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Ann Thorac Surg 2005;80:409-417
© 2005 The Society of Thoracic Surgeons
Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York
Accepted for publication January 10, 2005.
* Address reprint requests to Dr Fong, Department of Surgery, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021 (Email: fongy{at}mskcc.org).
Presented at the Fortieth Annual Meeting of The Society of Thoracic Surgeons, San Antonio, TX, Jan 26–28, 2004.
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Abstract |
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134.5 gene preferentially replicate in and kill malignant cells. The 134.5 gene codes for ICP 34.5, a protein that enhances viral replication, and is homologous to growth arrest and DNA damage protein 34 (GADD34), a radiation-inducible DNA repair gene. We hypothesized that radiation therapy may potentiate efficacy of oncolytic viral therapy by upregulating GADD34 and promoting viral replication. METHODS: The A549 and H1299 lung cancer cell lines were infected with NV1066, an oncolytic herpes simplex virus, at multiplicities of infection (number of viral particles per tumor cell) of 0.1 to 0.5 in vitro with radiation (2 to 10 Gy) or without radiation. Viral replication was determined by plaque assay, cell-to-cell spread was determined by flow cytometry, cell kill was determined by lactate dehydrogenase assay, and GADD34 induction was determined by real-time reverse transcription–polymerase chain reaction and Western blot method. Evidence of synergistic cytotoxicity dependence with GADD34 induction is further confirmed by small inhibitory RNA inhibition of GADD34 expression.
RESULTS: Using both the isobologram method and combination index method of Chou and Talalay, significant synergism was demonstrated between radiation therapy and NV1066 both in vitro and in vivo. As a result of such synergism, a dose reduction for each agent (2- to 6,000-fold) can be accomplished for a wide range of therapeutic effect levels without sacrificing tumor cell kill. This effect is correlated with increased GADD34 expression and inhibited by transfection of small inhibitory RNA directed against GADD34.
CONCLUSIONS: These data provide the cellular basis for the clinical investigation of combined use of radiation therapy with oncolytic herpes simplex virus therapy in the treatment of lung cancer to achieve synergistic efficacy while minimizing dosage and toxicity.
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Introduction |
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The Thoracic Surgery Directors Association (TSDA) Resident Research Award, sponsored by Medtronic, Inc, was established in 1990 to encourage resident research in cardiothoracic surgery. Abstracts submitted to The Society of Thoracic Surgeons (STS) Program Committee representing research performed by residents were forwarded to the TSDA to be considered for this award. The abstracts were selected by the TSDA Executive Committee consisting of Jeffrey P. Gold, MD, President, John Brown, MD, President-Elect, John H. Calhoon, MD, Secretary/Treasurer, Douglas J. Mathisen, MD, Immediate Past President, Bartley P. Griffith, MD, Councillor-at-Large, George L. Hicks, MD, Councillor-at-Large, and Leslie Kohman, MD, Councillor-at-Large. The Fourteenth Annual TSDA Resident Research Award was given to Prasad S. Adusumilli, a resident at the Memorial Sloan Kettering Cancer Center in New York, New York. He received a monetary award of $2,500 and an engraved desktop award. The TSDA, with support by Medtronic, Inc, makes this award annually, using the above selection procedure. The resident author of the selected study is recognized at the STS meeting.
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Lung cancer is the leading cause of cancer deaths in the United States [1]. An estimated 173,770 new patients were expected to be diagnosed with lung cancer in the year 2004, one third with advanced disease that is unresectable [1]. All patients with unresectable lung cancer die of this malignancy despite current therapies. Therefore, ongoing investigation is directed at finding effective novel therapies for this common cancer. Currently, radiation therapy (RT) plays an important role in the treatment of inoperable non–small-cell lung cancer in achieving local control and in the relief of symptoms of metastatic disease [2]. However, higher doses of radiation needed to achieve local control [3, 4] are associated with significant toxicity [5]. Thus, therapies are also sought that may synergize with RT to increase the tumor response and to decrease toxicities.
Herpes simplex virus (HSV)-mediated oncolysis and gene therapy have emerged as promising treatment modalities against cancer [6–11]. Oncolysis results from the replicative life cycle of the virus, which lyses infected tumor cells and releases viral progeny for further infection and killing of neighboring cancer cells. NV1066 is one such multimutated replication-competent oncolytic HSV-1 virus, which carries a gene for the marker protein enhanced green fluorescent protein (EGFP). We have previously demonstrated the efficacy of NV1066 and its predecessor NV1020 in the treatment of lung cancer both in vitro and in vivo [12, 13]. NV1066 has deletions from the internal repeat sequences, which result in deletion of one copy of each of the genes for ICP0, ICP34.5, and ICP4 [14, 15]. These deletions attenuate the virus and help to ensure that it preferentially replicates within cancer cells. One of these genes, HSV-1 134.5, which codes for ICP34.5, has significant homology at its carboxy-terminus to a radiation-inducible gene, GADD34 (growth arrest and DNA damage repair gene) [16–19]. Therefore, we hypothesized that radiation-induced upregulation of GADD34 in tumor cells would functionally complement the 134.5 deletions in NV1066 and increase viral tumoricidal activity.
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Material and Methods |
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Viruses
NV1066 is a replication-competent attenuated HSV-1 oncolytic virus with deletion of a single copy of the viral gene 134.5. G207 and NV3616 are replication-competent attenuated HSV-1 mutant viruses that have been characterized previously [19–21]. G207 is attenuated by deletion of a 1-kilobase coding sequence from both 134.5 loci and a deletion of ICP6, a gene coding for the enzyme ribonucleotide reductase. R3616 contains the same mutation in the 134.5 loci, but the ICP6 gene is intact, preserving ribonucleotide reductase function. Viral stocks were propagated on Vero cells, harvested by freeze-thaw lysis and sonication, and titered by standard plaque assay.
In Vitro Cytotoxicity Assay
A549 and H1299 cells were plated in 24-well flat-bottom plates (Becton Dickinson, Franklin lake, NJ) in 1 mL of media. Cells were treated with media alone (control wells), radiation alone (137Cs source irradiator, 224 cGy/min), NV1066 alone, or combination therapy using both RT and NV1066. NV1066 infection was carried out at multiplicities of infection (MOI: number of viral plaque forming units per tumor cell) of 0.1, 0.2, 0.3, 0.4, or 0.5 in a total volume of 100 µL of medium. Combination therapy was performed using serial dilutions of RT (2, 4, 6, 8, and 10 Gy) and NV1066 (MOI 0.1, 0.2, 0.3, 0.4, and 0.5) in a 1:20 ratio for both cell lines. These ratios were determined by estimating the dose necessary to kill 50% of the cells for each therapy in initial experiments and by using these doses to determine the ratio of combination therapy. Typically cells were plated overnight, radiated in the morning, and infected with virus within 2 hours of radiation. Percent survival for each group was determined on each day for 7 days after treatment using a standard lactate dehydrogenase release bioassay. Results were expressed as surviving fraction, from the measured absorbance of treated cellular lysates, compared with that of untreated, control cellular lysates. All samples were tested in triplicate. Experiments were repeated at least three times to ensure reproducibility. Cytotoxicity assays were also performed with A549 and H1299 cells using G207 or NV3616 virus with and without radiation.
Quantitative Analysis of Synergy Between Radiotherapy and NV1066
The combination effects of two therapies in terms of synergy, additivity, or antagonism were analyzed by the median effect plot using the multiple therapeutic effect analysis of Chou and Talalay [22]. This method defines the expected additive effect of two (or more) agents and then quantifies synergism or antagonism by determining how much the combination effect differs from the expected additive effect. Data were also analyzed by the isobologram technique, which is dose oriented. Both the median plot effect and isobologram method are described in detail elsewhere [23]. Another calculation available using the combination index method is the dose-reduction index (DRI). The DRI is a determination of the fold of dose reduction allowed for each drug when given in synergistic combination as compared with the concentration of single agent that is needed to achieve the same effect level. A DRI greater than 1 signifies a favorable reduction in toxicity while still maintaining therapeutic efficacy.
In Vitro Viral Growth Analysis
The ability of NV1066 to replicate within A549 and H1299 cells in the presence or absence of radiation was evaluated by viral growth analysis, with 5 x 104 cells per well plated into six-well plates. Cells were then infected with either NV1066 (MOI 0.01, 0.05, or 0.1) alone, or with NV1066 and RT (2 Gy). Cells and media were harvested at 48, 72, 96, 120, and 144 hours after infection. After three cycles of freeze-thaw lysis, standard plaque assay was performed on Vero cells to evaluate viral titers. All samples were performed in triplicate.
Vector Spread Assay
Vector propagation as analyzed by EGFP expression was determined by fluorescence-activated cell-sorting analysis at a viral infective dose of MOI 0.1 and 0.5 after 0, 2.5, 5, or 10 Gy of radiation. Percentage of EGFP-positive live cells at 12, 24, 48, 72, 96, and 108 hours after radiation compared with control cells without radiation was plotted to derive the EGFP-expression trend. Cells were harvested with 0.25% trypsin in 0.02% EDTA, centrifuged, washed in phosphate-buffered saline solution, and brought up in 100 µL of phosphate-buffered saline solution. Five microliters of 7-amino-actinomycin D (BD Pharmingen, San Diego, CA) was added as an exclusion dye for cell viability. Data for EGFP expression were acquired on a FACS Calibur machine equipped with Cell Quest software (Becton Dickinson, San Jose, CA). Results are reported as percent of live cells expressing EGFP. All samples were performed in triplicate.
Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis for GADD34 in Cells Treated With Radiation
Samples containing 1 x 105 A549 and H1299 cells per well were plated in six-well plates and were incubated for 12 hours. Cells were treated with a radiation dose of 5 Gy. Each sample was performed in triplicate. After 48, 72, and 96 hours of incubation, the cells from each well of the plate were collected after washing with cold phosphate-buffered saline solution and frozen for RNA collection. The RNA from each sample was collected and isolated with an RNeasy protect kit (QIAGEN Inc, Valencia, CA) using the manufacturer’s protocol. GADD34 in each sample is measured quantitatively by real-time reverse transcription–polymerase chain reaction using an SYBR green fluorophore with a Bio-Rad iCycler iQ detection system (Bio-Rad Laboratories, Hercules, CA) and normalized by corresponding 18S ribosomal RNA. For GADD34, the following primers were applied: GADD34 forward, 5'-GGA GGA AGA GAA TCA AGC CA-3'; GADD34 reverse, 5'-TGG GGT CGG AGC CTG AAG AT-3'. For 18S RNA: 18S forward, 5'-GTA ACC CGT TGA ACC CCA TT-3'; 18S reverse, 5'-CCA TCC AAT CGG TAG TAG CG-3'. A comparison between each treatment sample and the control group, which did not receive any radiation, is made to determine GADD34 upregulation. For ease of interpretation, semilogarithmic plots of actual GADD34 values were omitted, and the results were represented as fold increase in the treatment sample compared with the control group.
GADD34 Small Inhibitory RNA Transfection
Duplex small inhibitory RNAs (siRNAs) targeting human GADD34 outside the viral homology domain were designed and tested for the ability to decrease GADD34 expression. After preliminary experiments, the following sequence targeting from codon 635 was chosen for further experiments: 5-GUCAAUUUGCAGAUGGCCATTUGGCCAUCUGCAAAUUGACTT-3. A549 and H1299 cells were plated at a concentration of 5 x 104 per well in 24-well plates 12 hours before transfection in appropriate medium without antibiotics. Standard siRNA transfection protocol as described before was used [24]. Cells in the wells that were exposed to only Oligofectamine transfection reagent (Invitrogen, Carlsbad, CA) were used as controls.
Western Blot
A549 cells (nontransfected and GADD34 siRNA-transfected) were incubated overnight and radiated in the morning with either 5 or 10 Gy. Cells with no radiation served as controls. Cells were lysed and collected with Cell Lysis Buffer (Cell Signaling Technology, Inc, Beverly, MD). Equal amounts of proteins were resolved on 10% sodium dodecylsulfate–polyacrylamide gels (Bio-Rad Laboratories) under reducing conditions and blotted on polyvinylidene difluoride membrane (Schleicher & Schuell Bioscience, Keene, NH). Expression of proteins was determined by using primary rabbit polyclonal anti-human GADD34 (Santa Cruz Biotechnology, Santa Cruz, CA) and primary goat polyclonal anti-human actin (Santa Cruz Biotechnology). A secondary antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) was used to visualize the expression level of GADD34 and actin on chemiluminescence film (Hyperfilm; Amersham Biosciences, Buckinghamshire, England) by application of an ECL Plus Western Blotting Detection System (Amersham Biosciences).
Establishment and Treatment of Flank Tumors
Athymic male mice were purchased from the National Cancer Institute (Bethesda, MD) and were provided with food and water ad libitum. All animals received humane care in accordance with the "Guide for the Care and Use of Laboratory Animals (NIH)," and the animal protocols were approved by the animal care committee of the institution. Mice were anesthetized with a mixture of 70 mg/kg ketamine and 10 mg/kg xylazine administered intraperitoneally. H1299 flank tumor establishment and tumor measurements were conducted by the antitumor division of Sloan-Kettering Cancer Institute core facility who were blinded to the treatment arms. Mice were examined daily until tumor nodules reached approximately 250 mm3 volume, at which time they were randomized into four groups (n = 6 per group): (1) untreated controls, (2) 5 Gy RT alone, (3) single intratumoral injection of 1 x 107 plaque-forming units (pfu) NV1066 alone, and (4) 5 Gy RT followed by a single intratumoral injection of 1 x 107 pfu NV1066 (24 hours later). Mice were shielded with lead when flank tumors were exposed to external beam radiation. The length and width of the tumors were measured every 3 days for 32 days. Tumor volume was calculated by the formula for an ellipsoid volume, [(4/3) x (
) x (length/2) x (width/2)2]. Animals were sacrificed if the greatest tumor dimension exceeded 4 cm or if there was skin ulceration.
Statistical Analysis
All data were expressed as mean ± standard error of the mean. Comparisons among groups were made using the two-tailed Student’s t test. The analysis of variance test, when appropriate, was used to identify statistical significance for multiple comparisons.
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Results |
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134.5 deletion HSV mutants G207, and NV3616 in A549 and H1299 cell lines (data not shown).
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Vector Spread After NV1066 Infection and Radiation
Radiation increased the expression of EGFP-positive cells, indicating better viral propagation among both cell lines. In A549 cells infected at an MOI of 0.5, radiation of 5 Gy increased EGFP-positive cells to 37% ± 4%, and 10 Gy increased EGFP-positive cells to 44% ± 5% when compared with cells infected without radiation, which increased only 23% ± 2% (p < 0.001) after 12 hours.
Real-Time Reverse Transcription-Polymerase Chain Reaction Analysis for GADD34
Samples containing RNA extracted from cells that were not treated with radiation served as negative controls, whereas RNA extracted from U937 cells radiated at 20 Gy was used as positive controls in real-time reverse transcription–polymerase chain reaction analysis for GADD34. In A549 cells, 5 Gy of radiation increased GADD34 by 1.12-fold (48 hours), 3.49-fold (72 hours), and 5.04-fold (96 hours; p = 0.02). In H1299 cells, 5 Gy of radiation increased GADD34 by 2.92-fold (48 hours), 2.04-fold (72 hours), and 1.69-fold (96 hours; Fig 2).
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Comment |
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134.5-deficient HSV oncolytic virus. The genotoxic response to DNA damage by radiation results in complex cellular events that direct DNA repair. A series of five growth arrest and DNA damage-inducible (GADD) genes have been identified in mammalian cells in response to ionizing radiation, medium-depletion, and alkylating agents [17, 18]. As noted in other studies [18, 19], the fact that RT results in high expression of GADD34 was confirmed in our study and is central to our strategy of combining radiation therapy with oncolytic viral therapy.
NV1066 is an attenuated multimutant HSV-1 that has demonstrated cytotoxic activity against a wide variety of tumor cell types, including lung cancer. Strategic deletions have been made within the genome of NV1066 to improve the safety of this vector. One such deleted gene, 134.5, is a neurovirulent gene that codes for the protein ICP34.5, which functions to preclude the shutoff of protein synthesis that occurs as part of the host defense response to cellular infection by viruses. Expression of ICP34.5 produces a cellular milieu greatly favoring viral replication. There is great homology between the carboxy terminus of the mammalian GADD34 and ICP34.5 proteins [17]. The mammalian GADD34 gene has also been shown to complement viral 134.5 mutants and to permit viral growth in cultured cells. Our hypothesis that RT and oncolytic viral therapy are synergistic is confirmed by the increased cytotoxicity and the increased viral proliferation seen with combination treatment. The hypothesis that elevated GADD34 is at least partly responsible for such enhanced tumoricidal activity was indeed confirmed by increased GADD34 expression with RT and by GADD34 inhibition experiments using siRNA. Moreover, similar synergistic tumoricidal activity with RT was demonstrated for G207 and NV3616, two other viruses with 134.5 deletions.
Recent attempts have been made to restore the 134.5 gene into viral mutants as deletion of this gene markedly reduces cytotoxicity [25, 26]. In particular, investigators have attempted to insert the 134.5 genes under control of a transcriptionally regulated promoter to facilitate selective gene expression in rapidly dividing cells. Although this approach is promising, insertion of the entire 134.5 gene may theoretically restore neurovirulence and virulence in other nonmalignant cells in addition to providing any improvements in tumor cell kill. Our approach of inducing GADD34 function to replace the ICP34.5 function may provide a superior safety profile. Using RT in this strategy provides a selective means of restoring the GADD34 phenotype in tumor cells without the potential risk of increasing neurovirulence or enhancing viral replication in nonmalignant cells. Furthermore, such combination therapy may prove efficacious for tumor cells that may be resistant to either virus or radiotherapy alone.
Synergy was examined in this study using the combination index and isobologram methods of Chou and Talalay [27, 28]. This type of analysis is one of the few methods available that determines synergy on the basis of an extrapolated equation. The possibility of predicting false-positive synergistic interactions, a problem inherent in many other methods, is minimized as the analysis takes into account both the potency and shapes of the dose–effect curves in precisely analyzing two therapeutic combinations. Synergistic therapeutic efficacy was demonstrated using two different cell lines with varying sensitivities to virus and radiotherapy. From these data, a significant DRI was also demonstrated. Ultimately, the DRI is the most important variable in determining the clinical applicability of combination therapy because potential toxicity can be reduced without sacrificing any therapeutic effect.
Higher doses of radiation have been to shown to achieve better local control in advanced lung cancer at the cost of enhanced toxicity. Enhanced acute toxicities can lead to treatment interruptions, delays, or alterations in the schedule of radiation delivery, resulting in reduction in total dose, thereby potentially compromising the effectiveness of the definitive treatment modality. Dose reduction achieved because of synergistic cytotoxicity may reduce treatment-related toxicities. Studies of malignant glioma in mice have demonstrated that radiation increases viral persistence, furthering the idea that the two therapies can be considered interactive [21, 26]. Our studies confirmed, in a very different tumor type, namely lung cancer, that a significantly greater reduction in volume of tumors can be achieved with such combined radiation and viral therapy. The significantly enhanced oncolytic effects of the combined treatment correlate with enhanced replication of virus in irradiated tumor cells.
In our study, the synergistic efficacy of radiation therapy with oncolytic viral therapy is demonstrated in both p53-positive and p53-negative cell lines. Previous studies have demonstrated in many cancer cell lines that expression of GADD34 is independent of p53 status of the tumor [16, 29, 30]. Therefore, the synergistic efficacy with radiotherapy and oncolytic HSV therapy is independent of p53 status of the tumor, an important limitation in other novel therapies for lung cancer.
The data from these studies provide a cellular basis for the synergistic actions of RT and oncolytic viral therapy. These data are strong support for future clinical investigation of such combined therapy for lung cancer that aim to increase efficacy while minimizing toxicity.
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Discussion |
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DR ADUSUMILLI: Thank you very much, Dr Roth, for your comments and question. Our laboratory has in fact performed biodistribution studies in a colorectal cancer liver metastasis animal model using radiolabeled herpes simplex virus. In naïve mice, excellent viral uptake is demonstrated selectively by tumor cells irrespective of the route of administration. In mice with prior immunity (induced by prior administration of wild-type virus), no difference in viral uptake is noted when the virus is administered regionally. Preimmunity resulted in decreased viral uptake after systemic administration. This might be overcome with combination therapies such as in the current study, in which we have demonstrated that radiation therapy enhances viral replication by several-fold even after a very low dose of viral infectivity. In addition, oncolytic virus can be administered regionally in thoracic malignancies as demonstrated in this presentation and in several other animal models from our laboratory. In human beings, virus can be administered via bronchoscopy to centrally located tumors, by computed tomographic-guided needle injection to peripheral tumors, and by direct instillation into the pleural cavity in patients with disseminated lung cancer or mesothelioma.
DR THOMAS A. D’AMICO (Durham, NC): Prasad, are there other ways to stimulate the induction of GADD34, or is your strategy really limited to locally advanced lung cancer with no mechanism to ameliorate the systemic disease?
DR ADUSUMILLI: Thank you Dr D’Amico. GADD34, growth arrest and DNA damage repair gene, is shown to be upregulated in cancer cells by several other cellular stress response causative agents such as chemotherapeutic agents in addition to radiation therapy. We have shown from our laboratory that GADD34 induction by chemotherapeutic agents such as gemcitabine, mitomycin C, and cisplatin results in synergistic anticancer activity by oncolytic herpes viruses in the treatment of lung cancer, mesothelioma, gastric, and transitional cell bladder cancer. We anticipate that such clinically relevant combination therapies can complement current adjuvant modalities in the treatment of advanced thoracic malignancies.
DR THOMAS K. WADDELL (Toronto, Ontario, Canada): Prasad, is it possible to use this phenomenon to target the viral killing to areas that are treated with radiation? In other words, once the cells are activated, the GADD34 is upregulated, do they simply become viral producers, that then systemically more virus is produced throughout the whole animal, or is the production of virus limited to the local area which has undergone radiation?
Dr ADUSUMILLI: Thank you Dr Waddell. GADD34 upregulation enhances viral replication in the irradiated cells. The cell-to-cell spread is mostly by direct contact. The virus used in this study, NV1066, carries a transgene for enhanced green fluorescent protein (EGFP). As a result, the infected cells emit green fluorescence, which can be detected and quantified by flow cytometry. In this study we also conducted viral propagation studies by measuring the percentage of EGFP-expressing cancer cells after viral infection with and without radiation. These studies have shown enhanced viral propagation in cells that are radiated and infected. In other animal experiments when the virus injected either systemically or locally into the tumor, after viral replication, there is minimal persistence or propagation of virus in the systemic circulation as the virus does not infect or replicate in normal cells. This is confirmed by other investigators such as Advani et al. In the malignant glioma flank tumor animal model, they have shown by immunohistochemistry that enhanced viral presence is mostly confined to the areas adjacent to the needle track of injection after radiation, which confirms the fact that the viral propagation is mainly local rather than systemic.
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Acknowledgments |
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