Author Affiliations: Department of Medicine II, Division of Gastroenterology, Rostock University Medical Center, E.-Heydemann-Str. 6, 18057 Rostock, Germany (Witteck L and Jaster R)

Corresponding Author: Robert Jaster, MD, Department of Medicine II, Division of Gastroenterology, Rostock University Medical Center, E.-Heydemann-Str. 6, 18057 Rostock, Germany (Tel: +49-381-4947349; Fax: +49-381-4947482; Email: jaster@med.uni-rostock.de)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60032-7

Published online November 9, 2015.

 

Acknowledgements: We gratefully acknowledge the excellent technical assistance of Mrs. Katja Bergmann.

Contributors: WL and JR designed the research, performed the experiments and analyzed the data. JR wrote the manuscript. Both authors reviewed and contributed to the final manuscript. JR is the guarantor.

Funding: None.

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells (PSCs), which play a key role in pancreatic fibrosis.

 

METHODS: Cultured rat PSCs were exposed to small molecule kinase inhibitors. Proliferation and cell death were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine and cytotoxicity, respectively. Levels of mRNA were determined by real-time PCR, while protein expression and phosphorylation were analyzed by immunoblotting. Interleukin-6 levels in culture supernatants were quantified by ELISA. Zymography assays were performed to monitor collagenase activity in culture supernatants.

 

RESULTS: The MEK inhibitor trametinib and the dual phosphatidylinositol 3-kinase/mTOR inhibitor dactolisib, but not the multi-kinase inhibitor regorafenib, efficiently inhibited PSC proliferation. Trametinib as well as regorafenib suppressed the expression of two autocrine mediators of PSC activation, interleukin-6 and transforming growth factor-β1. Dactolisib-treated cells expressed less α1 type I collagen and lower levels of α-smooth muscle actin, a marker of the myofibroblastic PSC phenotype. Simultaneous application of dactolisib and trametinib displayed additive inhibitory effects on cell growth without statistically significant cytotoxicity. Activity of matrix metalloproteinase-2 was not affected by any of the drugs.

 

CONCLUSION: We suggest the combination of two drugs, that specifically target two key signaling pathways in PSC, Ras-Raf-MEK-ERK (trametinib) and phosphatidylinositol 3-kinase-AKT-mTOR (dactolisib), as a concept to modulate the activation state of the cells in the context of fibrosis.

 

(Hepatobiliary Pancreat Dis Int 2015;14:642-650)

 

KEY WORDS: stellate cell biology; pancreatic cancer; chronic pancreatitis; trametinib; dactolisib

 

Pancreatic stellate cells (PSCs) are the main source of extracellular matrix (ECM) proteins in the diseased pancreas. In response to profibrogenic mediators such as ethanol metabolites and various cytokines/growth factors, the cells undergo a transition from a quiescent to an activated stage.^{[1, 2]} Quiescent PSCs (roughly 4% of all pancreatic cells^[3]) are phenotypically characterized by the presence of vitamin A-containing fat droplets^{[3, 4]} and have recently been suggested to exhibit stem cell properties.^[5] Activated PSCs proliferate at a high rate, synthesize large amounts of collagen type I and other ECM proteins and express the myofibroblastic marker α-smooth muscle actin (α-SMA).^{[3, 4]} PSC activation is part of a physiological wound healing response after pancreatic injury and not pathological per se. Under persistent pathophysiological conditions, however, PSC activation is sustained and not terminated, resulting in an overshooting matrix deposition. This is typically the case in chronic pancreatitis and pancreatic cancer (PC), where pancreatic tissue is replaced by connective tissue, leading to organ fibrosis.^{[1, 2]} Although the pathophysiological mechanisms are not entirely understood yet, it is well established that paracrine mediators (e.g., derived from inflammatory cells and tumor cells) as well as autocrine loops play a key role in the maintenance of PSC activation in chronic pancreatitis and PC.^{[1, 2]} Important examples of such mediators include transforming growth factor β1 (TGF-β1), the main stimulator of ECM synthesis in PSC, and the proinflammatory cytokine interleukin-6 (IL-6), which both may act in an autocrine and paracrine manner.^[6-9]

 

Numerous studieshave shown that the stroma response not only accompanies PC but plays an active role in its progression (reviewed in^[10]). Thus, the connective tissue forms a fibrotic wall around tumor cells, thereby creating a barrier against chemotherapeutics and immune cells. Cytokines, chemokines and growth factors which are secreted by stroma cells have also been implicated in chemoresistance, suppression of apoptosis and accelerated proliferation of PC cells.^[11-15] Targeting the stroma, therefore, has been suggested as a complementary strategy to treat PC, one of the deadliest human malignancies with very limited therapeutic options. Accordingly, efforts have been made to develop specific antifibrotic therapies,^[10] which, however, are not established in the clinics yet. Moreover, two recent studies^[16,17] have put the simple concept of depleting the stroma for an improved cancer treatment into question. In a genetic mouse model of PC, reduction of the stroma through a gene deletion of the profibrogenic ligand sonic hedgehog was unexpectedly associated with more aggressive tumors and increased tumor angiogenesis.^[16] In a transgenic mouse model, a more invasive tumor growth was observed when α-SMA-positive myofibroblasts were specifically deleted.^[17] Together, both studies indicated that at least some components of the tumor stroma can act to restrain tumor growth. Most recently, the original concept has therefore been refined by suggesting a stroma modulation (rather than a stroma depletion), in a way that pro-tumorigenic interactions between stroma and cancer cells are blocked and anti-tumorigenic effects of the stroma remain preserved.^[18] One prerequisite to achieve this goal is to gain more mechanistic insights into key effector functions of activated PSCs.

 

We and others have previously shown that two intracellular signaling cascades, Ras-Raf-MEK (mitogen-activated protein kinase kinase)-ERK (extracellular signal-regulated kinase) and phosphatidylinositol 3-kinase (PI3K)-AKT (protein kinase B)-mTOR (mammalian target of rapamycin), play a crucial role in PSC activation in response to mitogens and profibrogenic mediators.^[19-21] Furthermore, we have also tested clinically available small molecule kinase inhibitors (SMIs) and found that two anticancer drugs, the multi-kinase inhibitors sorafenib and sunitinib, efficiently blocked activation of the AKT pathway and displayed distinct antifibrogenic effects.^[22] Here, we have advanced our studies by investigating the effects of two more recent and specific drugs, the MEK inhibitor trametinib^[23] and the dual PI3K/mTOR inhibitor dactolisib,^[24] on cultured rat PSC. For comparison, an additional multi-kinase inhibitor, the potent antineoplastic agent regorafenib,^[25] was included into the investigations.

 

 

Regorafenib, trametinib and dactolisib were purchased from Selleckchem (Houston, TX, USA). Unless stated otherwise, all other reagents were obtained from Sigma-Aldrich (Deisenhofen, Germany).

 

PSCs were isolated from the pancreas of healthy male Lewis inbred rats (Charles River Laboratories, Sulzbach, Germany) essentially as described before.^[19] Briefly, the organ was digested with collagenase, and PSC were separated by Nycodenz® (Nycomed, Oslo, Norway) density gradient centrifugation. Immediately after isolation, PSCs were resuspended in cryopreservation medium [fetal calf serum (FCS) supplemented with 10% dimethyl sulfoxide (DMSO)] and stored at -150 �� until required. After thawing, the cells were cultured in Iscove's modified Dulbecco's medium (IMDM; Biochrom, Berlin, Germany) supplemented with 17% FCS, 1% non-essential amino acids (dilution of a 100× stock solution), 10⁵ U/L penicillin and 100 mg/L streptomycin (all reagents from PAA Laboratories, Pasching, Austria). Compared to our previous studies where freshly isolated PSCs were cultured immediately,^[19] the cycle of freezing and thawing only insignificantly affected the number of vital cells for the experimental studies. On the other hand, it proved very useful to standardize experimental conditions, e.g., when comparing different preparations of PSCs. After approximately 5 days in primary culture, proliferating PSCs reached subconfluency and were harvested by trypsination. Unless indicated otherwise, the cells were subsequently plated in 12-well plates or 24-well plates at average seeding densities of 6×10⁴ and 3×10⁴ cells per well, respectively, and further grown and treated according to the experimental requirements. All experiments were performed with cells passaged no more than two times. Trypan blue staining was used to distinguish live from dead cells and to determine absolute cell counts.

 

Cell proliferation was assessed by quantifying incorporation of 5-bromo-2'-deoxyuridine (BrdU) into newly synthesized DNA, employing the BrdU labelling and detection enzyme-linked immunosorbent assay (ELISA) kit (Roche Diagnostics, Mannheim, Germany). Therefore, cells were plated in 96-well plates at equal seeding densities (2×10³ cells per well) and allowed to adhere overnight. Subsequently, regorafenib, trametinib and dactolisib (solved in DMSO; final solvent concentration here and in all further experiments ≤0.1%) were added as indicated. After 24 hours, BrdU labelling was initiated by adding labelling solution at a final concentration of 10 µmol/L. Another 24 hours later, labelling was stopped, and BrdU uptake was measured according to the manufacturer's instructions.

 

Cytotoxic effects of the investigated SMI were analyzed using the CytoTox-Glo Cytotoxicity Assay according to the instructions of the manufacturer (Promega, Madison, WI, USA). Therefore, PSCs were seeded into 96-well plates at equal seeding densities (2×10³ cells per well) and allowed to adhere overnight, before regorafenib, trametinib and dactolisib were added as indicated. After 48 hours, CytoTox-Glo Cytotoxicity Assay reagent was added to all wells, and incubation continued for 15 minutes at room temperature. Luminescence (originating from dead cells) was measured using a Glomax Multi Detection System (Promega) for 96-well plates. Afterwards, the remaining viable cells were lysed by incubation with lysis reagent for another 15 minutes, and luminescence was recorded again. Since the second luminescence signal corresponds to all cells, the ratio of the first and the second luminescence signals indicates the rate of cell death in the original sample.

 

Protein extracts of PSCs (growing in 24-well plates and pretreated as indicated) were prepared and subjected to immunoblotting analysis as described before,^[19] using polyvinylidene fluoride membrane (Merck Millipore, Darmstadt, Germany) for protein transfer. Unless specified otherwise, primary antibodies were obtained from New England BioLabs (Frankfurt, Germany). The following antibodies were employed: anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #2118), anti-phospho-AKT (P-AKT; #4060), anti-phospho-ERK1/2 (P-ERK1/2; #4370), anti-AKT protein (#4691), and anti-ERK1/2 (#06-182, Merck Millipore). The blots were developed using LI-COR reagents for an Odyssey® Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) as described previously.^[26] Signal intensities were quantified by means of the Odyssey® software version 3.0 and the raw data processed as described in the corresponding figure legend.

 

Quantitative determinations of IL-6 protein levels were performed using a rat IL-6-specific ELISA (BioLegend, San Diego, CA, USA). Therefore, PSC growing in 12-well plates were pretreated by SMI application as indicated. Cell culture supernatants were collected and stored at -80 �� until use. The ELISA was performed according to the manufacturer's instructions.

 

PSCs were plated in 24-well plates, grown to subconfluency, washed free of FCS and cultured for 24 hours in serum-free culture medium supplemented with SMI as indicated. Afterwards, the supernatants were collected and mixed 3:1 with 4× zymography sample buffer (250 mmol/L Tris-HCl, pH 6.8, 10% SDS, 40% glycerol, 0.1% bromophenol blue). Subsequently, samples and matrix metalloproteinase-2 (MMP2) zymography standard were loaded onto 10% precast polyacrylamide gel with gelatin (Bio-Rad Laboratories, Hercules, CA, USA). After electrophoresis, the gels were soaked in zymogram renaturation buffer (Bio-Rad Laboratories) with gentle shaking (60 minutes at room temperature; one change of detergent solution), before they were incubated overnight at 37 �� in zymogram development buffer (Bio-Rad Laboratories). Afterwards, lysis bands were visualized using Coomassie G-250 SimplyBlue SafeStain according to the instructions of the manufacturer (Thermo Fisher Scientific, Darmstadt, Germany). Finally, band intensities were quantitated with the Odyssey® Infrared Imaging System (software version 3.0), taking advantage of the fluorophore properties of protein-bound Coomassie stain.

 

PSC growing in 12-well plates were treated with SMI for 24 hours as indicated, and total RNA was isolated using TriFast reagent (PEQLAB Biotechnologie, Erlangen, Germany). All further steps were performed with reagents from Thermo Fisher Scientific. First, any traces of genomic DNA were removed employing the DNA-free kit. Afterwards, 1 µg of RNA was reverse transcribed into cDNA by means of TaqMan Reverse Transcription Reagents and random hexamer priming. Relative quantification of target cDNA levels by real-time PCR was performed in a Viia 7 sequence detection system (Thermo Fisher Scientific). Therefore, TaqMan Universal PCR Master Mix and rat-specific TaqMan Gene Expression Assays with fluorescently labelled MGB probes were used. The following assays were employed: Rn01759928_g1 (Acta2; α-smooth muscle actin; α-SMA), Rn00432360_m1 (cyclin D1; Ccnd1), Rn00589996_m1 (cyclin-dependent kinase inhibitor 1A; Cdkn1A), Rn00561420_m1 (Il-6), Rn00572010_m1 (Tgf-β1), Rn01463848_m1 (α1 type I collagen, Col1a1), and Rn01527840_m1 (hypoxanthine guanine phosphoribosyl transferase; Hprt; housekeeping gene control). PCR conditions were: 95 �� for 10 minutes, followed by 40 cycles of 15 seconds at 95 ��/1 minute at 60 ��. The relative expression of each mRNA (n≥5 independent samples per experimental condition) compared with HPRT was calculated according to the equation ?Ct=Ct_target-Ct_HPRT. The relative amount of target mRNA in control cells and cells treated with SMI as indicated was expressed as 2^-??Ct, where ??Ct_treatment=?Ct_SMI-?Ct_control.

 

All data were stored and analyzed using the IBM SPSS Statistics 20.0. Values were expressed as mean±standard error (SE) of the mean for the indicated number of separate cultures per experimental protocol. Statistical significance was checked using one-way ANOVA followed by Bonferroni's post-hoc test. If data did not meet the assumptions for ANOVA, analysis of variance was performed employing the Kruskal-Wallis test, before subgroups were tested using the Mann-Whitney U test. P<0.05 (Bonferroni-adjusted as indicated in the figure legends) was considered to be statistically significant.

 

 

SMI application was associated with significant effects on DNA synthesis (P_{Kruskal-Wallis}<0.001). The subsequent analysis of subgroups revealed a dose-dependent inhibition of BrdU incorporation by trametinib and dactolisib at nanomolar concentrations (Fig. 1A). Furthermore, two combinations of trametinib and dactolisib were found to be more effective than each substance at the corresponding concentration alone, suggesting additive effects of MEK and PI3K inhibition. Unexpectedly, micromolar concentrations of the multi-kinase inhibitor regorafenib did not diminish but even enhance DNA synthesis.

 

To distinguish between SMI effects on cell proliferation and cell death (which both may affect BrdU incorporation), cytoxicity of the drugs was evaluated. As a positive control, the pro-apoptotic agent staurosporine was included. Indeed, drug treatment of PSC affected cell survival (P_{Kruskal-Wallis}=0.013). However, as shown in Fig. 1B (subgroup analyses), staurosporine was the only substance that displayed a significant cytotoxic effect. We noticed that application of dactolisib at concentrations ≥30 nmol/L (alone and in combination with trametinib) also was associated with increased rates of cell death, but this effect was not statistically significant.

 

Together, these data suggest that inhibition of cell proliferation, rather than induction of cell death, reduces BrdU incorporation in response to trametinib and dactolisib treatment.

 

To gain insights into the molecular action of regorafenib, trametinib and dactolisib, we focused on the intracellular signal transduction pathways Ras-Raf-MEK-ERK and PI3K-AKT, which both play a key role in PSC activation and represent targets of the SMI tested in this study.^[19-25] Specifically, we asked how the three drugs affect activation of AKT and ERK in PSC, using levels of P-AKT and P-ERK1/2 as surrogate markers (Fig. 2A-D). In accordance with previously published data,^[22] serum restimulation of serum-starved PSCs induced a rapid phosphorylation of both AKT and ERK1/2. Global analysis of variance revealed that both AKT and ERK activation were significantly affected in SMI-treated PSC at any time point of FCS stimulation (AKT: P_{Kruskal-Wallis}≤0.009; ERK: P_{Kruskal-Wallis}≤0.03). Further analysis showed that dactolisib significantly inhibited phosphorylation of AKT but not ERK, while trametinib strongly inhibited activation of ERK but even enhanced phosphorylation of AKT. Interestingly, the multi-kinase inhibitor regorafenib blocked AKT activation as efficient as dactolisib, but did not affect the levels of P-ERK.

 

Since lower doses of dactolisib were used in the biological assays as well (Fig. 1), we tested the dose-dependency of the dactolisib effect. At the time point of 5 minutes after FCS-restimulation, PSCs pretreated with 100 and 30 nmol/L dactolisib displayed significantly lower P-AKT levels than untreated controls (Fig. 2E). The effect of the higher dose of the drug, however, was much stronger and corresponded to the more pronounced biological activity (Fig. 1A).

 

Employing real-time PCR, we characterized the gene expression profiles of PSCs after 24 hours of SMI treatment. Fig. 3 shows the results for selected genes that were chosen based on their established role in PSC function and ECM metabolism, and the results of pilot experiments. In all cases, global analysis of variance indicated the presence of significant differences among the groups (P_{Kruskal-Wallis}≤0.001). Further analysis revealed for each of the three inhibitors a specific action profile.

 

Despite its stimulatory effect on BrdU incorporation, regorafenib inhibited expression of cyclin D1 and induced expression of the cell cycle inhibitor CDKN1A. Furthermore, regorafenib-treated PSCs displayed increased mRNA levels of the PSC activation marker α-SMA and α1 type I collagen. In contrast, expression of the profibrogenic mediator TGF-β1 was diminished and expression of IL-6 was virtually blocked. Trametinib displayed similar effects on PSC expression as regorafenib, except for that CDKN1A and α1 type I collagen mRNA levels were not significantly affected by the drug. The action profile of dactolisib differed strongly from the other two SMIs: The drug reduced the mRNA levels of α-SMA and α1 type I collagen, but triggered the expression of TGF-β1. Cyclin D1, CDKN1A and IL-6 levels remained unchanged. In summary, each of the three drugs simultaneously displayed effects on gene expression that favor PSC activity (proliferation, synthesis of ECM) and that inhibit the functions of these cells. When combined, however, trametinib and dactolisib displayed a more homogenous action profile. Expression of cyclin D1, α1 type I collagen and IL-6 was reduced, while the mRNA levels of the other three genes remained unaffected.

 

To verify the striking effects of regorafenib and dactolisib, IL-6 was also assayed on the protein level by measuring the cytokine concentration in supernatants of PSC cultures (Fig. 4). Indeed, lower IL-6 levels (compared to control cells) were detected when PSCs were exposed to regorafenib, trametinib or a combination of trametinib and dactolisib. These inhibitory effects, however, were less pronounced than on the level of mRNA.

 

Supernatants of SMI-treated PSCs were also assayed for collagenase activity by zymography. In accordance with previously published data,^[27] strong lytic bands were detected in the molecular range of MMP2 (Fig. 5A), the dominant gelatinase secreted by activated PSCs. A quantitative assessment (Fig. 5B) showed no significant differences between the samples, suggesting that MMP2 secretion/activity was not affected by the SMI treatment.

 

 

Stroma cells are increasingly acknowledged as an important target for the treatment of stroma-rich tumors, such as PC.^{[10, 28]} Specifically, PSCs have been implicated in the progression of PC through paracrine effects favoring tumor growth and metastasis as well as through the secretion of ECM proteins that mediate chemoresistance and provide protection against anti-tumor immune responses.^{[11, 15]} On the other hand, stroma depletion alone may be deleterious and result in more aggressive tumors with increased vascularity and even heightened proliferation.^{[16, 17]} Antifibrotic therapy will, therefore, have to consider both sides of the coin, the stroma and the tumor cells themselves.

 

In this study, we took advantage of three SMIs that have been developed as antineoplastic agents. None of these substances has been shown to be beneficial in the treatment of PC, and we do not claim direct clinical relevance of our in vitro findings. The results of our studies, however, do show that two of the drugs, the MEK inhibitor trametinib and the dual PI3K/mTOR inhibitor dactolisib, are effective inhibitors of PSC proliferation and may combine antineoplastic with antifibrotic effects. Each of the two substances targets a key signaling pathway in PSC activation, Ras-Raf-MEK-ERK (trametinib) and PI3K-AKT-mTOR (dactolisib), respectively.^{[19-21, 23, 24]} Consistently, a combination of trametinib and dactolisib was particularly effective regarding the inhibition of DNA synthesis. On the other hand, the rate of cell death was increased by trend only. At the level of gene expression, the same combination diminished the levels of cyclin D1, α1 type I collagen and IL-6, which is also in line with an antifibrotic efficiency.

 

An unexpected result was the transient increase of P-AKT levels in trametinib-treated PSC. Although similar findings have previously been made in a colorectal cancer cell line,^[29] such an effect cannot immediately be explained from the known action profile of trametinib as an MEK inhibitor, suggesting molecular follow-up studies.

 

The failure of regorafenib to inhibit PSC proliferation was surprising for several reasons. Thus, we have previously shown that two other multi-kinase inhibitors, sorafenib and sunitinib, display dose-dependent antiproliferative effects in cultured PSCs.^[22] Furthermore, the growth-stimulatory effect of regorafenib was restricted to primary PSCs, since cells of an immortalized rat PSC line^{[30, 31]} responded to the drug by growth retardation (data not shown). Finally, regorafenib blocked AKT signaling as effective as dactolisib. The latter observation suggests that regorafenib may also inhibit additional signaling pathways that, in primary PSC, transduce antimitotic signals, thereby overriding the antiproliferative effect of AKT inhibition. Interestingly, regorafenib nevertheless also displayed effects which we interpret as antifibrotic, such as inhibition of IL-6 and TGF-β1 expression.

 

Taken together, the results of this study show that clinically available inhibitors of Ras-Raf-MEK-ERK and PI3K-AKT-mTOR signaling exert specific antifibrotic effects on cultured rat PSC. A combination of two antineoplastic agents, trametinib and dactolisib, displayed strong growth-inhibitory effects without significant cytotoxicity. These data suggest a particular antifibrotic efficiency of drug combinations that simultaneously target the two key signaling pathways in PSC proliferation, Ras-Raf-MEK-ERK and PI3K-AKT-mTOR (Fig. 6). When combined, trametinib and dactolisib efficiently inhibited DNA synthesis already at relatively low doses, an aspect that is potentially interesting with respect to side effects of the drugs in vivo. We conclude that the antifibrotic efficiency of trametinib and dactolisib should be further evaluated in the context of PC-associated fibrosis, using animal models of the disease.

 

 

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