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Simvastatin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer
Pancreatology 13 (2013) 502e507
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Pancreatology journal homepage: www.elsevier.com/locate/pan
Original article
Simvastatin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer Volker Fendrich a, *,1, Moritz Sparn a,1, Matthias Lauth b, Richard Knoop a, Lars Plassmeier a, Detlef K. Bartsch a, Jens Waldmann a a b
Department of Surgery, Philipps-University Marburg, Baldingerstrasse, D-35043 Marburg, Germany Institute of Molecular Biology and Tumor Research, Philipps-University Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2013 Received in revised form 10 August 2013 Accepted 12 August 2013
Background and aims: Pancreatic cancer is among the most dismal of human malignancies. There are no chemopreventive strategies for pancreatic cancer or its precursor lesions, pancreatic intraepithelial neoplasia (PanINs). Recent evidence suggests that statins have potential chemopreventive abilities. In this study, we used a genetically engineered mouse model of pancreatic cancer to evaluate the chemopreventive potential of this drug. Methods: Simvastatin was injected i.p. in LsL-KrasG12D; Pdx1-Cre or LsL-KrasG12D;LsL-Trp53R172H;Pdx1-Cre mice. After five months, animals were sacrificed. The effect of simvastatin was evaluated by histopathological analyses, immunostaining, and real-time PCR. Results: After five months of treatment, simvastatin was able to significantly delay progression of mPanINs in LsL-KrasG12D; Pdx1-Cre mice. Furthermore, formation of invasive pancreatic cancer in LsLKrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice was partially inhibited by simvastatin. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-KrasG12D; LsL-Trp53R172H;Pdx1-Cre untreated control mice. In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p ¼ 0.004) developed murine pancreatic cancer during the study. Using real-time PCR we found a significant up-regulation of Hmgcr as sign of blocking HMG-CoA reductase, a key enzyme in the cholesterol biosynthesis. This shows our ability to achieve effective pharmacologic levels of simvastatin during pancreatic cancer formation in vivo. Conclusion: Using a transgenic mouse model that recapitulates human pancreatic cancer, this study provides first evidence that simvastatin is an effective chemopreventive agent by delaying the progression of PanINs and partially inhibit the formation of murine pancreatic cancer. Copyright Ó 2013, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
Keywords: Pancreatic cancer PanINs Chemoprevention Statins Simvastatin
1. Introduction Pancreatic cancer is a devastating and almost uniformly lethal malignancy that accounts for approximately 33,000 deaths in the United States every year, rendering it the fourth most common cause of cancer-related mortality [1]. Though the past decades have seen intense research efforts aimed at better understanding of underlying etiologic and pathophysiological mechanisms, this * Corresponding author. Tel.: þ49 64215869141; fax: þ49 64215863851. E-mail addresses: [email protected], [email protected] (V. Fendrich). 1 These authors contributed equally to this work.
increased knowledge could so far not successfully be translated into better clinical treatment strategies and improved patient survival. In fact, during the past 30 years the overall median 5-year survival rates for pancreatic cancer have improved only marginally and are currently around 5% [1,2]. Notably, there is now strong evidence that invasive pancreatic adenocarcinoma proceeds through a morphologic spectrum of noninvasive ductal lesions known as pancreatic intraepithelial neoplasia (PanIN), and that histologic progression of these lesions toward invasive cancer is associated with the progressive accumulation of genetic abnormalities [3]. The studies of this disease have unfortunately been hampered by a number of unique challenges. For instance, at the
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time of diagnosis, pancreatic cancer is usually at an advanced stage and has often metastasized. As a result, the stepwise tumorigenic progression has been inaccessible for study and the precursor cell types still remain an area of ongoing studies. The preclinical study of PanINs has recently been made possible by the generation of genetically modified animal models, which recapitulate human PanINs and invasive pancreatic cancer on a genetic and histomorphologic level [4,5]. Very recently, our group evaluated the chemopreventive efficacy of enalapril and aspirin, either as single agents or in combination, for prevention of pancreatic cancer using the Hingorani mouse model [6]. Our evaluations focused on delaying progression of pancreatic intraepithelial neoplastic (PanIN) lesions and reducing cancer incidence using two promising and attractive candidate chemopreventive agents aspirin and enalapril which are moderately priced, widely available to the public and have known and readily managed toxicity profiles. With regard to agent efficacy, histopathological progression to PanIN was delayed as early as 3 months with all interventions. Efficacy at 3 months was reflected in more normal ducts, fewer PanIN lesions, and fewer advanced grade PanINs. With regard to triple mutant mice, progression to cancer occurred in 60% of controls over 5 months; aspirin, Enalapril or the combination resulted in 71%, 61%, and 48% reductions, respectively [6]. Statins inhibit 3-hydroxy-3-methylglutyral CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis [7]. Statins are commonly used as cholesterol-lowering medications and have shown effectiveness in the primary and secondary prevention of heart attack and stroke [8]. HMG-CoA reductase controls the conversion of HMG-CoA to mevalonic acid, which is the precursor for the biosynthesis of several fundamental endproducts including cholesterol, isoprenoids, dolichol, ubiquinone, and isopentenyladenine [7]. All of these end-products play crucial roles in promoting carcinogenesis, particularly isoprenoids such as farnesal and geranylgeranyl which are key intermediate products involved in protein prenylation, a posttranslational lipid modification [8]. Ras and Ras-related GTP-binding proteins require prenylation to enable cellular localization and function. Thus, in addition to lowering cholesterol levels, targeting protein prenylation by statin drugs such as simvastatin is crucial and possible mechanism for inhibiting oncogenic Kras activity and cancer [9]. The extensive evidence has led to widespread use of these drugs, leading to epidemiological, in vitro and in vivo data that statins may prevent different types of cancer [10e19], including pancreatic cancer [20]. In this study we show for the first time that the widely used drug simvastatin delay progression of Pancreatic Intraepithelial Neoplasia and even cancer formation in a genetically engineered
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mouse model of pancreatic cancer. Our findings suggest that statins might be effective chemopreventive agents for pancreatic cancer. 2. Material and methods 2.1. Mice Conditional LsL-Trp53R172H [21], LsL-KrasG12D and Pdx1-Cre [4] strains were interbred to obtain LsL-KrasG12D; Pdx1-Cre double mutant animals or LsL-KrasG12D;LsL-Trp53R172H;Pdx1-Cre triple mutant animals on a mixed 129/SvJae/C57Bl/6 background as described before [6]. All experiments were approved by the local Committees for Animal Care and Use. Animals were maintained in a climate-controlled room kept at 22 C, exposed to a 12:12-h lighte dark cycle, fed standard laboratory chow, and given water ad libidum. 2.2. Genotyping For genotyping, genomic DNA was extracted from tail cuttings using the REDExtract-N-AmpÔ Tissue PCR kit (SigmaeAldrich, Saint Louis, Missouri, USA). Three PCR reactions were carried out for each animal, to test for the presence of the oncogenic Kras (using LoxP) primers, p53 and Pdx1-Cre transgene constructs (using Cre-specific primers along with Gabra as positive control), respectively. Primers were obtained from Eurofins MWG (Ebersberg, Germany). 2.3. Drug treatment Simvastatin was prepared as previously described [22]. In brief, 25 mg of the prodrug were dissolved in 625 ml EtOH and 938 ml of NaOH was added. The mixture was incubated at 50 C for 2 h and it’s pH was normalized to 7.2 with HCl. Finally, the solution was sterile-filtered and the concentration of the stock solution was adjusted to 4 mg/ml. The application concentration was 1.25 mg/ ml. Simvastatin was injected once per day by intraperitoneal injection at a dosage of 10 mg/kg body weight. Drug treatment was initiated at the age of 5 weeks. LsL-KrasG12D; Pdx1-Cre or LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice were randomly assigned to receive either A) mock treatment, or B) Simvastatin. The formation of the different study groups is explained in Fig. 1. In cases, where littermates were available for drug treatment, only the first mouse was randomly assigned to one of the given treatment groups; the second littermate was then assigned to the ‘matched’ control arm, and so forth. This scheme was chosen in order to obtain the highest possible degree of consistency and to avoid randomization bias as far as possible. After five months animals were sacrificed and the pancreas was dissected.
Transgenic mice (n=44)
KrasG12D; Pdx1-Cre (n=24)
KrasG12D; Trp53R172H; Pdx1-Cre (n=20)
5 months treatment
5 months treatment
Control (n=12)
Control (n=10)
Simvastatin (n=12)
Simvastatin (n=10)
Fig. 1. Study design of the LsL-KrasG12D; Pdx1-Cre and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice.
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2.4. Histologic evaluation After completion of drug treatment the mice were euthanized and the pancreas was removed and inspected for grossly visible tumors and either preserved in 10% formalin solution (Sigmae Aldrich) for histology or processed for RNA extraction (see below). Formalin-fixed, paraffin-embedded tissues were sectioned (4 mm) and stained with H&E. Six sections (100 mm apart) of pancreatic tissues were histologically evaluated blinded to the experimental groups. mPanIN lesions were classified according to histopathologic criteria as recommended elsewhere [23,24]. To quantify the progression of mPanIN lesions in double mutant LsL-KrasG12D; Pdx1-Cre mice, the total number of ductal lesions and their grade were determined. About 100 to 130 pancreatic ducts of the entire fixed specimen (head, body, and tail of the pancreas) were analyzed for each animal. The relative proportion of each mPanIN lesion to the overall number of analyzed ducts was recorded for each animal [25]. In triple mutant LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice the mice were classified by having developed invasive pancreatic cancer or not. 2.5. RNA extraction and real-time RT-PCR PanINs and tumor cells from invasive carcinomas were microdissected. Tumor samples used for RNA isolation had a neoplastic cellularity between 85% and 100% after microdissection and were homogenized and lysed with 600 ml buffer RLT and whole RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) with oncolumn DNA digestion following the standard protocol provided by the manufacturer. The mRNA was reverse transcribed into cDNA with oligo-dT primers using the Superscript 1st Strand System for RT-PCR (Invitrogen, Carlsbad, CA, USA) at 42 C for 50 min. All PCRs were carried out on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) over 40 cycles, with denaturation for 15 s at 95 C and combined annealing/extension at 60 C for 1 min. Following an activation step at 95 C for 10 min, determination of mRNA expression was performed over 40 cycles with 15 s of denaturation at 95 C and annealing/extension/data acquisition at 60 C for 60 s using the Power SYBRÒ Green PCR kit (Applied
Biosystems). Primer sequences are available on request. Relative fold mRNA expression levels were determined using the 2(-DDCt) method [26]. All reactions were done in triplicates and results are presented as means and standard errors. 2.6. Statistical analysis Log-rank test was applied to identify significant differences. Differences in the mean of two samples were analyzed by an unpaired t test. Comparisons of more than two groups were made by a one-way ANOVA with post hoc Holm-Sidak analysis for pair wise comparisons and comparisons vs. control and by KruskaleWallis one-way analysis of variance. Statistical analysis for incidence PDAC was analyzed by Fisher’s Exact test. P-values <0.05 were considered statistically significant. Data were analyzed using SPSS software (Version 14; SPSS, Inc., Chicago, IL). 3. Results 3.1. Development of PanINs in LsL-KrasG12D; Pdx1-Cre and of pancreatic cancer in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice As previously described in the initial reports (Hingorani 203 and 2005), we observed development of low grade and high grade mPanINs in LsL-KrasG12D; Pdx1-Cre and fully invasive pancreatic cancers in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice (data not shown). The histologies resembled ductal adenocarcinomas of the pancreas or its precursor lesions observed in humans. 3.2. Simvastatin delays progression of mPanINs in LsL-KrasG12D; Pdx1-Cre transgenic mice After five months of treatment, Simvastatin was able to delay progression of mPanINs in LsL-KrasG12D; Pdx1-Cre mice significantly (Fig. 2). After histological evaluation of PanIN grade using serial sections, we found a significant delay of PanIN progression in LsL-KrasG12D; Pdx1-Cre mice treated with Simvastatin (Fig. 2DeF), compared to control LsL-KrasG12D; Pdx1-Cre mice (Fig. 2AeC). In the control group, only 15% of the ducts evaluated showed no signs of changes, whereas in mice treated with Simvastatin 59.5% of
Fig. 2. Simvastatin delay progression of PanINs in LsL-KrasG12D; Pdx1-Cre transgenic mice. After histological evaluation of PanIN grade (arrows) using serial sections, a significant delay of PanIN progression in LsL-KrasG12D; Pdx1-Cre mice treated with Simvastatin (DeF), compared to control LsL-KrasG12D; Pdx1-Cre mice (AeC) was detected.
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all ducts were normal (p ¼ 0.04). In both groups, we found the same percentage of low grade PanIN-1A (27.3% vs. 24.9%, p ¼ 0.608). Interestingly, all higher grading and progression of PanINs were significantly delayed by Simvastatin. In the control group we found 22.3% of all ducts as mPan-1B, 8.9% as mPanIN-2 and 2.5% as mPanIN-3, respectively. In contrast, mice treated with Simvastatin had only 3.5% mPanIN-1B (p ¼ 0.001), 0.3% mPanIN-2 (p ¼ 0.00) and no mPanIN-3 (p ¼ 0.028) (Fig. 3). After proving the potential of Simvastatin to delay the progression of precursor lesions of pancreatic cancer in genetically engineered mice, we next sought to evaluate their potential to inhibit the formation of invasive cancer.
3.3. Formation of invasive murine pancreatic cancer in LsLKrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice is partially inhibited by simvastatin After five months of treatment, LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice were euthanized and the abdomens were opened and inspected for cancer formation. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre untreated control mice (Fig. 4). In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p ¼ 0.004) developed murine pancreatic cancer during the study (Fig. 4).
3.4. Simvastatin treatment leads to an accumulation of HMGCoAreductase in precursor lesions and pancreatic cancer Given the clinical and histopathological evidence that Simvastatin delays the progression of PanINs and inhibits partially the formation of invasive pancreatic cancer we next explored the expression of the targeted enzyme in the tumor tissue. It is well known that statins are inhibiting hydroxy-methyl-glutaryl coenzyme A (HMGCoA) reductase [12]. Quantitative real-time PCR demonstrated significant upregulation of HMGCoA as a consequence of enzyme blocking in the malignant cells in LsL-KrasG12D; Pdx1-Cre and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice treated with Simvastatin (Fig. 5). These experiments confirmed our ability to achieve an effective pharmacologic blocking of the targeted enzyme in vivo.
70% 60% 50% 40%
Normal duct PanIN-1A PanIN-1B PanIN-2 PanIN-3
30% 20% 10% 0% Control
Simvastatin
Fig. 3. Simvastatin delay progression of PanINs in LsL-KrasG12D; Pdx1-Cre transgenic mice. Quantitative analysis of mPanINs in experimental groups after five months of treatment. Percentages of pancreatic ducts with no pathology (normal ducts), PanIN1A, PanIN-1B, PanIN-2, and PanIN-3 lesions in control mice (n ¼ 10) and treated mice with Simvastatin.
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4. Discussion The curative potential for pancreatic cancer is dependent on the stage of this disease at diagnosis. Unfortunately, for the majority of patients diagnosed with pancreatic cancer, symptoms do not develop until it is either unresectable or metastatic, and therefore incurable. Hence, the mortality rate approaches the incidence rate for pancreatic cancer with only 1e4% of all patients surviving five years [2]. The conditional KrasG12D model, first described by Hingorani et al. [4], is considered a very valuable tool to study PanIN biology. LSL-KrasG12D/þ;LSL-Trp53R172H/þ;Pdx-1-Cre animals [5] have a median survival of approximately 5 months, manifest widely metastatic pancreatic ductal adenocarcinoma that recapitulates the human spectrum. Now, for the first time, chemopreventive studies can be conducted in mice recapitulating the progression from PanINs to invasive pancreatic cancer [27]. Inhibitors of hydroxy-methyl-glutaryl coenzyme A (HMGCoA) reductase (so called “statins”) are widely used for treatment of hypercholesterolemia. The inhibition of HMG-CoA reductase, a key enzyme in the cholesterol biosynthesis, also results in the depletion of several important intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which modify and target small GTPases to their site of action. Accordingly, inhibition of farnesylation/geranylgeranylation became a plausible approach to modify cell proliferation in tumor tissues [9]. In this regard, PDAC is of particular interest, since more than 90% of human pancreatic cancers shows activating mutations in the K-ras protooncogene [3]. Suppression of this event via statin mediated inhibition of K-Ras farnesylation thus seems to be a promising therapeutic approach. In general, inhibition of HMGCoA can be demonstrated by reduced prenylation of target proteins or by increased feedback upregulation of cholesterol biosynthesis genes, such as HMGCoA itself. As seen in Fig. 5, we show that to use the second approach. Quantitative real-time PCR demonstrated significant upregulation of HMGCoA as a consequence of enzyme blocking in the malignant cells in LsL-KrasG12D; Pdx1-Cre and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice treated with Simvastatin (Fig. 5). These experiments confirmed our ability to achieve an effective pharmacologic blocking of the targeted enzyme in vivo. The other approach, analysing the prenylation status of kras after statin treatment was used in a study, which was published during the preparation of our manuscript [9]. In this study the colleagues analyzed membrane fractions of pancreatic tumor tissues for level of membrane-bound prenylated Kras protein using a Western blot approach. They found a marked decreased level of membrane-bound Kras protein in pancreatic tumor in mice treated with 100 ppm atorvastatin compared to the control mice. Using b-actin as an internal protein control, a phophorylated c-raf protein, a gene immediately downstream of Kras, was markedly decreased. A densitometer analysis demonstrated that with 100 ppm atorvastatin treatment, membrane-bound Kras protein and its down-stream phosphorylated-c-raf protein were significantly reduced in mice treated with atorvastatin. After very early in vivo experiments in laboratory animals indicated that pancreatic tumors and tumors of are sensitive to statins [28], the antiproliferative effects of statins in PDAC were evaluated in numerous epidemiological studies. Coogan et al. compared patients who had any of 10 types of cancer with controls admitted for noncancer diagnoses regarding the association between statin use and cancer. They identified 218 patients with PDAC of whom 208 did not use statins and 8 did so (OR 0.7; 95% CI: 0.3e 1.4) [20]. These results confirmed a large study from Graaf et al. which analyzed 3129 patients and more than 16.000 controls [29],
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Fig. 4. Formation of invasive pancreatic cancer in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice is inhibited by simvastatin (AeC) In more than 70% of control LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice invasive pancreatic cancer was identified. (DeF) In a large subset of simvastatin treated LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice, we found no invasive cancer after five months of treatment.
whereas a very recently published study did not provide support for a beneficial association between usage of Statin and pancreatic cancer [30]. The strongest evidence from an epidemiological study that statins are reducing the risk of pancreatic cancer came from Khurana et al. [31]. Statin use of more than 6 months was associated with a risk reduction of pancreatic cancer of 67% (adjusted odds ratio, 0.33; 95% confidence interval, 0.26e0.41; P < 0.01). The authors concluded that statins seem to be protective against the development of pancreatic cancer. However, two recently published meta analysis to assess the association between statin use and risk of pancreatic cancer suggest did not find a significant reduction of PDAC [13,32]. We now present for the first time, that the Simvastatin do not only inhibits the formation of invasive pancreatic cancer but even more important delays the progression of mPanINs in this genetically engineered mouse model. The most significant finding of our
Fig. 5. Simvastatin treatment leads to an accumulation of HMGCoA-reductase in precursor lesions and pancreatic cancer Quantitative real-time PCR of RNA obtained from control (Con) mice and simvastatin (Sim) treated mice demonstrates profound upregulation of HMGCoA as a consequence of enzyme blocking in the malignant cells in LsL-KrasG12D; Pdx1-Cre (KC) and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre (KPC) mice treated with simvastatin.
study is the fact that Simvastatin was able to delay the progression from low grade PanIN1A to PanIN1B and higher (Fig. 3). Why is that so important? Very recently, Rhim et al. found circulating pancreatic cancer cells and even invasion of the liver by these cells in the same transgenic mouse model of PDAC. When they evaluated the pancreas, surprisingly no invasive cancer but only preinvasive PanIN lesions were found. The author concluded that epithelial cells from higher grade PanIN lesions are able to form metastases within the liver already [33]. This very elegant study underlines the significance of our findings. Liao et al. published their elegant study showing that also atorvastatin inhibits pancreatic carcinogenesis and increased survival in the same mouse model we used [9]. They used five-weekold LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice and fed either a control diet or a diet supplemented with 100 ppm atorvastatin. Mice fed with atorvastatin showed a significant longer survival (171.9 vs. 144.9 days) compared to the control mice (p < 0.05). In contrast to our study they tested the effect of statins only in the LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice but not in LsLKrasG12D; Pdx1-Cre mice. The delay of mPanIN progression in the LsL-KrasG12D; Pdx1-Cre mice in our study shows the potential of statins to be an effective chemopreventive agent. Because the activation of an oncogenic Kras is the earliest event in PanIN formation our data now show that statins delays the progression from normal ducts to mPanIn1A and to mPanIN1B (Fig. 3). Because only PanIN3 are classified as carcinoma in situ, delaying the progression toward PanIN3 would have a major impact on cancer development. Therefore, our study is strengthening the conclusion of Liao et al. [9] that statins might be an effective chemopreventive agent for pancreatic cancer. In the presented study, we confirm the results from Liao et al. [9]. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-KrasG12D; LsL-Trp53R172H;Pdx1-Cre untreated control mice. In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p ¼ 0.004) developed murine pancreatic cancer. In conclusion, we identified Simvastatin with potential to delay the formation of pancreatic cancer and its precursor lesions. Our results support the hypothesis that statins might be a valid chemopreventive strategy in delaying PanIN progression.
V. Fendrich et al. / Pancreatology 13 (2013) 502e507
Grant support V.F was supported by a Research Grant of the University Medical Center Giessen and Marburg.
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Pancreatology journal homepage: www.elsevier.com/locate/pan
Original article
Simvastatin delay progression of pancreatic intraepithelial neoplasia and cancer formation in a genetically engineered mouse model of pancreatic cancer Volker Fendrich a, *,1, Moritz Sparn a,1, Matthias Lauth b, Richard Knoop a, Lars Plassmeier a, Detlef K. Bartsch a, Jens Waldmann a a b
Department of Surgery, Philipps-University Marburg, Baldingerstrasse, D-35043 Marburg, Germany Institute of Molecular Biology and Tumor Research, Philipps-University Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 January 2013 Received in revised form 10 August 2013 Accepted 12 August 2013
Background and aims: Pancreatic cancer is among the most dismal of human malignancies. There are no chemopreventive strategies for pancreatic cancer or its precursor lesions, pancreatic intraepithelial neoplasia (PanINs). Recent evidence suggests that statins have potential chemopreventive abilities. In this study, we used a genetically engineered mouse model of pancreatic cancer to evaluate the chemopreventive potential of this drug. Methods: Simvastatin was injected i.p. in LsL-KrasG12D; Pdx1-Cre or LsL-KrasG12D;LsL-Trp53R172H;Pdx1-Cre mice. After five months, animals were sacrificed. The effect of simvastatin was evaluated by histopathological analyses, immunostaining, and real-time PCR. Results: After five months of treatment, simvastatin was able to significantly delay progression of mPanINs in LsL-KrasG12D; Pdx1-Cre mice. Furthermore, formation of invasive pancreatic cancer in LsLKrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice was partially inhibited by simvastatin. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-KrasG12D; LsL-Trp53R172H;Pdx1-Cre untreated control mice. In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p ¼ 0.004) developed murine pancreatic cancer during the study. Using real-time PCR we found a significant up-regulation of Hmgcr as sign of blocking HMG-CoA reductase, a key enzyme in the cholesterol biosynthesis. This shows our ability to achieve effective pharmacologic levels of simvastatin during pancreatic cancer formation in vivo. Conclusion: Using a transgenic mouse model that recapitulates human pancreatic cancer, this study provides first evidence that simvastatin is an effective chemopreventive agent by delaying the progression of PanINs and partially inhibit the formation of murine pancreatic cancer. Copyright Ó 2013, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.
Keywords: Pancreatic cancer PanINs Chemoprevention Statins Simvastatin
1. Introduction Pancreatic cancer is a devastating and almost uniformly lethal malignancy that accounts for approximately 33,000 deaths in the United States every year, rendering it the fourth most common cause of cancer-related mortality [1]. Though the past decades have seen intense research efforts aimed at better understanding of underlying etiologic and pathophysiological mechanisms, this * Corresponding author. Tel.: þ49 64215869141; fax: þ49 64215863851. E-mail addresses: [email protected], [email protected] (V. Fendrich). 1 These authors contributed equally to this work.
increased knowledge could so far not successfully be translated into better clinical treatment strategies and improved patient survival. In fact, during the past 30 years the overall median 5-year survival rates for pancreatic cancer have improved only marginally and are currently around 5% [1,2]. Notably, there is now strong evidence that invasive pancreatic adenocarcinoma proceeds through a morphologic spectrum of noninvasive ductal lesions known as pancreatic intraepithelial neoplasia (PanIN), and that histologic progression of these lesions toward invasive cancer is associated with the progressive accumulation of genetic abnormalities [3]. The studies of this disease have unfortunately been hampered by a number of unique challenges. For instance, at the
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time of diagnosis, pancreatic cancer is usually at an advanced stage and has often metastasized. As a result, the stepwise tumorigenic progression has been inaccessible for study and the precursor cell types still remain an area of ongoing studies. The preclinical study of PanINs has recently been made possible by the generation of genetically modified animal models, which recapitulate human PanINs and invasive pancreatic cancer on a genetic and histomorphologic level [4,5]. Very recently, our group evaluated the chemopreventive efficacy of enalapril and aspirin, either as single agents or in combination, for prevention of pancreatic cancer using the Hingorani mouse model [6]. Our evaluations focused on delaying progression of pancreatic intraepithelial neoplastic (PanIN) lesions and reducing cancer incidence using two promising and attractive candidate chemopreventive agents aspirin and enalapril which are moderately priced, widely available to the public and have known and readily managed toxicity profiles. With regard to agent efficacy, histopathological progression to PanIN was delayed as early as 3 months with all interventions. Efficacy at 3 months was reflected in more normal ducts, fewer PanIN lesions, and fewer advanced grade PanINs. With regard to triple mutant mice, progression to cancer occurred in 60% of controls over 5 months; aspirin, Enalapril or the combination resulted in 71%, 61%, and 48% reductions, respectively [6]. Statins inhibit 3-hydroxy-3-methylglutyral CoA reductase (HMGCR), the rate-limiting enzyme in cholesterol synthesis [7]. Statins are commonly used as cholesterol-lowering medications and have shown effectiveness in the primary and secondary prevention of heart attack and stroke [8]. HMG-CoA reductase controls the conversion of HMG-CoA to mevalonic acid, which is the precursor for the biosynthesis of several fundamental endproducts including cholesterol, isoprenoids, dolichol, ubiquinone, and isopentenyladenine [7]. All of these end-products play crucial roles in promoting carcinogenesis, particularly isoprenoids such as farnesal and geranylgeranyl which are key intermediate products involved in protein prenylation, a posttranslational lipid modification [8]. Ras and Ras-related GTP-binding proteins require prenylation to enable cellular localization and function. Thus, in addition to lowering cholesterol levels, targeting protein prenylation by statin drugs such as simvastatin is crucial and possible mechanism for inhibiting oncogenic Kras activity and cancer [9]. The extensive evidence has led to widespread use of these drugs, leading to epidemiological, in vitro and in vivo data that statins may prevent different types of cancer [10e19], including pancreatic cancer [20]. In this study we show for the first time that the widely used drug simvastatin delay progression of Pancreatic Intraepithelial Neoplasia and even cancer formation in a genetically engineered
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mouse model of pancreatic cancer. Our findings suggest that statins might be effective chemopreventive agents for pancreatic cancer. 2. Material and methods 2.1. Mice Conditional LsL-Trp53R172H [21], LsL-KrasG12D and Pdx1-Cre [4] strains were interbred to obtain LsL-KrasG12D; Pdx1-Cre double mutant animals or LsL-KrasG12D;LsL-Trp53R172H;Pdx1-Cre triple mutant animals on a mixed 129/SvJae/C57Bl/6 background as described before [6]. All experiments were approved by the local Committees for Animal Care and Use. Animals were maintained in a climate-controlled room kept at 22 C, exposed to a 12:12-h lighte dark cycle, fed standard laboratory chow, and given water ad libidum. 2.2. Genotyping For genotyping, genomic DNA was extracted from tail cuttings using the REDExtract-N-AmpÔ Tissue PCR kit (SigmaeAldrich, Saint Louis, Missouri, USA). Three PCR reactions were carried out for each animal, to test for the presence of the oncogenic Kras (using LoxP) primers, p53 and Pdx1-Cre transgene constructs (using Cre-specific primers along with Gabra as positive control), respectively. Primers were obtained from Eurofins MWG (Ebersberg, Germany). 2.3. Drug treatment Simvastatin was prepared as previously described [22]. In brief, 25 mg of the prodrug were dissolved in 625 ml EtOH and 938 ml of NaOH was added. The mixture was incubated at 50 C for 2 h and it’s pH was normalized to 7.2 with HCl. Finally, the solution was sterile-filtered and the concentration of the stock solution was adjusted to 4 mg/ml. The application concentration was 1.25 mg/ ml. Simvastatin was injected once per day by intraperitoneal injection at a dosage of 10 mg/kg body weight. Drug treatment was initiated at the age of 5 weeks. LsL-KrasG12D; Pdx1-Cre or LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice were randomly assigned to receive either A) mock treatment, or B) Simvastatin. The formation of the different study groups is explained in Fig. 1. In cases, where littermates were available for drug treatment, only the first mouse was randomly assigned to one of the given treatment groups; the second littermate was then assigned to the ‘matched’ control arm, and so forth. This scheme was chosen in order to obtain the highest possible degree of consistency and to avoid randomization bias as far as possible. After five months animals were sacrificed and the pancreas was dissected.
Transgenic mice (n=44)
KrasG12D; Pdx1-Cre (n=24)
KrasG12D; Trp53R172H; Pdx1-Cre (n=20)
5 months treatment
5 months treatment
Control (n=12)
Control (n=10)
Simvastatin (n=12)
Simvastatin (n=10)
Fig. 1. Study design of the LsL-KrasG12D; Pdx1-Cre and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice.
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2.4. Histologic evaluation After completion of drug treatment the mice were euthanized and the pancreas was removed and inspected for grossly visible tumors and either preserved in 10% formalin solution (Sigmae Aldrich) for histology or processed for RNA extraction (see below). Formalin-fixed, paraffin-embedded tissues were sectioned (4 mm) and stained with H&E. Six sections (100 mm apart) of pancreatic tissues were histologically evaluated blinded to the experimental groups. mPanIN lesions were classified according to histopathologic criteria as recommended elsewhere [23,24]. To quantify the progression of mPanIN lesions in double mutant LsL-KrasG12D; Pdx1-Cre mice, the total number of ductal lesions and their grade were determined. About 100 to 130 pancreatic ducts of the entire fixed specimen (head, body, and tail of the pancreas) were analyzed for each animal. The relative proportion of each mPanIN lesion to the overall number of analyzed ducts was recorded for each animal [25]. In triple mutant LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice the mice were classified by having developed invasive pancreatic cancer or not. 2.5. RNA extraction and real-time RT-PCR PanINs and tumor cells from invasive carcinomas were microdissected. Tumor samples used for RNA isolation had a neoplastic cellularity between 85% and 100% after microdissection and were homogenized and lysed with 600 ml buffer RLT and whole RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) with oncolumn DNA digestion following the standard protocol provided by the manufacturer. The mRNA was reverse transcribed into cDNA with oligo-dT primers using the Superscript 1st Strand System for RT-PCR (Invitrogen, Carlsbad, CA, USA) at 42 C for 50 min. All PCRs were carried out on a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA) over 40 cycles, with denaturation for 15 s at 95 C and combined annealing/extension at 60 C for 1 min. Following an activation step at 95 C for 10 min, determination of mRNA expression was performed over 40 cycles with 15 s of denaturation at 95 C and annealing/extension/data acquisition at 60 C for 60 s using the Power SYBRÒ Green PCR kit (Applied
Biosystems). Primer sequences are available on request. Relative fold mRNA expression levels were determined using the 2(-DDCt) method [26]. All reactions were done in triplicates and results are presented as means and standard errors. 2.6. Statistical analysis Log-rank test was applied to identify significant differences. Differences in the mean of two samples were analyzed by an unpaired t test. Comparisons of more than two groups were made by a one-way ANOVA with post hoc Holm-Sidak analysis for pair wise comparisons and comparisons vs. control and by KruskaleWallis one-way analysis of variance. Statistical analysis for incidence PDAC was analyzed by Fisher’s Exact test. P-values <0.05 were considered statistically significant. Data were analyzed using SPSS software (Version 14; SPSS, Inc., Chicago, IL). 3. Results 3.1. Development of PanINs in LsL-KrasG12D; Pdx1-Cre and of pancreatic cancer in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice As previously described in the initial reports (Hingorani 203 and 2005), we observed development of low grade and high grade mPanINs in LsL-KrasG12D; Pdx1-Cre and fully invasive pancreatic cancers in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice (data not shown). The histologies resembled ductal adenocarcinomas of the pancreas or its precursor lesions observed in humans. 3.2. Simvastatin delays progression of mPanINs in LsL-KrasG12D; Pdx1-Cre transgenic mice After five months of treatment, Simvastatin was able to delay progression of mPanINs in LsL-KrasG12D; Pdx1-Cre mice significantly (Fig. 2). After histological evaluation of PanIN grade using serial sections, we found a significant delay of PanIN progression in LsL-KrasG12D; Pdx1-Cre mice treated with Simvastatin (Fig. 2DeF), compared to control LsL-KrasG12D; Pdx1-Cre mice (Fig. 2AeC). In the control group, only 15% of the ducts evaluated showed no signs of changes, whereas in mice treated with Simvastatin 59.5% of
Fig. 2. Simvastatin delay progression of PanINs in LsL-KrasG12D; Pdx1-Cre transgenic mice. After histological evaluation of PanIN grade (arrows) using serial sections, a significant delay of PanIN progression in LsL-KrasG12D; Pdx1-Cre mice treated with Simvastatin (DeF), compared to control LsL-KrasG12D; Pdx1-Cre mice (AeC) was detected.
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all ducts were normal (p ¼ 0.04). In both groups, we found the same percentage of low grade PanIN-1A (27.3% vs. 24.9%, p ¼ 0.608). Interestingly, all higher grading and progression of PanINs were significantly delayed by Simvastatin. In the control group we found 22.3% of all ducts as mPan-1B, 8.9% as mPanIN-2 and 2.5% as mPanIN-3, respectively. In contrast, mice treated with Simvastatin had only 3.5% mPanIN-1B (p ¼ 0.001), 0.3% mPanIN-2 (p ¼ 0.00) and no mPanIN-3 (p ¼ 0.028) (Fig. 3). After proving the potential of Simvastatin to delay the progression of precursor lesions of pancreatic cancer in genetically engineered mice, we next sought to evaluate their potential to inhibit the formation of invasive cancer.
3.3. Formation of invasive murine pancreatic cancer in LsLKrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice is partially inhibited by simvastatin After five months of treatment, LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice were euthanized and the abdomens were opened and inspected for cancer formation. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre untreated control mice (Fig. 4). In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p ¼ 0.004) developed murine pancreatic cancer during the study (Fig. 4).
3.4. Simvastatin treatment leads to an accumulation of HMGCoAreductase in precursor lesions and pancreatic cancer Given the clinical and histopathological evidence that Simvastatin delays the progression of PanINs and inhibits partially the formation of invasive pancreatic cancer we next explored the expression of the targeted enzyme in the tumor tissue. It is well known that statins are inhibiting hydroxy-methyl-glutaryl coenzyme A (HMGCoA) reductase [12]. Quantitative real-time PCR demonstrated significant upregulation of HMGCoA as a consequence of enzyme blocking in the malignant cells in LsL-KrasG12D; Pdx1-Cre and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice treated with Simvastatin (Fig. 5). These experiments confirmed our ability to achieve an effective pharmacologic blocking of the targeted enzyme in vivo.
70% 60% 50% 40%
Normal duct PanIN-1A PanIN-1B PanIN-2 PanIN-3
30% 20% 10% 0% Control
Simvastatin
Fig. 3. Simvastatin delay progression of PanINs in LsL-KrasG12D; Pdx1-Cre transgenic mice. Quantitative analysis of mPanINs in experimental groups after five months of treatment. Percentages of pancreatic ducts with no pathology (normal ducts), PanIN1A, PanIN-1B, PanIN-2, and PanIN-3 lesions in control mice (n ¼ 10) and treated mice with Simvastatin.
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4. Discussion The curative potential for pancreatic cancer is dependent on the stage of this disease at diagnosis. Unfortunately, for the majority of patients diagnosed with pancreatic cancer, symptoms do not develop until it is either unresectable or metastatic, and therefore incurable. Hence, the mortality rate approaches the incidence rate for pancreatic cancer with only 1e4% of all patients surviving five years [2]. The conditional KrasG12D model, first described by Hingorani et al. [4], is considered a very valuable tool to study PanIN biology. LSL-KrasG12D/þ;LSL-Trp53R172H/þ;Pdx-1-Cre animals [5] have a median survival of approximately 5 months, manifest widely metastatic pancreatic ductal adenocarcinoma that recapitulates the human spectrum. Now, for the first time, chemopreventive studies can be conducted in mice recapitulating the progression from PanINs to invasive pancreatic cancer [27]. Inhibitors of hydroxy-methyl-glutaryl coenzyme A (HMGCoA) reductase (so called “statins”) are widely used for treatment of hypercholesterolemia. The inhibition of HMG-CoA reductase, a key enzyme in the cholesterol biosynthesis, also results in the depletion of several important intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which modify and target small GTPases to their site of action. Accordingly, inhibition of farnesylation/geranylgeranylation became a plausible approach to modify cell proliferation in tumor tissues [9]. In this regard, PDAC is of particular interest, since more than 90% of human pancreatic cancers shows activating mutations in the K-ras protooncogene [3]. Suppression of this event via statin mediated inhibition of K-Ras farnesylation thus seems to be a promising therapeutic approach. In general, inhibition of HMGCoA can be demonstrated by reduced prenylation of target proteins or by increased feedback upregulation of cholesterol biosynthesis genes, such as HMGCoA itself. As seen in Fig. 5, we show that to use the second approach. Quantitative real-time PCR demonstrated significant upregulation of HMGCoA as a consequence of enzyme blocking in the malignant cells in LsL-KrasG12D; Pdx1-Cre and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice treated with Simvastatin (Fig. 5). These experiments confirmed our ability to achieve an effective pharmacologic blocking of the targeted enzyme in vivo. The other approach, analysing the prenylation status of kras after statin treatment was used in a study, which was published during the preparation of our manuscript [9]. In this study the colleagues analyzed membrane fractions of pancreatic tumor tissues for level of membrane-bound prenylated Kras protein using a Western blot approach. They found a marked decreased level of membrane-bound Kras protein in pancreatic tumor in mice treated with 100 ppm atorvastatin compared to the control mice. Using b-actin as an internal protein control, a phophorylated c-raf protein, a gene immediately downstream of Kras, was markedly decreased. A densitometer analysis demonstrated that with 100 ppm atorvastatin treatment, membrane-bound Kras protein and its down-stream phosphorylated-c-raf protein were significantly reduced in mice treated with atorvastatin. After very early in vivo experiments in laboratory animals indicated that pancreatic tumors and tumors of are sensitive to statins [28], the antiproliferative effects of statins in PDAC were evaluated in numerous epidemiological studies. Coogan et al. compared patients who had any of 10 types of cancer with controls admitted for noncancer diagnoses regarding the association between statin use and cancer. They identified 218 patients with PDAC of whom 208 did not use statins and 8 did so (OR 0.7; 95% CI: 0.3e 1.4) [20]. These results confirmed a large study from Graaf et al. which analyzed 3129 patients and more than 16.000 controls [29],
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Fig. 4. Formation of invasive pancreatic cancer in LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre transgenic mice is inhibited by simvastatin (AeC) In more than 70% of control LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice invasive pancreatic cancer was identified. (DeF) In a large subset of simvastatin treated LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice, we found no invasive cancer after five months of treatment.
whereas a very recently published study did not provide support for a beneficial association between usage of Statin and pancreatic cancer [30]. The strongest evidence from an epidemiological study that statins are reducing the risk of pancreatic cancer came from Khurana et al. [31]. Statin use of more than 6 months was associated with a risk reduction of pancreatic cancer of 67% (adjusted odds ratio, 0.33; 95% confidence interval, 0.26e0.41; P < 0.01). The authors concluded that statins seem to be protective against the development of pancreatic cancer. However, two recently published meta analysis to assess the association between statin use and risk of pancreatic cancer suggest did not find a significant reduction of PDAC [13,32]. We now present for the first time, that the Simvastatin do not only inhibits the formation of invasive pancreatic cancer but even more important delays the progression of mPanINs in this genetically engineered mouse model. The most significant finding of our
Fig. 5. Simvastatin treatment leads to an accumulation of HMGCoA-reductase in precursor lesions and pancreatic cancer Quantitative real-time PCR of RNA obtained from control (Con) mice and simvastatin (Sim) treated mice demonstrates profound upregulation of HMGCoA as a consequence of enzyme blocking in the malignant cells in LsL-KrasG12D; Pdx1-Cre (KC) and LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre (KPC) mice treated with simvastatin.
study is the fact that Simvastatin was able to delay the progression from low grade PanIN1A to PanIN1B and higher (Fig. 3). Why is that so important? Very recently, Rhim et al. found circulating pancreatic cancer cells and even invasion of the liver by these cells in the same transgenic mouse model of PDAC. When they evaluated the pancreas, surprisingly no invasive cancer but only preinvasive PanIN lesions were found. The author concluded that epithelial cells from higher grade PanIN lesions are able to form metastases within the liver already [33]. This very elegant study underlines the significance of our findings. Liao et al. published their elegant study showing that also atorvastatin inhibits pancreatic carcinogenesis and increased survival in the same mouse model we used [9]. They used five-weekold LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice and fed either a control diet or a diet supplemented with 100 ppm atorvastatin. Mice fed with atorvastatin showed a significant longer survival (171.9 vs. 144.9 days) compared to the control mice (p < 0.05). In contrast to our study they tested the effect of statins only in the LsL-KrasG12D; LsL-Trp53R172H; Pdx1-Cre mice but not in LsLKrasG12D; Pdx1-Cre mice. The delay of mPanIN progression in the LsL-KrasG12D; Pdx1-Cre mice in our study shows the potential of statins to be an effective chemopreventive agent. Because the activation of an oncogenic Kras is the earliest event in PanIN formation our data now show that statins delays the progression from normal ducts to mPanIn1A and to mPanIN1B (Fig. 3). Because only PanIN3 are classified as carcinoma in situ, delaying the progression toward PanIN3 would have a major impact on cancer development. Therefore, our study is strengthening the conclusion of Liao et al. [9] that statins might be an effective chemopreventive agent for pancreatic cancer. In the presented study, we confirm the results from Liao et al. [9]. Invasive murine pancreatic cancer was identified in 9 of 12 (75%) LsL-KrasG12D; LsL-Trp53R172H;Pdx1-Cre untreated control mice. In contrast, transgenic mice treated with Simvastatin, only 4 out of 10 (40%, p ¼ 0.004) developed murine pancreatic cancer. In conclusion, we identified Simvastatin with potential to delay the formation of pancreatic cancer and its precursor lesions. Our results support the hypothesis that statins might be a valid chemopreventive strategy in delaying PanIN progression.
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Grant support V.F was supported by a Research Grant of the University Medical Center Giessen and Marburg.
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