Invest New Drugs DOI 10.1007/s10637-014-0090-9

PRECLINICAL STUDIES

PRIMA-1, a mutant p53 reactivator, induces apoptosis and enhances chemotherapeutic cytotoxicity in pancreatic cancer cell lines Patricia Izetti & Agnes Hautefeuille & Ana Lucia Abujamra & Caroline Brunetto de Farias & Juliana Giacomazzi & Bárbara Alemar & Guido Lenz & Rafael Roesler & Gilberto Schwartsmann & Alessandro Bersch Osvaldt & Pierre Hainaut & Patricia Ashton-Prolla

Received: 9 January 2014 / Accepted: 13 March 2014 # Springer Science+Business Media New York 2014

Summary TP53 mutation is a common event in many cancers, including pancreatic adenocarcinoma, where it occurs in 50–70 % of cases. In an effort to reactivate mutant p53 protein, several new drugs are being developed, including PRIMA-1 and PRIMA-1Met/APR-246 (p53 reactivation and P. Izetti : J. Giacomazzi : B. Alemar : P. Ashton-Prolla Laboratório de Medicina Genômica, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil 90035-003 P. Izetti : B. Alemar : P. Ashton-Prolla Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500–Prédio 43323M, Porto Alegre, RS, Brazil 91501-970 P. Izetti : A. L. Abujamra : C. B. de Farias : R. Roesler : G. Schwartsmann Laboratório de Pesquisas em Câncer, Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil 90035-003 A. Hautefeuille International Agency for Research on Cancer, 150 Cours Albert Thomas, Lyon, France 69372 A. L. Abujamra : C. B. de Farias Instituto do Câncer Infantil do Rio Grande do Sul, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil 90035-003 A. L. Abujamra : C. B. de Farias : R. Roesler : G. Schwartsmann Instituto Nacional de Ciência e Tecnologia Translacional em Medicina, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil 90035-003 G. Lenz Laboratório de Sinalização e Plasticidade Celular, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500–Prédio 43431, Porto Alegre, RS, Brazil 91501-970

induction of massive apoptosis). PRIMA-1 has been shown to induce apoptosis in tumor cells by reactivating p53 mutants, but its effect in pancreatic cancer remains unclear. Here we investigated the effects of PRIMA-1 on cell viability, cell cycle and expression of p53-regulated proteins in PANC-1

R. Roesler Laboratório de Neurofarmacologia e Biologia de Tumores Neurais, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500–Sala 211, Porto Alegre, RS, Brazil 91501-970 G. Schwartsmann Serviço de Oncologia Clínica, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil 90035-003 A. B. Osvaldt Serviço de Cirurgia do Aparelho Digestivo, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre, RS, Brazil 90035-003 P. Hainaut International Prevention Research Institute, 95 Cours Lafayette, Lyon, France 69006 P. Ashton-Prolla Departamento de Genética, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500–Prédio 43323M, Porto Alegre, RS, Brazil 91501-970 P. Izetti (*) Laboratório de Medicina Genômica, Hospital de Clínicas de Porto Alegre, Rua Ramiro Barcelos, 2350, 90035-903 Porto Alegre, RS, Brazil e-mail: [email protected]

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and BxPC-3 (mutant TP53), and CAPAN-2 (wild-type TP53) pancreatic cell lines. Treatment with PRIMA-1 selectively induced apoptosis and cell cycle arrest in p53 mutant cells compared to CAPAN-2 cells. The growth suppressive effect of PRIMA-1 was markedly reduced in p53 mutant cell lines transfected with p53 siRNA, supporting the role of mutant p53 in PRIMA-1 induced cell death. Moreover, treatment with the thiol group donor N-acetylcysteine completely blocked PRIMA-1-induced apoptosis and reinforced the hypothesis that thiol modifications are important for PRIMA-1 biological activity. In combination treatments, PRIMA-1 enhanced the anti-tumor activity of several chemotherapic drugs against pancreatic cancer cells and also exhibited a pronounced synergistic effect in association with the Mdm2 inhibitor Nutlin3. Taken together, our data indicate that PRIMA-1 induces apoptosis in p53 mutant pancreatic cancer cells by promoting the re-activation of p53 and inducing proapoptotic signaling pathways, providing in vitro evidence for a potential therapeutic approach in pancreatic cancer. Keywords Pancreatic cancer . p53 . PRIMA-1 . Apoptosis

Introduction Pancreatic cancer is the fourth-leading cause of cancer deaths in the United States and the only of the most commonly diagnosed cancers for which both the incidence and death rates are increasing, despite advances in cancer therapy [1]. The impairment of drug delivery caused by the low density of vasculature within pancreatic tumors, the multiple subsets of genes undergoing genetic changes [2] and the existence of cancer stem cells that aberrantly activate developmental signaling pathways [3] make pancreatic cancers highly resistant to treatment. Some recent therapeutic agents and different combination chemotherapy regimens such as FOLFIRINOX (5-Fluorouracil/Irinotecan/ Oxaliplatin) have shown some promise in increasing the response rate to pancreatic adenocarcinoma treatment [4], but the results are still modest and new therapies capable of extending survival and down-staging inoperable tumors are needed. Sequential stages of disease progression have been well characterized at the molecular level, involving activating mutations in oncogenes (e.g. K-RAS) and inactivation of tumour-suppressor genes (e.g. TP53, DPC4, p16/CDKN2A and BRCA2) [5], and the development of molecular profiling technologies has fueled efforts to personalize cancer treatment and target specific somatic alterations present in these cancers [6]. TP53 is the most frequently mutated gene identified in human cancer and is inactivated in 50–70 % of pancreatic adenocarcinomas, ranking this disease among those with the highest frequency of TP53 inactivation [7, 8]. Most TP53

mutations are missense and result in accumulation of mutant p53 protein, with potentially gain-of-function and dominantnegative properties. Mutations in TP53 clearly occur in the progression of precursors lesions to pancreatic adenocarcinoma [9] and the p53 mutant phenotype, rather than genetic loss of TP53, associates with promotion of metastasis in murine models [10]. The development of new therapies that are able to selectively kill cancer cells is a very attractive strategy for resistant diseases such as pancreatic cancer and targeting the mutant p53 protein could represent a unique model for the treatment of this particular cancer. PRIMA-1 (p53 reactivation and induction of massive apoptosis) is a small molecule that has the ability to convert mutant p53 to an active conformation, restoring DNA binding and transcriptional activity [11, 12]. PRIMA-1 and its methylated form PRIMA-1Met/APR-246 have been shown to induce p53-mediated apoptosis and cellcycle arrest in different types of cancer, such as leukaemia [13], breast [14], head and neck [15], melanoma [16] and thyroid carcinoma [17], and to suppress the growth of tumor xenografts carrying mutant p53 proteins in vivo [16, 18]. More recently, PRIMA-1 was also linked to autophagy induction in breast and colon cancer cells [19]. Given the number and complexity of roles that p53 plays in tumorigenesis and aggressive tumor biology, the use of PRIMA-1 to restore the suppressive activity of mutant p53 could be a powerful strategy to control pancreatic cancer progression and to improve the effectiveness of existing chemotherapeutic regimens. We have tested this hypothesis by examining the effects of PRIMA-1, alone or in combination with different types of drugs, on the growth and survival of pancreatic adenocarcinoma cell lines with different p53 mutation status. Our results demonstrate that PRIMA-1 suppresses the growth of pancreatic cancer cells expressing mutant p53 in vitro by reactivating the p53 pathway, inducing p53-dependent growth arrest and apoptosis, and enhancing the antitumor activity of several chemotherapeutic agents currently used in this type of cancer.

Material and methods Cell culture, transfections and drugs The mutant p53 (mtp53) expressing pancreatic cancer cell lines PANC-1 (p.R273H) and BxPC-3 (p.Y220C) and the wild type p53-expressing Capan-2 cells (wtp53) were used in this study. The three cell lines were obtained from ATCC (Manassas, VA, USA) and were sequenced to confirm mutation and wild type status. PANC-1 and Capan-2 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with L-glutamine and 4.5 g/L of D-Glucose (Gibco®, Life Technologies, USA) and BxPC-3 cells were grown in RPMI-1640 Medium (LGC Biotecnologia), all supplemented with 10 % fetal bovine

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serum (Life Technologies, USA), gentamicin (4 mg/ml; Nova Pharma, Brazil), and Fungizone (250 mg/kg; Invitrogen, USA). Cells were kept at a temperature of 37 °C, a minimum relative humidity of 95 %, and an atmosphere of 5 % CO2 in air. All cell lines were tested with the VenorGeM Mycoplasma PCR-based Detection Kit and confirmed to be free of Mycoplasma spp. contamination (Sigma-Aldrich, USA). For siRNA silencing of TP53 mRNA in PANC-1, cells were plated in six-well plates at 3×105 cells per well and transfected with 5nM p53-specific siRNA (Cell Signaling, USA) or a scrambled siRNA control (Cell Signaling, USA) using Hiperfect Transfection Reagent (Qiagen, Germany) in optiMEM medium (Gibco, Invitrogen, UK). Two subsequent transfections at 1 day intervals were performed to obtain maximum TP53 mRNA inhibition. PRIMA-1 was obtained from Sigma-Aldrich® (USA) and dissolved in 100 % dimethyl sulphoxide (DMSO) for cellular treatments. Direct sequencing of TP53 mutations Genomic DNA was extracted from untreated cells and the exonic and flanking intronic regions of the TP53 gene were amplified by PCR according to the protocol used at the International Agency for Research on Cancer (IARC) (http://www-p53.iarc.fr/ p53sequencing.html). The resulting PCR products were sequenced and the locations and types of mutation were determined and confirmed by a second PCR reaction and sequencing. MTT assay Cell viability was measured by MTT assay (Sigma-Aldrich, USA) after treatment with PRIMA-1. Briefly, cells were seeded in each well of 96-well plates in 100 μl culture medium and incubated overnight at 37 °C in an atmosphere of 5 % CO2. The next day, the medium was removed and cells washed with PBS and treated with vehicle control (DMSO, dimethylsulfoxide) or different concentrations of PRIMA-1 for 12 to 48 h; the medium was replaced with MTT solution diluted in medium once the treatment was completed. The plates were further incubated at 37 °C under 5 % CO2 for 4 h and then left at room temperature until completely dry. DMSO was then added and the absorbance was read at 492 nm using a microplate enzyme-linked immunoassay reader (ELISA). The relative growth activity was determined as the percentage absorbance of treated cells compared to that of vehicle treated cells (control). Cell apoptosis and death assay The Annexin V-FITC apoptosis detection kit (BD Biosciences, USA) was used to detect apoptosis and propidium iodide (PI) to detect DNA fragmentation or cell death. Cells were grown in 6-well plates overnight and the next day, the medium was removed and cells washed and treated with 25–100 μM PRIMA-1 for 6, 24 and 48 h. Once the treatment was completed, cells were harvested and washed twice with ice-cold PBS, resuspended in 1×

binding buffer, and stained with Annexin V-FITC and PI, according to the manufacturer’s instructions. The percentage of Annexin V-FITC-positive and PI-positive cells was determined from the fluorescence of 10,000 cells by a BD FACSCanto (BD Biosciences, USA). Data were analysed using the CellQuest™ software (BD Biosciences, USA). BrdU incorporation assay BrdU incorporation assay (BD Pharmingen, USA) was used to evaluate the synthesis of DNA. Cells were seeded in a 6-well plate, followed by treatment with PRIMA-1 at 25, 50 and 75 μM or vehicle control for 12, 16, 24 and 36 h. Cells were incubated with BrdU 10 mM for 2 h before completion of the treatment schedule, followed by fixation with cold ethanol 70 %. Later, cells were washed with PBS and incubated with 2 M HCl for 20 min at room temperature. After this, cells were washed twice with PBS and incubated with 0.1 M sodium borate (Na2B4O7, pH 8.5), for 3 min at room temperature. Finally, cells were incubated with the anti-BrdU FITC-conjugated antibody for 20 min in the dark, co-stained with PI and analyzed by flow cytometry. Western blotting Cells were treated with vehicle control, 50 and 75 μM PRIMA-1 before harvesting after 24 h treatment. Whole-cell lysates were prepared using RIPA-like buffer. For the detection of p53, Mdm2, p21/waf1, Bax, cleaved caspase3 and actin, western blotting was performed according to standard procedures. Briefly, the proteins were separated using polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane using a Trans-Blot transfer system from Bio-Rad®, blocked overnight in PBS containing 1 % nonfat dry milk and 0.1 % Tween 20, and incubated for 1 h with primary antibodies. After rinsing the membrane to remove unbound primary antibody, the membrane was incubated with a secondary antibody for 1 h and rinsed. Proteins were detected by ECL chemiluminescence and bands were quantified using ImageJ densitometry software. The values obtained from densitometric analysis were used to make relative comparisons of band intensity between lanes. Clonogenic assay Cells were treated with 10–25 μM of PRIMA-1 or vehicle control (DMSO) for 48 h, harvested, and then plated in triplicate culture dishes. The cells were then cultured in drug-free medium for 10–14 days until colony counts had stabilized. Colonies were counted after fixation with 70 % ethanol and staining with 0.5 % crystal violet (Sigma, USA), and the number of colonies in each drugtreatment group was expressed as a percentage of the number of colonies in vehicle-treated control dishes. Only colonies containing 50 or more cells were scored. Statistical analysis Values were expressed as mean ± standard error of the mean (SEM) number of cells. The mean values for

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control cells were taken as 100 % in MTT analyses. Drug interaction was assessed at different concentration ratios using the combination index (CI) [20, 21], where CI1 indicate synergistic, additive and antagonistic effects, respectively. Data were treated with one-way analysis of variance (ANOVA) and non-parametric tests if needed. Values of p