Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

Dulbecco's modified Eagle's medium, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid (Hepes), penicillin and streptomycin were from Gibco (Grand Island, NY). Neurotensin, 12-O-tetradecanoylphorbol-13-acetate (TPA), thapsigargin, epidermal growth factor (EGF), and wortmannin were obtained from Sigma-Aldrich (St. Louis, MO). [2-[1-(3-dimetylaminopropyl)-1H-indol-3-yl]-maleimide] (GF109203X), 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (tyrphostin AG1478), 2'-amino-3'-methoxyflavone (PD98059, and N-[(2R)-2 (hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM6001/Galardin) were from Calbiochem (San Diego, CA). 7-Methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a] pyrimidin-4-one (TGX-221) was obtained from Cayman Chemical (Ann Arbor, MI). Transforming growth factor α (TGFα) was obtained from Bachem (Bubendorf, Switzerland). 4-Quinazolinamine, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-4-morpholin)propoxy] (gefitinib) was a gift from Astra Zeneca (Cheshire, UK), and cetuximab was kindly provided by Merck KgaA (Darmstadt, Germany). [6-³H]thymidine (20-30 Ci/mmol) and myo-[2-³H]inositol (15.0 Ci/mmol) were from Amersham Biosciences (Buckinghamshire, UK). Antibodies against phosphorylated Akt^Ser473, total Akt, dually phosphorylated ERK^{Thr202/Tyr204}, phospho-EGF receptor^Tyr1173, and phospho-Shc ^Tyr239/240 were obtained from Cell Signaling Technology (Boston, MA). Anti-ERK and anti-Shc antibodies were obtained from Upstate (Billerica, MA). EGFR antibody (1005) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies were purchased from Bio-Rad Laboratories (Hercules, CA) and Licor Biosciences (Lincoln, NE). All other chemicals were of analytical quality. Stock solutions of test compounds were prepared in DMSO (TPA, thapsigargin, wortmannin, PD98059, GM6001, TGX221, gefitinib) or 0.9% NaCl (neurotensin, GF109203X). EGF was dissolved in 4 mM HCl, and TGFα in 4 mM HCl containing 1% bovine serum albumin from Sigma Aldrich (St. Louis, MO). Cetuximab was dissolved in phosphate-buffered saline (PBS). When solutions containing DMSO were used, the final concentration of DMSO was kept as low as possible.

Human colorectal cancer cell lines HCT116 and HT29, and pancreatic adenocarcinoma cell line Panc-1 were obtained from ATCC (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium containing 1 g/l glucose (or 4.5 g/l for Panc-1) supplemented with 10% horse serum (10% fetal bovine serum for Panc-1 cells), penicillin (67 μg/ml), streptomycin (100 μg/ml) and 2 mM glutamine (4 mM for Panc-1). Cells were plated onto Costar plastic culture wells (Corning Life Sciences, Acton, MA) at a density of 50 000 cells/cm² (25 000 cells/cm² in the case of Panc-1 cells) in serum-containing medium. The cultures were kept in 95% air/5% CO₂ at 37°C. After 24 hours the medium was replaced with serum-free medium and the cells were cultured for 24 hours before stimulation with agonists.

Neurotensin, TPA, and inhibitors of PKC and EGF receptor were added to serum-starved HCT116 cells as described in the figure legends, and [³H]thymidine was added 12 hours after stimulation. Serum-starved HT29 and Panc-1 cells were stimulated for 21 hours with neurotensin and EGF before [³H]thymidine was added. The cells were harvested after three hours pulsing with [³H]thymidine, and DNA synthesis was measured as the amount of radioactivity incorporated into DNA as previously described [34]. Briefly, medium was removed, and cells were washed twice with 0.9% NaCl. The cellular material was dissolved with 1.5 ml of 0.5 N NaOH for 3 hours at 37°C, collected, mixed with 1.5 ml H₂O, and precipitated with 0.75 ml 50% trichloroacetic acid (TCA). The acid-precipitable material was transferred to glass fiber filters (GF/C Whatman, GE Healthcare, UK) and washed twice with 5.0 ml 5% TCA, followed by liquid scintillation counting of the filters in a Packard Tri-Carb liquid scintillation counter.

Cells were labelled with [³H]inositol, 2.5 μCi/ml for 24 hours in serum-free medium. Medium was removed 30 minutes before agonist stimulation and replaced with Krebs-Ringer-Hepes buffer pH 7.4, containing 10 mM glucose and 15 mM LiCI. HCT116 cells were stimulated with neurotensin for 30 minutes, and the reaction was stopped by removing buffer and adding 1 ml ice-cold 0.4 M perchloric acid. Samples were harvested and neutralized with 1.5 M KOH, 60 mM EDTA, 60 mM Hepes, in the presence of Universal indicator. The neutralized supernatants were applied on columns containing 1 ml Dowex AG 1-X8 resin (Bio-Rad Laboratories, Hercules, CA), and inositol phosphates were eluted with 10 ml 1 M ammonium formate/0.1 M formic acid.

Aliquots with ~30 000 cells (total cell lysate prepared in Laemmli or RIPA buffer) were electrophoresed on 6-12% (w/v) polyacrylamide gels. This was followed by protein electrotransfer to nitrocellulose membranes and immunoblotting with antibodies against phospho-Akt, total Akt, phospho-ERK1/2, total ERK, phospho-EGFR, total EGFR, phospho-Shc, and total Shc, respectively. Immunoreactive bands were visualized with enhanced chemiluminescence using LumiGLO (KPL Protein Research Products, Gaithersburg, MD), or infrared imaging using Odyssey Infrared Imaging System supplied by Licor Biosciences (Lincoln, NE), respectively.

Results are expressed as means ± standard error of the mean (S.E.M). DNA synthesis data were analyzed by one-way ANOVA, and post tests using Bonferroni correction to compare groups, using GraphPad Prism (version 5.01, GraphPad Software, San Diego, California, USA). Results were considered significant when p < 0.05.

Neurotensin has been reported to act as a mitogen in certain colon cell lines [10, 22]. We found that neurotensin dose-dependently induced DNA synthesis in HCT116 cells, reaching a two- to three-fold increase as compared to basal levels (Figure 1A, 2C). In contrast, addition of EGF only slightly increased DNA synthesis, which is in agreement with previous data and might be explained by an autocrine production of EGFR ligands by these cells, masking the effects of exogenously added EGF [35‐37]. Furthermore, concomitant stimulation of HCT116 cells with neurotensin and EGF did not induce any synergistic or additive effect on DNA synthesis. In HT29 cells, EGF dose-dependently (data not shown) stimulated DNA synthesis, whereas neurotensin had no significant effects, neither alone nor in combination with EGF (Figure 1B). In Panc-1 cells, both neurotensin and EGF stimulated DNA synthesis, as reported previously [25], (Figure 1C).

The high affinity NTSR1 receptor is known to activate PLC [15]. Neurotensin was previously shown to elevate intracellular Ca²⁺ in HCT116 cells [38], and in our experiments neurotensin strongly and dose-dependently stimulated accumulation of inositol phosphates in these cells (Figure 2A). This strongly implicates PLC in the mechanisms of the cellular response of HCT116 cells to neurotensin. We next pretreated HCT116 cells with the PKC inhibitor GF109203X, and Figure 2B shows that this blocker strongly reduced DNA synthesis. It was also noted that the stimulatory effect of neurotensin on DNA synthesis was of the same magnitude as the effect of the direct PKC activator tetradecanoylphorbol acetate (TPA) (Figure 2C). Together, the results suggest a major role of the PLC/PKC pathway in the stimulation of DNA synthesis by neurotensin in these colon cancer cells.

Neurotensin induced a marked, rapid, and sustained phosphorylation of ERK in HCT116 cells (Figure 3A), which appeared to plateau at a concentration of 3-10 nM (Figure 3B). Direct activation of PKC by TPA also stimulated ERK phosphorylation (Figure 3C). The phosphorylation of ERK in response to neurotensin and TPA was strongly reduced by pretreatment of the cells with GF109203X (Figure 3C). In contrast, EGF-stimulated ERK phosphorylation was not affected by the PKC blocker (Figure 3C). In agreement with previous data [23] neurotensin stimulated ERK phosphorylation in a PKC-dependent manner in Panc-1 cells (Figure 4A), whereas in HT29 cells, ERK phosphorylation was only slightly attenuated by the PKC inhibitor (Figure 4B). Thus, in agreement with previous results from other cells where neurotensin stimulated ERK phosphorylation and DNA synthesis in a PKC-dependent manner [23‐25], our data indicate that neurotensin-induced ERK phosphorylation in HCT116 cells is PKC-dependent.

EGF induced a marked phosphorylation of Akt in HCT116 cells, indicating activation of the phosphoinositide 3-kinase (PI3K) pathway (Figure 3C). Neurotensin also stimulated phosphorylation of Akt, although not as strongly as EGF (Figure 3C). The effect of neurotensin on Akt first appeared after 3 min, while ERK phosphorylation was evident already at 1 min (Figure 3A). Furthermore, unlike the data indicating a PKC-mediated activation of ERK, neurotensin-induced phosphorylation of Akt was not affected by inhibition of PKC and was not mimicked by TPA (Figure 3C).

We next examined the ability of neurotensin to induce tyrosine phosphorylation of EGFR in HCT116 cells. Figure 5A shows that treating the cells with neurotensin or EGF resulted in phosphorylation of the EGFR. Although the effect of neurotensin was clearly less than that of EGF, the phosphorylation induced by both these agonists was blocked by pretreatment with the EGFR tyrosine kinase inhibitor gefitinib (Figure 5A). Moreover, we found that neurotensin stimulated phosphorylation of Shc (Figure 5B), which is an adaptor protein that binds to, and is phosphorylated by, active RTKs [39, 40]. Taken together, these results suggest that the EGFR can be transactivated by neurotensin in HCT116 cells.

Pretreatment with gefitinib strongly attenuated neurotensin-induced phosphorylation of Akt in HCT116 cells (Figure 5C). In these experiments, TGFα was used as the EGFR ligand, and the effect of TGFα on Akt phosphorylation was completely abolished by gefitinib. Neurotensin also induced Akt phosphorylation in HT29 and Panc-1 cells (Figure 4). Whereas this effect was abolished by pretreatment with gefitinib in HT29 cells (Figure 4B), neither gefitinib nor the PKC inhibitor GF109203X inhibited neurotensin-stimulated Akt phosphorylation in Panc-1 cells (Figure 4A).

Evidence from many cell types indicates that transactivation of the EGFR induced by GPCRs may be mediated by the activation of cell surface proteinases, resulting in subsequent shedding of EGFR ligands [41, 42], or by intracellular mechanisms involving kinases such as Src and Pyk2 [43, 44]. To explore further the mechanism of the gefitinib-sensitive Akt phosphorylation induced by neurotensin, we examined the effect of cetuximab, an antibody which binds to the extracellular domain of the EGFR and thereby blocks the ability of ligand-induced activation. As expected, EGF-stimulated phosphorylation of both Shc and Akt was completely inhibited by cetuximab (Figure 6A). Cetuximab pretreatment also blocked neurotensin-stimulated Shc phosphorylation, suggesting the involvement of a ligand-dependent mechanism. Neurotensin-induced phosphorylation of Akt was also inhibited by cetuximab, but only partially. We next pretreated the cells with GM6001, a broad-spectrum inhibitor of matrix and metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). Pretreatment with GM6001 did not affect the effect of neurotensin on ERK, but markedly reduced neurotensin-induced phosphorylation of Akt (Figure 6B). These results support a role of release of EGFR ligand(s) in neurotensin-stimulated phosphorylation of EGFR and Akt. However, since neither cetuximab nor GM6001 completely abolished the effect of NT on Akt phosphorylation, it seems likely that additional mechanisms are operating. As expected, the effect of exogenous EGF was insensitive to GM6001 (Figure 6B).

The results above suggest that neurotensin-stimulated phosphorylation of Akt in HCT116 cells is mediated, at least in part, through transactivation of the EGFR. In search for mechanisms that mediate the release of EGFR ligands in HCT116 cells, we next examined the role of intracellular Ca²⁺. Thapsigargin, which increases the intracellular Ca²⁺-level by inhibiting the SERCA pump [45], induced phosphorylation of Shc, ERK and Akt (Figure 7A). Furthermore, like the effect of neurotensin, the effect of thapsigargin on Shc phosphorylation was abolished by pretreatment with cetuximab, while the effect on Akt phosphorylation was attenuated, which suggests the involvement of Ca²⁺ in the response of the PI3K pathway to neurotensin. Further experiments showed that the effects of neurotensin and thapsigargin on Akt phosphorylation were sensitive to chelating Ca²⁺-inhibitors (data not shown). However, we have so far not been able to show that this effect is selective, as EGF-stimulated Akt phosphorylation was also attenuated by Ca²⁺-inhibitors. In contrast to the findings in HCT116 cells, thapsigargin did not stimulate phosphorylation of Akt in Panc-1 cells (data not shown). However, in these cells neurotensin-stimulated Akt phosphorylation was abolished by pretreating the cells with TGX-221, an inhibitor of PI3Kβ [46] (Figure 7B). This indicates that PI3Kβ is involved in neurotensin-induced activation of Akt in Panc-1 cells.

The above results suggest a role for the PLC/PKC pathway in the DNA synthesis induced by neurotensin in HCT116 cells. Furthermore, consistent with a role of ERK in the mitogenic response, pretreatment of the cells with the MEK inhibitor PD98059 (50 μM) strongly reduced both basal and neurotensin-induced DNA synthesis (Figure 8A). Although stimulation with EGF only slightly affected DNA synthesis in the cells (Figure 1), we examined the possibility that activation of the EGFR pathway might play a role in neurotensin-induced mitogenic stimulation. We found that inhibition of the EGFR tyrosine kinase activity by gefitinib or AG1478 resulted in a reduction of both basal and neurotensin-induced DNA synthesis (Figure 8B). Furthermore, a role for the PI3K pathway in the neurotensin-induced mitogenesis was likely since the DNA synthesis was reduced by the PI3K inhibitor wortmannin (Figure 8C).