- Open Access
AduPARE1A and gemcitabine combined treatment trigger synergistic antitumor effects in pancreatic cancer through NF-κB mediated uPAR activation
- Maria Victoria Maliandi†1, 2,
- Ana Mato-Berciano†1, 2,
- Luciano Sobrevals1, 2,
- Gaël Roué1,
- Anabel José1, 2 and
- Cristina Fillat1, 2Email author
© Maliandi et al. 2015
Received: 4 February 2015
Accepted: 15 July 2015
Published: 31 July 2015
Combined treatment of oncolytic adenoviruses with chemotherapeutic agents is foreseen as a therapeutic option for cancer. Here we have investigated the potential to use gemcitabine in combination with the oncolytic adenovirus AduPARE1A to treat pancreatic cancer and evaluate the underlying mechanism.
We treated pancreatic cancer cell lines BxPC-3 and PANC-1 with AduPARE1A and gemcitabine individually or in combination and analyzed cell viability, combination index, apoptosis and viral production. We also investigated the effects of the combination on tumor growth and mice survival in two xenograft models. Furthermore, we analyzed uPAR promoter activity from different uPAR-controlled adenovirus and studied NF-κB mediated effects.
Synergistic cell killing from the combination AduPARE1A/Gemcitabine was observed in BxPC-3 and PANC-1 cells. Moreover, the combination treatment produced therapeutic benefits over either individual modality in two mouse models bearing orthotopic tumors, showing reduced tumor progression and significant prolonged mouse survival. Mechanistic studies showed that the synergistic cell death was not due to an increase in viral replication but occurred through an enhancement of apoptotic cell death. Gemcitabine stimulation increased the transcription of uPAR-controlled transgenes through the induction of NF-κB acting on the uPAR promoter. Interestingly, NF-κB gemcitabine-mediated induction of AduPAR adenoviruses interfered with the activation of NF-κB regulated genes, probably as a result of an intracellular competition for NF-κB DNA binding. Consequently, AduPARE1A infection sensitized cells to gemcitabine-induced apoptosis in the combined treatment.
These data highlights the potential of the combination as a treatment modality for pancreatic cancer patients.
Pancreatic cancer is a particularly devastating disease, ranked as the fourth leading cause of cancer-related death and a 5-year survival rate of only 6 % .
Replication-selective oncolytic adenoviruses are designed to replicate and spread into the tumor, resulting in cancer cell lysis. Oncolytic mutants are being evaluated in the clinic and emerge as a novel approach to treat cancer. Clinical trials in pancreatic cancer patients with ONYX-015, a p53 binding defective oncolytic adenovirus, proved that the administration was safe, although clinical efficacy was not observed when administered alone and only minor responses and stable disease were observed in combination with gemcitabine .
We previously developed an oncolytic adenovirus that drives the E1A gene under the control of the urokinase-type plasminogen activator receptor (uPAR) promoter, designated AduPARE1A, and showed its selective replication and its strong antitumor activity in pancreatic cancer models [3, 4]. Despite its anticancer activity demonstrated in the preclinical studies as a monotherapy, multimodal strategies to enhance antitumor efficacy are essential for successful clinical outcome.
Gemcitabine has been the standard chemotherapy treatment in patients with pancreatic cancer since 1997 with modest improvements in median overall survival . More recently, a 2-months increase in overall survival has been observed in the combination treatment of gemcitabine and nab-paclitaxel . Thus most likely patients that will enter clinical trials with oncolytic viruses are expected to be receiving gemcitabine. Combination treatment of oncolytic mutants and gemcitabine has already been explored for given viruses and improved anticancer effects have been reported [7–10]. However, the mechanism of action supporting the benefits of the combination have been quite different in line with the individual characteristics that every oncolytic adenovirus possesses. In fact, a major concern on the combination therapy is that the combination may impede the therapeutic function of each other.
In the current work we have examined the effects of the combination treatment AduPARE1A and gemcitabine, in pancreatic cancer in vitro and in vivo. We have observed a synergistic anticancer effect in vitro and a significant potentiation of tumor growth suppression in xenograft models. Mechanistic studies identified a NF-κB uPAR promoter activation mediated by gemcitabine induction. As a consequence there was increased sensitization to gemcitabine-induced apoptosis in the combined treatment.
Combination therapy of gemcitabine and AduPARE1A show synergistic effects
Pharmacological interaction between the above-mentioned treatments was assessed by Combination Index analysis (CI) in both PANC-1 and BxPC-3 cells. CI was calculated for 20–80 % fractional inhibition. In Bx-PC-3, at all the fractions analyzed, the CI was lower than one, indicating a potentiation effect of AduPARE1A when combined with gemcitabine, and vice versa, highlighting that both treatments were synergistic. In PANC-1 cells synergism started to show at 40 % of the inhibitory fraction (Fig. 1b).
In vivo multimodal gemcitabine and AduPARE1A therapy significantly improved mouse survival over either individual therapy in animals bearing orthotopic BxPC-3 and PANC-1 tumors
AduPARE1A and gemcitabine combined treatment decrease viral replication but increase apoptotic cell death
Gemcitabine induces the activity of uPAR-controlled adenoviruses
To evaluate whether the increase in E1A expression was the consequence of uPAR promoter activation mediated by gemcitabine stimuli, we decide to analyze the effects of gemcitabine on the expression of different transgenes also controlled by the uPAR promoter. BxcPC-3, PANC-1 and MIAPaCa-2 cells were transduced with AduPARTKT, a non-replicative adenovirus that expresses a modified form of the Herpes simplex thymidine kinase gene under the control of the uPAR promoter, in the presence or not of gemcitabine. Analysis of TK expression revealed increased levels of TK mRNA in the presence of gemcitabine (Fig. 4c). Cell transduction with AduPARLuc, a reporter adenovirus that contains the luciferase expression regulated by the uPAR promoter, showed that luciferase activity was significantly higher in cells that received AduPARLuc and gemcitabine (Fig. 4d). Furthermore, gemcitabine treatment also induced endogenous uPAR mRNA overexpression (Fig. 4e).
These findings indicate that gemcitabine enhances uPAR promoter transcriptional activity.
NF-κB activation mediates gemcitabine uPAR-induction
Collectively, these results suggest that higher amounts of AduPARE1A viral particles will interfere with NF-κB activation, facilitating gemcitabine-induced apoptosis, leading to in vitro synergistic cell death and improve antitumor response of the combination treatment.
Oncolytic adenoviruses are now under evaluation in clinical trials in patients with pancreatic cancer [15–17]. These agents are attractive treatment options either alone or in combination with traditional cytotoxic drugs. Previous work from our group has shown the antitumor efficacy and tumor selectivity of AduPARE1A [3, 4]. The current study aimed to determine the efficacy to combine AduPARE1A and gemcitabine commonly used to treat pancreatic cancer. We found a synergistic cell killing effect in the combination treatment. In vivo, in two orthotopic xenograft models, combined treatment prolonged mouse survival compared to either therapy alone providing evidence of the efficacy of AduPARE1A plus gemcitabine for pancreatic cancer. Furthermore, we provide evidences on the mechanisms of such synergistic interaction.
The increased cell death in the combined treatment was not due to an improvement in viral replication, on the contrary, replication was attenuated by the presence of gemcitabine. The effect was more pronounced in PANC-1 cells, where much higher doses of gemcitabine were used. In fact, viral attenuation by DNA damaging agents has also been observed in the E1B19K oncolytic adenoviral mutants  and in replication-selective herpes mutants targeting pancreatic cancer cells . The G1/S cell-cycle block triggered by gemcitabine limits DNA cellular synthesis, hampering to support viral propagation .
Interestingly, the increased cell death in the combination treatment could be explained by an enhancement of gemcitabine-induced apoptosis since we observed an augmented activity of caspase-3. Several authors have postulated that increased E1A expression might be responsible for most of the synergistic effects in combination treatments of oncolytic adenovirus and DNA damaging agents, since E1A is a potent apoptotic inducer [8, 10, 19, 20]. The pro-apoptotic functions of E1A, have been linked to its capacity to upregulate Egr-1 which is directly involved in Bim transactivation. The alterations of Bim contribute to Bax activation and subsequent caspase activation . We observed an increase in E1A expression upon gemcitabine treatment, suggesting that at least part of the synergism could rely on the pro-apoptotic functions of E1A. However this seems unlikely since AduPARE1A oncolytic adenovirus retains all the viral functions and infected cells also express the anti-apoptotic E1B and E3 viral proteins, what may hamper or at least mitigate E1A activity. In fact, AduPARE1A alone was not able to trigger caspase-3 activation. Thus, other mechanisms might be operating in the AduPARE1A cooperation to gemcitabine-induced apoptosis. It should be considered that E1A has been shown to induce mesenchymal-to epithelial conversion resulting in the formation of gap junctions, what facilitates the cell-to cell transfer of gemcitabine toxic metabolites and the spread of gemcitabine cytotoxicity [22, 23].
We observed that the increase in E1A expression by the combined treatment was the consequence of a transcriptional activation of the uPAR promoter mediated by gemcitabine. Induction of transgene expression was extended to all uPAR-promoter controlled adenoviral vectors analyzed such as the AduPARTKT, increasing TK transcriptional levels and the AduPARLuc, enhancing luciferase activity. Furthermore, gemcitabine induced transcriptional activation of the endogenous uPAR gene. These data highlight that gemcitabine induction of uPAR adenoviruses enhances the biological effects of the controlled transgenes. Such increase in transgene expression could partially account by the gemcitabine enhancement of viral infectivity, as reported by some authors [10, 24]. However, since we observed a gemcitabine-mediated increase in the uPAR promoter transcriptional activity, most likely the increased transgene expression will result from the interaction of different transcription factors to cis acting regulatory sequences in the uPAR gene . One of the key factors regulating the uPAR promoter is NF-κB . NF-κB is constitutively active in pancreatic cancer and gemcitabine is able to induce NF-κB binding activity. In this line, we observed that gemcitabine induced-uPAR promoter activation was completely blocked by the NF-κB inhibitor BAY11-7082, and gemcitabine was unable to activate a uPAR promoter with NF-κB mutagenized binding sites. However, activation of NF-κB has been linked to reduced cytotoxicity and antiapoptotic effects, and it is proposed as a mechanism of gemcitabine resistance . Interestingly, what we observed is that in the combination treatment of AduPARE1A and gemcitabine, at high viral doses, where E1A is induced, AduPARE1A interferes with the activation of NF-κB regulated genes. E1A has been proposed to counteract gemcitabine induced NF-κB activation sensitizing cells to apoptosis , however in our studies the suppression of NF-κB activation would not only account by E1A since the same kind of interference was observed with AduPARTKT. These results support the notion that there will be an intracellular competition for NF-κB DNA binding, favoring AduPAR promoter activation. As a consequence E1A will be highly expressed. It could be speculated that in this scenario endogenous NF-κB- responsive antiapoptotic genes might be suppressed leading to reversal of gemcitabine resistance and enhancement of apoptosis. The proposed mechanism of synergistic activity between AduPARE1A and gemcitabine is a novel mechanism that could also extent to other viruses susceptible to NF-κB regulation. Furthermore, the remarkable in vivo anti-cancer effects could also result from the capacity of oncolytic adenovirus to disrupt tumor architecture thus facilitating gemcitabine penetration .
In summary in the current study we show that the combination of AuPARE1A and gemcitabine has a synergistic cell killing effect that holds potential as a novel therapy for pancreatic cancer treatment. We propose a model of the operating mechanism underlying the synergistic effects, through which gemcitabine will trigger an NF-κB activation to induce uPAR promoter activity, leading to an increase in E1A protein. The NF-κB transcription factor binding to the high number of uPAR sequences in the adenovirus will produce a sponge effect, interfering with NF-κB activation of antiapoptotic genes, thus sensitizing gemcitabine-induced apoptosis.
Human pancreatic tumor cell lines: BxPC-3, PANC-1 and MIAPaCa-2, and 293T cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Luciferase-expressing cells PANC-1-Luc and BxPC-3-Luc were established as previously described [3, 29]. Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10 % fetal bovine serum (FBS), penicillin (100 U/ ml), streptomycin (100 μg/ ml), and glutamine (2 mM) (GIBCO-Invitrogen). Every 2 months cells were plated from a frozen vial of the original batch but were not authenticated by the authors. Interspecies contamination was tested by PCR routinely.
Chemicals and antibodies
Gemcitabine (Hospira UK Limited) was dissolved in water to a concentration of 10 mg/ml. BAY 11-7082 (Calbiochem, La Jolla, CA) was dissolved in dimethyl sulfoxide, stored at −20 °C, and protected from light. The following antibodies were used in western blot analysis: Rabbit anti-Cleaved Caspase-3 (Asp175) (Ref.9661, Cell Signaling Technology, MA, USA), Rabbit anti-PARP (Cat. No.11835238001, Roche), Rabbit anti-GAPDH (Ref.ABS16, Merck Millipore, Germany), HRP-conjugated Goat anti-Rabbit (DakoCytomation, Denmark).
Replication-defective adenoviruses AduPARLuc express the firefly luciferase gene under the control of uPAR promoter. This virus has already been described . The oncolytic AduPARE1A was constructed by inserting the uPAR promoter (450-bp fragment) upstream the E1A adenoviral gene . AduPARTKT encodes a modified Herpes Simplex virus thymidine kinase gene, as described . Experiments with AduPARE1A and AduPARTKT were conducted with CsCl purified viral preparations and results are expressed as vp/ml. AduPARLuc experiments were performed with homogenates from viral infected cells subjected to three rounds of freeze-thaw lysis and viral doses are expressed as MOI.
A 450-bp fragment of the uPAR promoter ( −402/+48 region) and the luciferase gene present in the pAd-TrackuPARLuc  were excised and cloned into a pGEM-T vector (puPARLuc). The reporter plasmid puPARLuc-NF-κBmut was generated by directed mutagenesis. The NF-kB binding site in the uPAR promoter (located at −45 bp of transcription initiation site) 5'-GGGAGGAGT-3' , was mutated to 5'-GGATCCAGT-3', using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent, CA USA), as recommended by the manufacturer and using the mutagenesis primer: 5'-ctctttcgcaaaacgtctggatccagtccctggggccacaaaac-3'. puPARE1A was generated by cloning the uPARE1A construct in a pGEM-T vector. The NF-κBmutation in the puPARE1A to generate puPARE1A-NF-κBmut was carried out by directed mutagenesis as described above. 3xκB-Luc reporter plasmid was previously described .
Reporter gene assays
With Adenoviruses: Cells were transduced with AduPARLuc, 4 h later virus was removed and replaced with fresh medium containing or not BAY 11-7082 (Calbiochem) for 1 h and then exposed to gemcitabine for 24 h. Luciferase activity was measured in a luminometer (Synergy HT, Biotek) with luciferin as a substrate (Promega, Madison,USA). Results are expressed as light units (LU) and normalized to total protein levels.
With Reporter plasmids: 293T cells were seeded (40.000 cells/well) and co-transfected with luciferase reporter plasmids (150 ng/well): 3xκB-Luc, puPARLuc or puPARLuc-NF-κBmut or puPARE1A or puPARE1A-NF-κBmut and the β-gal reporter plasmid (30 ng/well). CalPhos mammalian transfection kit (Clontech, Takara Bio Company Inc.) was used to transfection, following the manufacturer guidelines. At 16 h post-transfection the medium was change and new fresh medium or medium containing 200 ng/ml of Gemcitabine was added. Twenty-four hours later, cell lysates were analyzed for luciferase activity using Luciferase Assay System (Promega), and β-galactosidase activity was used as a control of transfection efficiency.
Dose–response analysis and combination index analysis
BxPC-3 and PANC-1 cell lines were seeded in 96-well plates at a density of 3 × 103 cells per well. Cells were treated with different doses of gemcitabine (ng/mL), AduPARE1A (vp/cell) or the combination of both treatments, maintaining a constant ratio of gemcitabine:adenovirus of 1:800 or 1250:1 in BxPC-3 and PANC-1 respectively. Cell viability was measured 3 days later, using the MTT colorimetric assay (Ref.19265 USB, Affymetrix, CA USA). The induction of synergism, summation or antagonism between gemcitabine and AduPARE1A treatments was analyzed by Combination Index analysis, by the adapted method of Chou and Talay [32, 33]. Dose–response curves were obtained for combined or single treatments by a standard non-linear regression using GraphPad Prism Software (CA, USA), and IC50 and combination index values were calculated using Calcusyn Software (Biosoft, Cambridge UK). CI >1 indicates synergism, CI = 1 additivity and CI <1 indicates antagonism between treatments.
Quantitative RT-PCR analysis of gene expression
Total RNA was extracted from different cells lines using the RNeasy® Mini Kit (Qiagen). The cDNA was synthesized from total RNA using a reverse transcriptase and random hexamers (RETROscript® Kit, Ambion), according to the manufacturer’s instructions. The PCR reactions were performed in a total volume of 10 μl containing SYBR Green I Master Mix (Roche) with 10 μM of each primer and 1 μl cDNA. Quantitative PCR was performed in triplicate with the ViiA™ 7 System (Applied Biosystems). Primers sequences for the different genes are listed in Additional file 3: Table S1.
Western blot analysis
Cell lines were treated for 48 h with gemcitabine or/and infected with AduPARE1A. After treatment, floating and adherent cells were collected in lysis buffer (62.5 mM Tris HCl pH6.8, 2 % Sodium Dodecyl Sulfate and 10 % Glicerol) containing 1 % Complete Mini-Protease Inhibitor Cocktail (Roche Diagnosis, Switzerland), and 5 mM NaF. Cell lysates were boiled for 10 min at 98 °C. All protein extracts were quantified using BCA kit (Thermo scientific, MA USA) and 80 μg of protein was resolved in 12 % SDS-PAGE and transferred to PVDF or nitrocellulose membrane (Merk Millipore, Germany). Membranes were immunobloted with anti-cleaved caspase-3 (1/200, overnight at 4 °C) or anti-PARP (1/1000, 2 h at room temperature) and anti GAPDH (1/3000, 1 h at room temperature), rinsed with TBS-Tween and then incubated with HRP-conjugated goat anti rabbit (1/2000, 1 h at room temperature). Antibody labeling was detected by Enhanced Chemoluminiscence Kit (Amersham, GE Healthcare, Switzerland).
Viral genomes quantification
BxPC-3 and PANC-1 cells were infected with 4000 vp/cell and 200 vp/cell, respectively. Culture medium was removed at 4 h after infection and cells were treated, or not with 50 ng/ml and 500 μg/ml of gemcitabine (in BxPC-3 and PANC-1 respectively). Viral DNA was obtained from cell and supernatant (lysed by three freeze and thaw cycles) at 4 and 72 h after infection, using the UltraClean BloodSpin DNA Isolation Kit (Mo Bio Laboratories, CA USA) according to manufacturer’s instructions. Viral genomes were determined by Real Time PCR using SYBR Green I Master mix (Roche Diagnostics, Switzerland) and Hexon primers (described in Additional file 3: Table S1). The adenovirus copy number was interpolated in a standard curve, consisting of adenoviral DNA dilutions in a background of genomic DNA.
Bioluminescence assay and quantification
Animals were anesthetized with a mixture of isofluorane and oxygen preparation and the substrate d-Firefly-Luciferin (Xenogen, Alameda, CA) was administered i.p (32 mg/kg). Luciferase activity was visualized and quantified using an in vivo bioluminescent system (IVIS50; Xenogen) and Living Image 2.20.1 Software overlay on Igor Pro4.06A software (Wavematrics, Seattle, WA) was used, as previously described . Luciferase activity was quantified from non-saturated images, measuring the total amount of emitted light recorded by the CCD camera.
Male athymic nu/nu mice (6–8 weeks old, Harlan Iberica) were used to generate human orthotopic pancreatic tumors. Animal procedures met the guidelines of European Community Directive 86/609/EEC and were approved by the Local Ethical Committee.
Orthotopic human pancreatic cancer xenografts were generated as previously described . Briefly, 5 × 105 PANC-1-Luc and BxPC-3-Luc cells were injected into the pancreas of 8-week-old male athymic nude mice, in a final volume of 50 μl. When tumors reached 106–107 photons/s measured by in vivo bioluminescence, which corresponds to approximately 100 mm3, animals were randomly divided into four groups: Saline, GE, AduPARE1A and AduPARE1A + GE, and were i.v injected with AduPARE1A 5 × 1010 vp or PBS and or gemcitabine 160 mg/Kg or PBS following the protocol described in Fig. 2a. Tumor growth was monitored by bioluminescence analysis. Survival studies were performed and animals were sacrificed according to ethical guidelines.
Results are expressed as mean ± SEM of at least three independent experiments. Statistical differences were determined using Prism (version 5; GraphPad software) and were considered significant for P values less than 0.05. Survival studies were performed to analyze time-to-event probability using the SPSS software. The survival curves (Kaplan–Meier curves) obtained were compared for the different treatments. A log-rank test was used to determine the statistical significance of the differences in time-to-event. A p value of less than 0.05 was considered statistically significant.
We thank Anna Bigas for kindly providing the NF-κB-Luc reporter plasmid. This work was supported by grants from the Spanish Ministry of Economia y Competitividad BIO2011-30299-C02-01/02, BIO2014-57716-C2-2-R and receives partial support from the Generalitat de Catalunya SGR14/248. CIBER de Enfermedades Raras is an initiative of the ISCIII. CF group is partially financed by the Instituto de Salud Carlos III (IIS10/00014) and co-financed by Fondo Europeo de Desarrollo Regional (FEDER). A Mato-Berciano and A. José were recipient of a FPU fellowship from the Spanish Ministry of Education. This work was developed at the Centro Esther Koplowitz, Barcelona, Spain.
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