Department of Surgery, The University of Wisconsin Medical School, and the University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin 53792
Submitted 26 September 2002 ; accepted in final form 3 April 2003
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ABSTRACT |
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MAP kinase; signal transduction; neuroendocrine tumors
Growth of carcinoid and other neuroendocrine tumors has been shown to be dependent on growth factors and various downstream signaling pathways (33, 35). Therefore, we hypothesized that manipulation of certain cellular signaling pathways could potentially alter hormone secretion and growth of carcinoid tumors (33). It has been shown that the activation of the ras/raf-1 signal transduction pathway in other neuroendocrine tumors leads to a differentiation response (7, 10, 27). However, the biological consequences of raf-1 induction in GI neuroendocrine tumors, such as carcinoids, are unknown.
To study the effects of raf-1 activation in GI carcinoid cells in vitro, we used an established pancreatic carcinoid cell line, BON, derived from a metastasis of a human pancreatic carcinoid tumor (16, 32). BON cells synthesize and secrete several bioactive molecules including 5-HT (serotonin), chromogranin A, synaptophysin, neurotensin, and neuron-specific enolase (26, 38). Several studies have previously shown that hormone and peptide secretion by BON cells can be altered by treatment with IGF-1, phorbol esters, and D-glucose, and even by mechanical stimulation (19, 20, 33).
In the present study, we show that BON cells have minimal levels of phosphorylated MEK and MAP kinases ERK1/2 at baseline. Activation of raf-1 in these cells led to high levels of phosphorylated MEK and ERK1/2 within 48 h. Induction of raf-1 and phosphorylated MEK and ERK1/2 in BON cells resulted in morphological changes accompanied by a marked decrease in the number of neuroendocrine secretory granules by electron microscopy. Raf-1 activation also caused significant reductions in the levels of 5-HT, chromogranin A, and synaptophysin. Importantly, although neuroendocrine marker and hormone levels were significantly reduced, raf-1 activation did not stimulate proliferation of BON cells. Furthermore, by using MEK inhibitors, we demonstrate that these raf-1-mediated changes are dependent on MEK activation.
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MATERIALS AND METHODS |
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BON-raf cell line. BON cells were stably transduced with the
retroviral vector pLNCraf-1:ER
(31). This construct is an
estrogen receptor-raf-1 fusion molecule, and contains the ligand-binding
domain of the estrogen receptor fused to the raf kinase domain of c-raf-1
(6,
34). Derivation and
maintenance of this cell line has previously been described
(31). To induce raf-1 activity
in BON-raf cells, 1 µM
-estradiol was added to the media. An
equivalent dilution of ethanol, the carrier for the
-estradiol, was used
to treat control cells. For the MEK inhibitor studies, cells were pretreated
with 2550 µM PD-98059
(7) and/or 510 µM
U-0126 (17) or control (DMSO)
for 45 min before the addition of
-estradiol or carrier control.
Electron microscopy. BON and BON-raf cells were grown to 5075% confluency on 25 cm3 tissue culture dishes and then treated with control or estradiol for 48 h. Cells were then fixed with 3% glutaraldehyde in 0.1 M cacodylated buffer, dehydrated with varying concentrations of ethanol and 2-hydroxypropyl methacrylate, and imbedded in Eponate. For quantitation of neuroendocrine secretory granules, random images at similar magnifications from each treatment group were selected. The number of secretory granules per cell was counted in a blinded manner by three independent observers.
Western blot analysis. Cells were trypsinized and cellular pellets
were lysed in sample buffer (50 mM Tris, 0.15 M NaCl, 0.1% SDS, 1% Nonidet
P-40, 0.5% Na/deoxycholate, and 0.6 mM PMSF). Total cellular protein
concentrations were determined with bicinchoninic acid (BCA) assay (Pierce,
Rockford, IL). Cellular extracts (15 µg) were boiled with equal amounts
(1:1) of loading dye (2% SDS, 20% glycerol, 0.1 M TRIS, 5%
-mercaptoethanol, and 0.04% bromophenol blue) for 10 min and through 10%
SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Schleicher
and Schuell, Keene, NH) by electroblotting. Membranes were blocked for at
least 1 h in a milk solution (1x PBS, 5% dry milk, 0.05% Tween-20). The
following primary antibody dilutions were used: ERK1/2 and phospho-ERK1/2
(1:1,000; Cell Signal Technology, Beverly, MA); MEK and phospho-MEK (1:1,000;
Cell Signal Technology); chromogranin A (1:1,000; Zymed Laboratories, San
Francisco, CA); and synaptophysin (1:500; Santa Cruz Biotechnology, Santa
Cruz, CA). Primary antibody incubations were performed either overnight at
4°C or for 1 to 2 h at room temperature. After primary antibody
incubation, membranes were washed 1 x 10 and 2 x 5 min in PBS-T
wash buffer (1x PBS, 0.05% Tween 20). Membranes were incubated with a
1:1,000 dilution of goat anti-rabbit secondary antibody (Cell Signal
Technology) for 1 h, except for synaptophysin in which a dilution of goat
anti-mouse antibody (1:1,000; Pierce) was used. Membranes were washed 1
x 10 and 2 x 5 min in PBS-T wash buffer and developed by enhanced
chemiluminescence (Amersham, Arlington Heights, IL) according to the
manufacturer's directions, except for synaptophysin, in which the Super West
Pico chemiluminescence substrate (Pierce) was used.
5-HT assay. To determine serotonin levels in cellular extracts, we used a serotonin ELISA kit (Research Diagnostics, Flanders, NJ) per the manufacturer's instructions. 5-HT values were standardized for total protein content and quantitated relative to control cells. Samples from two independent experiments in triplicate were analyzed.
Northern blot analysis. Cells were trypsinized and total RNA was extracted by using the RNeasy Mini Prep Kit (Qiagen, Valencia, CA). RNA samples were then quantified and run on a formaldehyde-containing agarose gel. Passive transfer to a nylon membrane was performed by using the Northern Max protocol (Ambion, Austin, TX). Nonisotopically labeled chromogranin A and synaptophysin DNA probes were created by using the BrightStar psoralen-biotin labeling kit (Ambion). The blot was then hybridized with the DNA probe overnight at 43.5°C. The blot was washed and blocked per the Northern Max protocol and developed by using Ambion's BrightStar BioDetect Kit.
The Northern blot was stripped by using boiling 0.1% SDS and then reprobed with a psoralen-biotin labeled RNA probe for GAPDH. The same protocol as above was performed with the exception of the hybridization temperature increased to 68°C.
MEK in vitro kinase assay. Cells were trypsinized and cellular pellets were lysed in sample buffer (50 mM Tris, 0.15 M NaCl, 0.1% SDS, 1% Nonidet P-40, 0.5% Na/deoxycholate, and 0.6 mM PMSF). Total cellular protein concentrations were determined by BCA assay (Pierce). Cellular proteins (200 µg) were diluted with sample buffer to a concentration of 1 mg/ml. The protein samples were incubated with 5 µl of anti-MEK antibody (Cell Signal Technology) for2hat4°Con an end-over-end rotator. We then added 50 µl of protein A agarose (Sigma) and rotated the mix overnight at 4°C. The samples were centrifuged at 4°C for 5 min at 14,000 rpm. The immunoprecipitates were washed twice with ice-cold 1x lysis buffer (Cell Signal Technology) with 1 mM PMSF and twice with 1x kinase buffer (Cell Signal Technology). Samples were centrifuged for 2 min at 4°C at 14,000 rpm between each wash. Immunoprecipitated products were incubated for 30 min at 30°C with 200 µM ATP and 2 µg of inactive glutathione S-transferase (GST)-p42 MAPK (Upstate Cell Signaling) in 1x kinase buffer. An aliquot of this reaction (20 µl) was then incubated with 2 µg of nonphosphorylated Elk-1 fusion protein (Cell Signal Technology) and 200 µM ATP for 30 min at 30°C. A single reaction without ATP served as a measure of background kinase activity. We added 20 µlof4x SDS sample dye to each sample. Samples were then boiled for 5 min and centrifuged for 2 min. Final products were electrophoresed through 10% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membranes by electroblotting. Membranes were washed and blocked according to our Western blot protocol and then exposed to anti-Elk-1 antibody (1:1,000; Cell Signal Technology) in a 5% BSA solution (1x PBS, 0.1% Tween-20, 5% BSA) and dilution of goat anti-rabbit secondary antibody (1:1,000; Cell Signal Technology) in milk solution for 1.5 h. Membranes were washed 1 x 10 and 2 x 5 min in PBS-T wash buffer and developed by enhanced chemiluminescence (Amersham) per the manufacturer's directions.
Growth assays. The methylthiazoletetrazolium (MTT) assay (Sigma) was performed. Briefly, BON and BON-raf cells were seeded in triplicate onto 24-well plates in phenol red-free DMEM/F-12K with 10% FCS at 3 x 105 for 24 h. Cells were then treated with estradiol or control. At 2-day intervals, medium was removed and replaced with a 250-µl medium containing MTT (0.5 mg/ml) and incubated at 37°C for 2 h. DMSO (750 µl; Sigma) was then added to each well, and absorbance at 540 nm was measured.
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RESULTS |
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Changes in morphology and neuroendocrine secretory granules. We have recently shown that induction of raf-1 in BON carcinoid cells results in striking morphological changes (31). BON-raf cells treated with estradiol are flatter and have sharper cellular borders and more cytoplasmic extensions under light microscopy (31). These morphology changes were visible in some cells as early as 12 h but appeared to affect all cells by 48 h. Although these changes resembled a morphological differentiation, examination under electronmicroscopy (EM) suggested otherwise. Neuroendocrine tumors such as GI carcinoids are characterized by the presence of numerous neuroendocrine secretory granules that can be seen on EM. BON cells treated with control or estradiol and BON-raf cells treated with control have abundant neuroendocrine secretory granules under EM (Fig. 2). In looking at numerous fields, rarely did any individual BON cell lack secretory granules. Significantly, induction of raf-1 by estradiol-treatment of BON-raf cells resulted in a 10-fold reduction in the number of neuroendocrine secretory granules at 48 h (P = 0.012) (Fig. 3).
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Effects of raf-1 induction on neuroendocrine hormone levels. To determine whether the reduction in neuroendocrine secretory granules translated into lower levels of hormone, we carried out both Western analysis and ELISA. BON cells normally produce high levels of 5-HT, chromogranin A, and synaptophysin (26, 38). As shown in Figs. 4 and 5, BON and BON-raf cells both express high levels of these molecules. Although estradiol treatment did not alter the levels of neuroendocrine markers in BON cells, raf-1 activation through estradiol treatment in BON-raf cells led to a significant decrease in the levels of chromogranin A and synaptophysin (Fig. 4). Furthermore, induction of raf-1 in BON-raf cells resulted in a significant reduction in 5-HT levels by ELISA (Fig. 5). These reductions in neuroendocrine markers and hormone levels were detected as early as 24 h after raf-1 induction.
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To determine whether the raf-1-induced reduction in neuroendocrine markers were the result of changes at the mRNA level, we performed Northern analysis on BON and BON-raf cells for chromogranin A and synaptophysin. As shown in Fig. 6, there were no significant differences in the levels of chromogranin A and synaptophysin messages in the cells before and after raf-1 activation. Thus raf-1-mediated reduction in neuroendocrine marker proteins and hormones does not appear to be due to changes at the mRNA level.
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Raf-1-mediated hormone suppression and morphology changes are dependent on MEK activation. To determine whether the raf-1-induced effects were dependent on MEK activation, we used the well-characterized MEK inhibitors PD-98059 and U-0126 (17). As demonstrated above, induction of raf-1 in BON-raf cells leads to a significant reduction in 5-HT levels (Fig. 7). However, treatment of these cells with either MEK inhibitor, alone or in combination, blocked raf-1-induced 5-HT suppression (Fig. 7). Similarly, the MEK inhibitors completely inhibited raf-1-mediated reduction in chromogranin A and synaptophysin protein levels (data not shown). Furthermore, treatment with PD-98059 and/or U-0126 blocked the BON-raf cellular morphology changes associated with raf-1 activation (Fig. 8). This blockade of raf-1 mediated hormone reduction and morphology changes by the MEK inhibitors was detectable within 12 h but much more noticeable by 48 h when the effects of raf-1 are more prominent. Of note, these inhibitors had no effect on native BON cells (data not shown).
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Interestingly, although both MEK inhibitors blocked raf-1-mediated hormone suppression and morphology changes, treatment of BON-raf cells with PD-98059 and U-0126, alone or in combination, did not inhibit phosphorylation of ERK1/2. As shown in Fig. 9, the levels of phosphorylated ERK1/2 were similar in estradiol-treated BON-raf cells in the absence or presence of the MEK inhibitors. Furthermore, because ERK1/2 phosphorylation persists after blocking the raf-1-induced effects by treatment with the MEK inhibitors, phosphorylation of ERK1/2 alone does not appear to be sufficient to induce BON cell morphology changes and hormone reduction. In addition, these results also suggest that raf-1 can activate ERK1/2 through a MEK-independent pathway in BON carcinoid cells.
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Because of the persistence of ERK1/2 phosphorylation, we planned to definitively prove that the MEK inhibitors blocked the raf-1-mediated morphology changes and hormone suppression in BON cells through direct inhibition of MEK, as opposed to nonspecific effects. As shown in Fig. 9, BON-raf cells pretreated with the MEK inhibitors for 45 min, followed by treatment with estradiol showed evidence of persistent ERK1/2 phosphorylation at 48 h. We then performed a MEK in vitro kinase assay on the same cellular extracts used for the Western blotting to determine whether MEK was indeed inhibited at 48 h. Immunoprecipitated MEK was used to phosphorylate an inactive GST-MAPK fusion protein, which, when activated, was able to phosphorylate Elk-1. As shown in Fig. 10, after 48 h of estradiol treatment and in the absence of MEK inhibitors, there was an increase in phosphorylation of Elk-1 due to MEK activity (lane 1). However, pretreatment with the MEK inhibitors followed by estradiol addition, markedly reduced the ability of immunoprecipitated MEK to generate phosphorylated Elk-1 in a coupled in vitro kinase assay (lanes 2 and 3). Thus these results show that ERK1/2 phosphorylation persists in estradiol-treated BON-raf cells despite adequate inhibition of MEK activity by MEK inhibitors. Therefore, raf-1 appears to be able to activate ERK1/2 through a MEK-independent pathway in BON carcinoid cells.
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Effect of Raf-1 induction on BON cell proliferation. Activation of ras and/or raf-1 has been shown to promote cellular growth in a variety of tumors (14). To determine whether raf-1 activation in BON cells affects cellular proliferation, we used MTT assays. As shown in Fig. 11, there were no differences in proliferation rates of BON or BON-raf cells treated with control or estradiol over a 10-day period before reaching confluency. Similar results were obtained by cell counts with Trypan blue exclusion (data not shown).
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DISCUSSION |
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Activation of raf-1 has been shown to result in growth inhibition, phenotypic differentiation, and downregulation of the RET protooncogene in medullary thyroid cancer cells (8, 10). In small cell lung cancer cells, Ravi et al. (28) showed that raf-1 activation resulted in growth suppression, loss of soft agar cloning ability, and cell cycle arrest. In the present study, we found that activation of raf-1 in human BON carcinoid cells led to high levels of phosphorylated MEK and ERK1/2 within 48 h. Induction of raf-1 and phosphorylated MEK and ERK1/2 resulted in a striking morphological change resembling differentiation (31). Surprisingly, there was an >10-fold reduction in the number of neuroendocrine secretory granules seen by electron microscopy after raf-1 induction. Raf-1 activation also caused significant reductions in the levels of 5-HT, chromogranin A, and synaptophysin. It is possible that raf-1 activation in BON cells leads to a more undifferentiated phenotype, resembling progenitor neuroendocrine stem cells. Interestingly, others have demonstrated that neuroendocrine cells have the capacity to transdifferentiate. Schmied et al. (30) have shown that human islet cells can transdifferentiate into an undifferentiated phenotype. Importantly, although neuroendocrine marker and hormone levels were significantly reduced, raf-1 activation did not stimulate proliferation of BON cells.
Treatment of the BON-raf cells with MEK inhibitors PD-98059 and U-0126 led to two important observations. First, raf-1-mediated morphological changes and hormone suppression are dependent on MEK activation. This finding seems predictable given that MEK is the best characterized substrate for raf-1. However, several studies (2, 13, 24, 29) have recently observed raf-1-mediated effects in the absence of MEK activation through induction of other target signaling factors including MEK kinase 1, cell cycle proteins such as retinoblastoma protein, p53, cdc25, and apoptosis signal-regulated kinase 1. Moreover, studies using raf-1 knockout mice have shown that MEK kinase activity is not necessary for raf-1 function (18).
Second, because ERK1/2 phosphorylation persists after blocking the raf-1-induced effects by treatment with the MEK inhibitors, phosphorylation of ERK1/2 alone does not appear to be sufficient to induce BON cell morphology changes and hormone reduction. Thus the morphological changes and hormone suppression seen in BON cells may be due to raf-1/MEK signaling through an alternative downstream pathway other than ERK1/2. In addition, the persistence of ERK1/2 phosphorylation also suggests that raf-1 can activate ERK1/2 through a MEK-independent pathway in BON carcinoid cells. This observation is further supported by the MEK in vitro kinase assay showing that the persistence of ERK1/2 phosphorylation occurs in the setting of adequate MEK inhibition by the MEK inhibitors. Although these findings are unique, other reports have illustrated ERK1/2 activation by raf-1 through MEK-independent pathways. Interestingly, Navas et al. (24) recently reported that receptor interacting protein-2 (RIP2) is involved in caspase activation and tumor necrosis factor receptor and Fas signaling and that it can be induced by raf-1 and can phosphorylate ERK1/2. Therefore, in the tumor necrosis factor signaling pathway, RIP2 takes the place of MEK to couple raf-1 to ERK1/2.
Our findings are especially interesting given the fact that other recent studies have focused on understanding the mechanisms that regulate hormone production in BON cells.
Kim et al. (20) showed that mechanical stimulation of BON cells leads to increased levels of 5-HT secretion. They determined that the mechanism of this enhanced hormone release was stimulation of a G protein-coupled receptor leading to mobilization of intracellular calcium (20). The same group also discovered that exposure of BON cells to high concentrations of D-glucose activates other signaling pathways to increase levels of 5-HT secretion (19). Furthermore, Zhang et al. (38) showed that phorbol ester treatment of BON cells results in a persistent release and cellular depletion of chromogranin A. They further demonstrated that these effects were mediated through the PKC pathway (38). Although raf-1 activation also led to marked cellular depletion of chromogranin A and other hormones, we did not see enhanced secretion of hormones as seen with phorbol ester treatment. In fact, when testing BON-raf cellular supernatants for 5-HT after raf-1 activation, we saw slightly diminished levels of 5-HT.
Although our present study is the first to examine the role of raf-1 in GI carcinoid cells, others have looked at pathways associated with raf-1. Bold et al. (4) studied the effects of NGF and its associated receptor trkA. They demonstrated that NGF acts as a mitogen for BON cells without any effect on cellular phenotype or hormone production (4). Interestingly, they hypothesized that trkA could be signaling through raf-1 and MAP kinases. However, our findings showed that activation of raf-1 led not only to changes in cellular morphology, but also to the reduction in hormone production. These differences could be due to the intensity of the raf-1 signals generated by overexpression of raf-1 versus a ligand-receptor interaction. It is also possible that trkA could be signaling through parallel pathways, such as phosphatidylinositol 3-kinase, and that BON cell growth and differentiation are controlled by a combination of these signaling pathways.
In conclusion, we have demonstrated that overactivation of raf-1 suppresses hormone production by human carcinoid tumor cells. Further characterization of this pathway will determine whether modulation of the raf-1 signaling could play a potential role in the management of patients with carcinoid tumors. Furthermore, these finding may permit development of components of the raf-1 pathway as therapeutic targets in the treatment and palliation of GI neuroendocrine tumors.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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