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Identification of a novel Raf-1 pathway activator that inhibits
gastrointestinal carcinoid cell growth
Mackenzie R. Cook, Scott N. Pinchot, Renata Jaskula-Sztul, Jie Luo, Muthusamy
Kunnimalaiyaan, and Herbert Chen
Endocrine Surgery Research Laboratory, University of Wisconsin, and the University of Wisconsin
Carbone Cancer Center, Madison, Wisconsin 53792-7375
Abstract
Carcinoids are neuroendocrine tumors (NETs) that secrete hormones, including serotonin, resulting
in the malignant carcinoid syndrome. In addition to the significant morbidity associated with the
syndrome, carcinoids are frequently metastatic at diagnosis and untreated mortality at 5 years tops
70%. Surgery is the only curative option and the need for other therapies is clear. We have previously
shown that activation of Raf-1 inhibits carcinoid cell proliferation.
We investigated the ability of Leflunomide (LFN), an FDA approved medication for the treatment
of rheumatoid arthritis, and its active metabolite Teriflunomide (TFN) as a potential anti-NET
treatment. LFN and TFN inhibit the in vitro proliferation of gastrointestinal carcinoid cells and induce
G2/M phase arrest. Daily oral gavage of nude mice with subcutaneous xenografted carcinoid tumors
confirms that LFN can inhibit NET growth in vivo. Treatment with TFN suppresses the cellular levels
of serotonin and chromogranin A, a glycopeptide co-secreted with bioactive hormones. Additionally
TFN reduces the level of Achaete-Scute Complex-Like 1 (ASCL1), a NET marker correlated with
survival. These effects are associated with the activation of the Raf-1/MEK/ERK1/2 pathway and
blockade of MEK signaling reversed the effects of TFN on markers of the cell cycle and ASCL1
expression.
In summary, LFN and TFN inhibit carcinoid cell proliferation in vitro and in vivo and alter the
expression of NET markers. This compound thus represents an attractive target for further clinical
investigation.
Keywords
Leflunomide; Teriflunomide; Carcinoid; Raf-1; Achaete-Scute Complex-Like 1
Introduction
A compound originally reported to be effective in Rheumatoid arthritis, Leflunomide (LFN)
(Arava, SU-101) has proven to be remarkably safe in human patients (1,2). Approved by the
FDA in 1998, LFN is nearly completely converted to its main active metabolite, Teriflunomide
(TFN) in first pass metabolism (1-3). While its current indications are limited to rheumatologic
conditions, recent studies have focused on the ability of LFN and TFN to inhibit the
proliferation of a variety of human malignancies, including an in vivo model of colon cancer.
Corresponding author and requests for reprints to: Herbert Chen, H4/722 Clinical Science Center, 600 Highland Avenue, Madison,
WI 53792-7375, Office: (608) 263-1387, FAX: (608) 263-7652, Chen@surgery.wisc.edu.
The authors have no potential conflicts of interest to disclose.
NIH Public Access
Author Manuscript
Mol Cancer Ther. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
Mol Cancer Ther. 2010 February ; 9(2): 429. doi:10.1158/1535-7163.MCT-09-0718.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
While capable of inducing apoptosis in some cell lines, it does not appear that this is a general
effect of either LFN or TFN, as they are also capable of inducing cell cycle arrest without
necrosis or apoptosis. This is consistent with the known safety profile of LFN in vivo (4-8).
The recent focus on oncologic applications spurred our interest in the effect of LFN and TFN
in carcinoid cancer (9).
Derived from the diffuse enterochromaffin cells of the gastrointestinal tract, carcinoid cancer
is a subtype of neuroendocrine tumors (NETs). They characteristically secrete a variety of
bioactive hormones, including serotonin, that are implicated in the malignant carcinoid
syndrome(10). These cancers also characteristically express high levels of chromogranin A
(CgA), an acidic glycopeptide co-secreted with hormones and considered a clinical marker of
disease. High serum levels of CgA have been associated with a poor clinical prognosis in
carcinoid tumors (11-13). The basic Helix-Loop-Helix transcription factor, Achaete-Scute
Complex-Like 1 (ASCL1) is similarly highly expressed in NETs. Important in the development
of normal NE cells and lost in the normal adult tissue, ASCL1 expression is associated with
poor prognosis in small cell lung cancer, a related NET (14).
Gastrointestinal carcinoids are often clinically silent until hepatic metastases are present and
the patient begins to experience the debilitating flushing, wheezing and diarrhea of the
malignant carcinoid syndrome (15). It is important to note that in addition to this significant
morbidity, the 5 year untreated mortality is approximately 70% (15-20). Surgical resection
may be potentially curative, though is difficult with metastatic disease and optimal resection
still results in significant mortality at 5 years (16). Adjuvant therapies, including chemotherapy
and radiation, have shown limited success and patients become rapidly resistant to octreotide
(15,17,19,20). The severity of carcinoid disease and the lack of effective therapies highlights
the need for targeted treatment options.
The Raf-1 pathway has been described as having anti-carcinoid effects and validated as a
potential target in the treatment of carcinoid disease. We and others have shown that humans
NETs lack phosphorylated ERK 1/2 (extracellular signal-regulated kinase 1/2), a marker of
Raf-1 pathway activation. Activation of the Raf-1/MEK/ERK1/2 pathway suppresses levels
of ASCL1 as well as CgA and serotonin. In a model of medullary thyroid cancer, a related
NET, the ability of Raf-1 pathway activation to suppress in vivo NET growth was confirmed
(21-25). While several excellent reviews of the Raf-1 pathway exist, we will briefly summarize
the important points. Active, phosphorylated, Raf-1 phosphorylates MEK1/2 (Mitogen-
activated protein kinase 1/2) and subsequently ERK 1/2 which then targets multiple key cellular
pathways (26).
In this paper we describe, for the first time, the activity of LFN and TFN in NET cells. We
show that LFN and its active metabolite, TFN, are capable of suppressing growth of human
carcinoid cells in vitro, by inducing cell cycle arrest and that this finding can be replicated in
vivo. We also show that these compounds can inhibit the cellular expression of CgA and
serotonin, compounds linked to the poor prognosis of NETs. Furthermore, we show that these
compounds suppress ASCL1, a well characterized marker of NE malignancy, at the protein
and the mRNA level through a mechanism that is predominately dependent upon the Raf-1/
MEK/ERK1/2 pathway.
Methods
Cell Culture and Treatment
Human GI carcinoid cancer cells (BON), graciously provided by Drs. B. Mark Evers and
Courtney M. Townsend, Jr. (University of Texas Medical Branch, Galveston, TX, USA), and
NCI-H727 human bronchopulmonary carcinoid tumor cells (American Type Culture
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Collection, Mannassas, VA, USA) were maintained in DMEM F12 and RPMI1640 (Life
Technologies, Rockville, MD, USA), respectively, supplemented with 10% fetal bovine serum
(Sigma-Aldrich, St. Louis, MO, USA), 100 IU/mL penicillin and 100 μg/mL streptomycin
(Life Technologies) in a humidified atmosphere of 5% CO2 in air at 37°C
LFN and TFN (Calbiochem, San Diego CA) were dissolved in DMSO at 100mM and stored
at −80C. Fresh dilutions in media were prepared for each experiment and new media dilutions
were added every 48 hours for multi-day experiments. The MEK1/2 inhibitor UO126
(Promega, Madison WI) was stored as a stock solution in DMSO at −20°C and fresh dilutions
in media were prepared for each experiment.
Cells were plated and allowed to adhere overnight. Either LFN or TFN at concentrations
ranging from 0-125 μM was then added and cells incubated for 48, 96 hours or 168 hours.
When U0126 was used, cells were pretreated with 10 μM U0126 for 1 hour prior to addition
of 100 μM TFN.
Cell Proliferation Assay
Carcinoid tumor cell proliferation was measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5
diphenyl tetrazolium bromide (MTT) rapid colorimetric assay as previously described (27).
Briefly, BON cells were seeded on 24-well plates and incubated overnight under standard
conditions to allow cell attachment. Cells were then treated with LFN or TFN (Calbiochem)
in quadruplicate with up to 125 μM and incubated for up to 6 days.
The MTT assay was performed by replacing the standard medium with 250 μL of serum-free
medium containing 0.5 mg/ml MTT and incubating at 37°C for 4 hours. After incubation, 750
μl of dimethyl sulfoxide (Fischer Scientific, Pittsburgh, PA) was added to each well and mixed
thoroughly. The multiwell plates were then measured at 540 nm using a spectrophotometer
(μQuant; Bio-Tek Instruments, Winooski, VT).
Cell Cycle Analysis by Flow Cytometry
BON cells were treated with TFN (0, 50, and 100 μM) for 48h. After treatment, the cells were
harvested, washed with ice cold 0.9% saline buffered with phosphate to a pH of 7.4 (1x PBS),
and viability was determined using trypan blue exclusion (Mediatech, Herndon, VA). For DNA
content analysis, cells (1 × 106) were fixed with ice cold 70% ethanol, washed with 1x PBS,
incubated with 0.2 mg/ml RNAse-A and stained with 10 μg/ml Propidium iodide (PI) staining
solution. FACS analysis was performed on a flow cytometer at 488 nm (FACSCalibur flow
cytometer; BD Biosciences), results were analyzed with ModFit LT 3.2 software (Verity,
Topsham, ME).
Tumor Xenograft Studies
Male nude (Nu/Nu) mice (Charles River Laboratories, Wilmington, MA) were injected
subcutaneously with 1×106 gastrointestinal carcinoid cells suspended in 100 μL Hanks
Balanced Salt Solution (Mediatech Inc. Manassas VA). Palpable tumors were allowed to
develop, and stratified randomization was used to assign mice to either a control or treatment
group. Mice were treated with either 35 mg/kg Leflunomide suspended in 1.5% carboxymethyl
cellulose, or 1.5 % carboxymethyl cellulose alone, by daily oral gavage. This dose was chosen
based upon previously published doses in a mouse model of colon cancer(5). Tumor size was
measured with vernier calipers every four days and tumor volume calculated using the formula,
0.52 x [(length) x (width)2], where width was defined as the shorter tumor dimension. After
28 days of treatment, animals were sacrificed. All animal care and treatment was performed
in compliance with our animal care protocol approved by the University of Wisconsin–
Madison animal care and use committee.
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Immunoblot Analysis
After treatment as described, cells were washed with ice cold 1X PBS, and total protein lysates
prepared. Total protein concentration was measured with a bicinchoninic acid assay kit (Pierce
Protein Research Products, Rockford, IL). Denatured cellular extracts were resolved by
10%-12% SDS-PAGE (Invitrogen), transferred onto a nitrocellulose membrane (Bio-Rad
Laboratories, Hercules, CA) and incubated overnight in the appropriate primary antibody. The
antibody dilutions were as follows: 1:1,000 for phosphorylated ERK 1/2thr202/tyr204, Total ERK
1/2, phosphorylated MEK1/2ser217/221, phosphorylated Raf-1ser338, phosphorylated Glycogen
Synthase Kinase-3βSer9 (GSK-3β), phosphorylated AKTSer473 and cyclin B1 (Cell Signaling
Technology, Beverly, MA), 1:2000 for mammalian Achaete-Scute Homologue-1 for detection
of ASCL1 (BD PharMingen, San Diego, CA), CgA (Zymed Laboratories, San Francisco, CA),
1:1000 (Cell Signaling) and 1:10,000 for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) (Trevigen, Gaithersburg, MD). Horseradish peroxidase-conjugated goat anti-rabbit
or anti-mouse secondary antibodies (Pierce) were used depending on the source of the primary
antibody. Immunstar (Bio-Rad), or SuperSignal West Pico or Femto (Pierce) kits were used
per the manufacturer’s instructions for detection.
Serotonin Enzyme Linked Immunosorbent Assay (ELISA)
To determine serotonin levels in cellular extracts of carcinoid cells treated with TFN for 48 h,
we utilized a serotonin enzyme-linked immunosorbant assay (ELISA) kit as per the
manufacturer’s instructions (Fitzgerald, Concord, MA). The multiwell plates were then
measured at 405 nm using a spectrophotometer (μQuant; Bio-Tek Instruments, Winooski, VT).
Serotonin concentrations were calculated based on the manufacturer’s standard curve.
Quantitative real-time reverse transcriptase Polymerase Chain Reaction (qPCR)
BON cells were treated with TFN (0, 50, and 100 μM) for 48h. Total RNA was harvested using
a Qiagen RNeasy minikit (Qiagen, Valencia, CA), per the manufacturers directions. Integrity
was assured and concentration determined using a Nanodrop spectrophotometer (Nanodrop,
Wilmington DE). Exactly 2 μg of total RNA were then converted to complementary DNA
(cDNA) using the iScript cDNA synthesis kit (Biorad) according to the manufacturers
directions.
The qPCR reactions were performed on the Biorad iCycler, conditions were: 3 minutes at 95°
C, 35 cycles of: 30 seconds at 95°C, 25 seconds at 60°C and 30 seconds at 72°C followed by
1 minute at 95°C and 1 minute at 55°C. Primers were obtained from Integrated DNA
Technologies (Coralville, IA) and sequences used were: ASCL1(F: 5′ TCC CCC AAC TAC
TCC AAC GAC 3′, R: 5′ CCC TCC CAA CGC CAC TG 3′), GAPDH (F: 5′ ACC TGC CAA
ATA TGA TGA C 3′, R: ACC TGG TGC TCA GTG TAG 3). Results were normalized to
GAPDH from the same sample. Expression of ASCL1 calculated for each treatment using the
formula 2(Ct(GAPDH)-Ct(ASCL1)) as outlined in the iCycler Applications Guide (Biorad).
Expression was then plotted as average ± standard error of the mean (SEM).
Statistical Analysis
Densitometric analysis of Western blotting results was performed using Quantity One software
v. 4.6.3 (Biorad). Analysis of MTT growth curves was performed using a one way analysis of
variance (ANOVA) testing and Bonferroni post-hoc testing. In the analysis of the nude mouse
xenograft experiment, we utilized a Pearson’s chi-square test. Student’s t-test was utilized to
compare ELISA and qPCR results. In this work, p < 0.05 was considered statistically significant
and analyses were performed with SPSS software v. 10.0 (SPSS, Chicago, IL). Unless
specifically noted, all data are represented as mean ± SEM.
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Results
Leflunomide and Teriflunomide inhibit in vitro proliferation of GI carcinoid cells
As recent studies have described attributed anti-proliferative effects to LFN and TFN, we first
sought to examine the ability of LFN and TFN to inhibit carcinoid proliferation in vitro (4,6).
BON cells were treated with increasing doses of either LFN or TFN and the MTT assay was
performed at 2, 4 and 6 days. Dose dependent, statistically significant growth inhibition was
observed after treatment with both LFN FIGURE 1A and TFN FIGURE 1B at 4 and 6 days (p
≤ 0.05 vs. control). These data suggest that LFN and its major metabolite can inhibit the in
vitro proliferation of human GI carcinoid cells.
Treatment with LFN and TFN additionally resulted in the suppression of in vitro proliferation
after 2 days of treatment, though this did not reach statistical significance in all treatment groups
(Data not shown). In total, these data suggest that LFN and TFN can inhibit in vitro proliferation
of gastrointestinal carcinoid cells.
Teriflunomide induces cell cycle arrest and suppresses in vivo carcinoid growth
TFN has been published to induce cell cycle arrest and apoptosis, thus we performed Western
blot analysis for markers of cell cycle progression and apoptosis as well as PI exclusion flow
cytometry to determine the mechanism of in vitro growth suppression (4,6).
Treatment with increasing concentrations of TFN resulted in a dose dependent induction of
cyclin B1, a marker of G2/M phase transition FIGURE 2A (28, 29). We did not observe any
cleavage of poly (ADP)-ribose polymerase (PARP), a marker of apoptosis (data not shown).
These protein results suggest that the mechanism of growth inhibition is cell cycle arrest. We
next sought to confirm this finding using PI exclusion flow cytometry. Treatment with
increasing doses of TFN for 48 hours resulted in the progressive accumulation of S-phase
products FIGURE 2B. These data, in total, suggest that arrest prior to the G2/M transition is
the mechanism of TFN induced in vitro growth suppression.
Approved by the FDA in 1998, LFN is currently in clinical use and has proven to be remarkably
safe in human patients (2,3). With this in mind, we wanted to investigate the ability of LFN,
converted to TFN in first pass metabolism, to suppress the in vivo growth of a NET xenograft
(1,30). Treatment of subcutaneous GI carcinoid xenografts with daily oral gavage of LFN
resulted in the statistically significant suppression of tumor growth after 24 days of treatment
(p < 0.02) FIGURE 2C. These data suggest that the growth inhibitory effects observed in
vitro can be replicated in vivo with oral LFN.
Leflunomide and Teriflunomide can alter the expression of neuroendocrine markers
We then looked to investigate the effect of LFN and TFN on CgA, ASCL1 and serotonin. These
markers are of particular interest as they have been associated with poor prognosis and the
malignant carcinoid syndrome (10-12,14,22,31,32). In order to determine if LFN and TFN
were able to modulate the expression of NE markers, we treated BON cells for 48 hours with
increasing doses of LFN or TFN. Treatment with 125 μM LFN resulted in the 61% suppression
of CgA and the 42% suppression of ASCL1 protein FIGURE 3A. Treatment with 125 μM TFN
resulted in the 65% suppression of CgA and the 84% suppression of ASCL1 protein FIGURE
3B. Densitometric analysis was used to compare the 125 μM dose of LFN and TFN to their
respective controls.
The ability of TFN to alter the expression of cellular serotonin, a hormone important in the
development of the carcinoid syndrome, was next investigated using an ELISA. Treatment
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with TFN reduced the cellular expression of serotonin by 77% after 48 hours of treatment (p
< 0.005) FIGURE 3C.
As the expression of ASCL1 could potentially be modulated at several levels, we next
investigated the levels of ASCL1 mRNA after treatment with TFN (33,34). Using qPCR, we
observed that treatment with 50 and 100 μM TFN for 48 hours resulted in the suppression of
ASCL1 levels by 54% and 62%, respectively (p < 0.05) FIGURE 3D.
We next sought to confirm that the above results could be generalized. Treatment of NCI-H727
bronchopulmonary carcinoid cells with increasing doses of either LFN, FIGURE 3E, or TFN,
FIGURE 3F, resulted in the dose dependent inhibition of ASCL1 protein expression, by
western blot analysis. Treatment with 100μM LFN and TFN resulted in a 43% and 71%
reduction in ASCL1 protein expression, respectively. These data suggest that the ability of
LFN and TFN to alter the expression of a neuroendocrine tumor marker associated with poor
prognosis is not limited to a single cell line.
These data, in total, suggest that LFN and TFN can suppress the expression of NET markers
that are associated with the carcinoid syndrome as well a poor survival in NETs (14,31).
Teriflunomide activates the Raf-1/MEK/ERK pathway
The Raf-1/MEK/ERK1/2 pathway has been described by our group as being an important
regulator of NET cellular proliferation and hormone production (13,23-25). A relationship
between phosphorylated ERK1/2 and the regulation of the G2/M checkpoint has additionally
been described (35,36). Given these known associations, and the effects described above, we
hypothesized that TFN was inducing Raf-1 pathway activation. To examine the status of Raf-1
signaling, BON cells were treated with increasing doses of TFN and Western blotting
performed for key components of the pathway.
A dose dependent induction of phosphorylated Raf-1 as well as phosphorylated MEK1/2 and
phosphorylated ERK 1/2 was observed in BON cells treated for 48 hours with TFN FIGURE
4A. These results suggest that the Raf-1/MEK/ERK1/2 pathway is intact and that TFN is
capable of efficiently inducing pathway activation. The levels of total ERK 1/2 remained
unchanged, suggesting that pathway activation and not translation of additional protein was
responsible for the observed results.
Suppression of ASCL1 and induction of cell cycle arrest is a direct result of Raf-1 pathway
activation
In order to show that the effects on NET cells are directly due to Raf-1/MEK/ERK pathway
activation, we used U0126, an inhibitor of phosphorylated MEK1/2, to disrupt activation of
this pathway. The induction of phosphorylated ERK 1/2 seen with 100 μM TFN can be totally
blocked by pretreatment with 10 μM U0126. This suggests that TFN induces the
phosphorylation of ERK 1/2 via MEK1/2. Importantly, the reduction of ASCL1 and induction
of cyclin B1 are similarly reversed by pretreatment with U0126 FIGURE 4B.
As the regulation of ASCL1 was shown to be at the level of mRNA, we next performed qPCR
on BON cells treated with either TFN alone or TFN after pre-treatment with UO126. While
100 μM TFN potently suppresses the level of ASCL1 mRNA, pre-treatment with U0126 is
able to block this suppression FIGURE 4C. These data suggest that TFN mediated suppression
of ASCL1 is dependent upon the Raf-1/MEK/ERK pathway at both the protein and mRNA
level. Additionally, these results suggest that TFN induced cell cycle arrest may also be
modulated by this pathway.
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In order to demonstrate that the effects on protein phosphorylation were pathway specific, we
next investigated the phosphorylation state of two other anti-carcinoid pathways, GSK-3β
FIGURE 4D and Akt FIGURE 4E (37-39). No change in the phosphorylation state of these
proteins was noted after treatment with TFN. These data suggest that the effects on protein
phosphorylation may be specific to the Raf-1/MEK/ERK1/2 pathway.
Discussion
Carcinoid tumors are the second most common source of isolated hepatic metastases, after
colorectal cancer, and carry an untreated 5 year mortality in excess of 70% (16). The most
effective chemotherapeutic combination tried to date has resulted in a less than 6% volume
response rate(40). This lack of efficacy extends to other adjuvant therapies as well as the
palliative option, octreotide, to which patients rapidly become resistant (10). The need for novel
targeted therapies is therefore clear.
We present data that suggests that LFN and TFN are novel potential therapeutic options for
the treatment of carcinoid disease. LFN and TFN are capable of suppressing in vitro cell
proliferation and TFN is shown to induce cell cycle arrest prior to the G2/M phase transition.
Additionally, in animal studies, we show that the in vitro growth inhibitory effects of LFN can
be replicated in vivo with daily oral dosing of LFN. These drugs can additionally suppress the
levels of CgA, ASCL1 and serotonin, all markers of NE malignancy. The suppression of these
three markers is an important point as serotonin is a mediator of the malignant carcinoid
syndrome and the expression of CgA and ASCL1 have been correlated with poor prognosis
(10-12,14,31,32). These effects on carcinoid cell markers appear to be mediated predominately
through the Raf-1/MEK/ERK1/2 pathway, a pathway that has been extensively studied as a
potential anti-carcinoid target (13,23-25).
The Raf-1 pathway has been traditionally thought of as a tumorigenic pathway and has been
noted to be either mutated or over-expressed in hepatocellular carcinoma, non-small cell lung
cancer, melanoma and papillary thyroid carcinoma (41,42). In tumors of NE origin, however,
there is minimal phosphorylation of ERK1/2 at baseline, suggesting that the Raf-1 pathway
does not play an essential oncogenic role. Activation of Raf-1 signaling in a gastrointestinal
carcinoid cell, with an estrogen inducible construct, results in the suppression of ASCL1, CgA
and serotonin(13,23). Additionally, in a medullary thyroid cancer xenograft model, activation
of the Raf-1 pathway resulted in the suppression of in vivo tumor growth (24). This work
suggests that Raf-1 pathway activation, if accomplished pharmacologically, could be a potent
strategy for the inhibition of carcinoid cancer and other NETs.
Our group has described ZM336372 and Tautomycetin (TTM) as two compounds that appear
to activate the Raf-1 pathway in vitro and inhibit the growth of carcinoid cancer and MTC
(25,43). Significant obstacles, however, exist between these compounds and clinical
applicability. TTM is a natural compound that must be isolated from Streptomyces
spiroverticillatus, limiting the quantity that can be produced at any one time. The exceptionally
poor solubility of ZM336372 limits its clinical utility and attempts to produce more soluble
sister compounds have met with limited success. In addition to these limitations, neither
compound has been described in vivo and thus ability to achieve necessary concentrations with
acceptable toxicity is unknown.
In contrast, we present LFN as a Raf-1 pathway activator in NETs that is FDA approved and
known to be safe in humans. Serum concentrations of TFN in human rheumatoid arthritis
patients treated with daily oral LFN are greater than 200μM, making the doses used in vitro
comparable to those attainable in humans(1,2). Additionally, peak serum TFN concentrations
in an in vivo model of oral LFN administration approach 500μM(44). Our animal data suggests
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that it is possible, even with oral dosing, to achieve blood concentrations of TFN sufficient to
slow the rate of gastrointestinal carcinoid cell proliferation. It is possible that higher doses
would result in more significant tumor inhibition. It is interesting that tumor size in the
treatment groups did not appear to diverge until after 14 days of treatment, perhaps suggesting
a role for the anti-angiogenic effects of LFN described by others (5). A larger upcoming study
designed to confirm and extend the in vivo data presented here will examine higher doses and
allow for histologic examination of the xenografted tumors.
LFN therefore, more than ZM336372 and TTM, represents a potential therapy for NETs
targeting the Raf-1 pathway. We conclude that LFN is worthy of additional study, including a
larger animal study and potentially an initial human trial.
Acknowledgments
Financial support:
Howard Hughes Medical Institute (MRC)
NIH – RO1 CA121115 (HC)
NIH – RO1 CA109053 (HC)
American College of Surgeons: George H. A. Clowes Jr. Memorial Research Career Development Award (HC)
Carcinoid Cancer Foundation Research Award (HC)
Abbreviations list
LFN Leflunomide
TFN Teriflunomide
NETs Neuroendocrine Tumors
MTC Medullary Thyroid Cancer
ASCL1 Achaete-Scute Complex-Like 1
CgA Chromogranin A
GI Gastrointestinal
PI Propidium Iodide
MTT 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide
ELISA Enzyme Linked Immunosorbent Assay
qPCR Quantitative Real-Time Polymerase Chain Reaction
ERK1/2 Extracellular regulated kinase 1/2
MEK1/2 Mitogen-activated protein kinase kinase 1/2
GSK-3βGlycogen Synthase Kinase – 3 Beta
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Figure 1.
LFN and TFN inhibit the proliferation of GI carcinoid cells. BON GI carcinoid cells were
treated with the indicated concentrations of either (A) LFN or (B) TFN for up to 6 days. Cell
viability was determined by the MTT colorimetric assay, treatments were significantly
different from control at 6 days (p ≤ 0.05). Experiments were performed in quadruplicate and
data is plotted as mean ± SEM.
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Figure 2.
TFN induces cell cycle arrest in vitro and suppresses in vivo proliferation of gastrointestinal
carcinoid cells. BON gastrointestinal carcinoid cells were treated with the indicated doses of
TFN and incubated for 48 hours. (A) A dose dependent accumulation of cyclin B1 was observed
by western blot, with GAPDH serving as a loading control. (B) Propidium Iodide exclusion
flow cytometry showed a dose dependent accumulation of S-phase products. Together, these
data suggest that TFN can induce in vitro cell cycle arrest prior to the G2/M transition. (C)
Daily oral gavage with 35mg/kg LFN of nude mice with subcutaneous xenografted NETs
resulted in the progressive inhibition of growth, statistically significant after 24 days (p < 0.02).
This result suggests that the observed in vitro growth suppression can be replicated in vivo.
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Figure 3.
LFN and TFN suppress important markers of NE malignancy. Treatment of BON
gastrointestinal carcinoid cells with either (A) LFN or (B) TFN results in a 61% and 65%
suppression of CgA and a 42% and 84% suppression of ASCL1 protein when the highest dose
is compared to control, respectively. GAPDH is presented as a loading control and
densitometry is performed on samples normalized to their respective GAPDH and subsequently
compared to control values. (C) Treatment with TFN additionally suppresses cellular content
of serotonin after 48 hours of treatment by 77% (p < 0.005). (D) Treatment with 50 and 100
μM TFN resulted in a 54% and 62% reduction in ASCL1 mRNA, respectively (p <0.05).
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Results were normalized to the GAPDH expression of the respective sample. Treatment of
NCI-H727 bronchopulmonary carcinoid cells with increasing doses of (E) LFN or (F) TFN
resulted in the 43% and 71% suppression of ASCL1 protein when the highest dose is compared
to control. Taken together, these data suggest that LFN and TFN can suppress key NET markers
implicated in the malignant carcinoid syndrome and correlated to poor prognosis, additionally
this effect may not be limited to a single cell line.
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Figure 4.
TFN suppresses ASCL1 via the Raf-1/MEK/ERK1/2 pathway. (A) Treatment of BON cells
for 48 hours with TFN resulted in a dose dependent increase in the level of phosphorylated
Raf-1, MEK and ERK 1/2 with no overall change in the level of total ERK. (B) Pretreatment
with the MEK1/2 inhibitor U0126 resulted in a reversal of TFN induced ASCL 1 suppression
at the protein level as well as reversal of the effects on cyclin B1, a marker of G2/M arrest.
GAPDH is presented as a loading control. (C) Similar pretreatment reversed the effects of TFN
on the level of ASCL1 mRNA, as measured by qPCR normalized to the GAPDH for the
respective sample. These data together suggest that TFN activates the Raf-1/MEK/ERK
pathway and that the suppression of ASCL1 protein and mRNA previously observed is
regulated via this pathway. Additionally, the observed cell cycle arrest may be mediated by
the same pathway. (D) No change in the phosphorylation state of Glycogen Synthase
Kinase-3β (GSK-3β) or (E) Akt was observed after carcinoid cell were treated with TFN. This
suggests that the modulation of protein phosphorylation may be specific to the Raf-1/MEK/
ERK pathway.
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