|Home | About | Journals | Submit | Contact Us | Français|
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.
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. 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.
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 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.
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).
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).
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.
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.
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.
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).
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.
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.
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.
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 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.
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.
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.
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.
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 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.
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)
The authors have no potential conflicts of interest to disclose.