOXA_for GTC CGA GGT GCT CCA GTT;

NOXA_rev AAA CGT GCA CCT CCT GAG A (Ampli-

con 298 bp; Accession number NM 21127; 6) and

GAPDH_for CGT CTT CAC CAC CAT GGA GA;

GAPDH_rev CGG CCA TCA CGC CAC AGT TT as

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-

sciences).

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.

Pat

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

shown).

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-

ples.

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

4).

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-

pression.

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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