Advances in adenovirus-mediated p53 cancer gene therapy

Introduction: The tumor suppressor p53 gene regulates diverse cellular processes, such as cell-cycle arrest, senescence, apoptosis and autophagy, and it is frequently inactivated by genetic alterations in ～ 50% of all types of human cancers. To restore wild-type p53 function in p53-inactivated tumors, adenovirus-mediated p53 gene therapy has been developed as a promising antitumor strategy in preclinical experiments and clinical studies.

Areas covered: This review focuses on the clinical relevance of replication-deficient adenovirus vectors that carry the wild-type p53 gene (Ad-p53; Advexin, Gendicine and SCH-58500) in clinical studies of patients with various cancers and the future perspectives regarding conditionally replicating adenovirus vectors expressing the wild-type p53 gene (CRAd-p53; AdDelta24-p53, SG600-p53, OBP-702) in preclinical experiments. Moreover, the recent advances in our understanding of the molecular basis for the p53-mediated tumor suppression network induced by Ad-p53 and CRAd-p53 vectors and the combination therapies for promoting the therapeutic potential of adenovirus-mediated p53 gene therapy are discussed.

Expert opinion: Exploration of the molecular mechanism underlying the p53-mediated tumor suppression network and the effective strategy for enhancing the p53-mediated cell death signaling pathway would provide novel insights into the improvement of clinical outcome in p53-based cancer gene therapy.     

Failure of wild-type p53 gene therapy in human cancer cells expressing a mutant p53 protein.

The introduction of exogenous wild-type p53 into human cancer cells bearing p53 mutation does not necessarily result in inhibition of tumor growth. We have demonstrated this in MDA-MB468 breast cancer cells which are hemizygous for p53 mutation and also in KM12SM colorectal carcinoma cells which are heterozygous for p53 mutation. The wtp53 transfectants decreased three- to four-fold the number of colonies compared with controls. Most wtp53-expressing cells died by apoptosis at early passages, but some cells were able to form colonies and their proliferation rate was similar to control transfectants. This reversion was observed in three of the six MDA-MB-468 clones selected. When MDA-wtp53 transfectants were implanted orthotopically in nude mice only one clone showed prolonged tumor latency. No differences were found in either tumor proliferation or apoptosis in tumors. Integration and expression of exogenous wtp53 was assessed in early and late passages in vitro, and in tumors growing in vivo. Consistently, we found mutations in the exogenous wtp53 gene of MDA-MB468 transfectants. Excision of the exogenous gene was an alternative to abrogate the wtp53 function that was extremely efficient in KM12 cells, although they maintained resistance to geneticin. These results were corroborated by the functional assay in yeast. In conclusion, wtp53 is inactivated in these cancer cells by different mechanisms. The presence of mutated p53 may confer genome instability and mutator ability, which allows cells to escape the effects of the exogenous wtp53 and contributes to the failure of wtp53 gene therapy.     

p53基因与骨肉瘤治疗及其预后的相关性

骨肉瘤的发生是多基因、多步骤、多阶段、多重损伤并存的过程,众多情况下认为是由于细胞核 DNA分子受破坏,细胞不能修复受损的 DNA分子,其继续存在并复制可激活癌基因和(或)抑癌基因异常,发展成为骨肉瘤.在细胞抑癌基因中 p53基因研究最广泛, 50%人类肿瘤与 p53基因突变有关.动物试验证实存在 p53基因突变的转基因鼠中除了淋巴瘤和肺腺癌外,骨肉瘤的发生率最高.p53基因的突变和失活影响着骨肉瘤的发生、发展、预后.随着少数 p53基因治疗进入临床实验阶段,目前 p53的基因治疗研究也出现了一些新的策略.随着分子生物学手段的不断发展和完善,基因治疗的安全性和有效性的提高, p53基因治疗将在骨肉瘤的治疗中体现出不可估量的作用.     

The role of natural killer cells in adenovirus-mediated p53 gene therapy

Adenovirus-mediated gene therapy is a promising new approach for treatment of ovarian cancer. In animal models, complete elimination of cancer cells is often achieved, although the therapeutic gene has not been delivered to all these cells. This is referred to as a bystander effect, because tumor cells near those that receive the therapeutic gene are also eliminated. Several mechanisms have been proposed for the bystander effect, including intercellular communication within the tumor via gap junctions, apoptosis, antiangiogenesis, cytokines or other soluble mediators, and immunological mechanisms. There are two well-documented antitumor effector cell populations in athymic nude mice: macrophages and natural killer (NK) cells. We hypothesize that peritoneal populations of NK cells in nude mice treated with adenoviruses are involved in the observed bystander effect in this in vivo model. We investigated the role of NK cells as immunological mediators for the bystander effect using the p53 tumor suppressor as the therapeutic anticancer gene. Most ovarian cancer cell lines tested were sensitive to lysis by NK cells, although different ovarian cancer cell lines exhibited different sensitivities to NK cell-mediated lysis. To determine the importance of NK cells in the overall efficacy and in the bystander effect of gene therapy, NK cells were depleted in mice by administration of anti-NK1.1 monoclonal antibodies. To study the efficacy of NK depletion, C57BL/6 (nu/nu) mice were given injections i.v. by a single tail vein injection or i.p. with increasing doses of anti-NK1.1 IgG. All doses of anti-NK1.1 antibody, from 100-500 micrograms, essentially eliminated cytotoxic NK activity. To assess the duration of depletion after a single dose of anti-NK1.1 IgG, a time-course experiment was performed. NK 1.1 antibody was effective in completely depleting cytotoxic NK cell activity in the mice for up to 7 days, whether given as 500 micrograms (i.p.) or 200 micrograms (i.v.). Flow cytometric analysis performed on peritoneal cell populations confirmed depletion of NK cells by approximately 80%. Finally, a survival study was performed, in which animals were depleted of NK cells. In this experiment, NK cell-depleted mice were injected with anti-NK1.1 IgG, and control mice were mice were treated with normal saline. Two days later, all mice were inoculated with a lethal i.p. dose of NIH:OVCAR-3 ovarian cancer cells. After 3 days, the mice were divided into two treatment groups; one treatment group received three consecutive daily i.p. injections of Ad-CMV-p53 (SCH58500), and the second treatment group received three consecutive daily i.p. injections of control adenovirus construct, rAd-null. All of the NK cell-depleted animals, whether treated with rAd-null or with Ad-CMV-p53 (SCH58500) were dead of disease by 116 and 138 days, respectively, after initiation of adenovirus treatment, and no statistically significant difference in survival was observed (P = 0.349). A significant survival advantage was seen in control (NK-competent) mice treated with rAd-null (P = 0.04), although all were dead of disease by day 184. Importantly, control NK-competent mice treated with Ad-CMV-p53 (SCH58500) showed no tumor growth or ascites production, and all animals survived. These results indicate that immunological mechanisms involving natural killer cells play an important role in the bystander effect involving adenovirus-p53 gene therapy for ovarian cancer.     

p53 gene therapy of human osteosarcoma using a transferrin-modified cationic liposome

Gene delivery via transferrin receptors, which are highly expressed by cancer cells, can be used to enhance the effectiveness of gene therapy for cancer. In this study, we examined the efficacy of p53 gene therapy in human osteosarcoma (HOSM-1) cells derived from the oral cavity using a cationic liposome supplemented with transferrin. HOSM-1 cells were exposed to transferrin-liposome-p53 in vitro, and the growth inhibition rate, expression of p53 and bax, and induction of apoptosis were measured 48 hours later. Treatment of HOSM-1 cells with transferrin-liposome-p53 resulted in 60.7% growth inhibition. Wild-type p53 expression and an increase in bax expression were observed following transfection with transferrin-liposome-p53, and 20.5% of the treated HOSM-1 cells were apoptotic. In vivo, the HOSM-1 tumor transplanted into nude mice grew to 5 to 6 mm in diameter. Following growth of the tumor to this size, transferrin-liposome-p53 was locally applied to the peripheral tumor (day 0) and then applied once every 5 days for a total of six times. During the administration period, tumor growth did not occur, and the mean tumor volume on the last day of administration (day 25) was 10.0% of that in the saline control group. These results suggest that p53 gene therapy via cationic liposome modification with transferrin is an effective strategy for treatment of osteosarcoma.     

Non-viral gene delivery for p53

Abnormality in the tumor suppressor gene p53 is one of the most common occurrences associated with human neoplasia. Consequently, restoration of wild-type p53 function is seen as a particularly promising approach for cancer gene therapy. In recent years, considerable research effort has centered upon developing and improving non-viral delivery systems as alternatives to viral vectors for gene delivery. These methods include the use of lipoplexes and polyplexes, and even delivery of naked DNA. Optimally effective cancer gene therapy requires treatment of metastatic as well as local disease, and to achieve this end, systemic delivery systems for therapeutic genes will be required. This review will discuss some of the recent advances in ways to improve targeting, transfection efficiency and stability for systemic, non-viral p53 gene therapy.     

A multifunctional PEI-based cationic polyplex for enhanced systemic p53-mediated gene therapy

We recently reported a novel coupling strategy involving salicylhydroxamic acid and phenyl(di)boronic acid molecules to attach the CNGRC peptide to PEI/DNA for CD13 targeting in tumors. Here, we doubly coupled Simian Virus (SV) 40 peptide-(nuclear localization signal)) and oligonucleotide-based (DNA nuclear targeting signal) nuclear signals to the same vector using peptide nucleic acid chemistry. This vector, CNGRC/PEG/PEI/DNA-betagal/NLS/DNTS, was predominantly localized in the cell nucleus, yielding about 200-fold higher betagal gene expression in vitro, more than 20-fold increase in tumor-specific gene delivery, and a robust betagal gene expression as demonstrated in stained tumor sections. For gene therapy purposes, we further engineered a similar targeting polyplex, CNGRC/PEG/PEI/DNA-p53/NLS/DNTS, with EBV-based episomal vector for sustained p53 gene expression. A distribution of vector DNA and apoptosis in p53-containing tumors was observed, yielding a significant tumor regression and 95% animal survival after 60 days. This multicomponent vector also co-targeted tumor and tumor-associated endothelial cells but not normal cells, and had more efficient therapeutic index than each vector administered as a single modality. The use of an efficient coupling strategy without compromising the vector's integrity for DNA condensation and endosomal escape; nuclear import; tumor-specific and persistent p53 gene expression clearly provides a basis for developing a single combinatorial approach for non-viral gene therapy.     

INGN 201 (Advexin): adenoviral p53 gene therapy for cancer

Mutations in the p53 gene are the most frequent genetic alterations in human tumours, occurring in approximately 50% of all cancers. The p53 protein is pivotal in maintaining genetic integrity after DNA damage, and alterations in the p53 pathway, including mutations in the p53 gene, greatly increase the probability of tumour formation. Gene therapy using adenoviral p53 has emerged as a novel treatment option, with the potential to be safe and effective in a wide range of cancer types. INGN 201 (Ad5CMV-p53, Advexin), a replication-impaired adenoviral vector that carries the p53 gene, has been evaluated in both preclinical and clinical trials. Results show that Advexin is a well-tolerated and efficacious treatment for numerous cancers, both as monotherapy and in combination with radiation and/or chemotherapy agents. In addition, there is now data to support the use of Advexin in cancer immunotherapy.     

Eradication of p53-mutated head and neck squamous cell carcinoma xenografts using nonviral p53 gene therapy and photochemical internalization.

Photochemical internalization (PCI) technology has been used for PEI-mediated p53 gene transfer in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts. Using luciferase as a reporter gene, PCI led to a 20-fold increase in transgene expression 48 h after transfection and sustained transgene expression for 7 days. Therefore, iterative p53 gene transfer was performed by means of a weekly single injection of PEIGlu4/p53 complexes alone or with PCI for 5 (group A) or 7 (group B) weeks. The efficiency of p53 gene therapy was evaluated by following tumor growth and expression of P53-related downstream proteins (P21, MDM2, Bcl2, Bax). Apoptosis induction was evidenced through caspase-3 activation and PARP cleavage. Using PCI, tumor growth inhibition was observed in all transfected animals. Further, successful tumor cure was achieved in 17% (group A) and 83% (group B) of animals. PCI-mediated p53 gene transfer led to higher P53 protein expression that was correlated with induction of Bax and P21 proapoptotic proteins, repression of Bcl2 as well as activation of caspase-3, and cleavage of PARP. The present study demonstrates that PCI enhances the in vivo efficiency of PEI-mediated p53 gene transfer and can be proposed for p53 gene therapy in HNSCC.     

瘢痕疙瘩家系p53基因的突变

目的:探讨瘢痕疙瘩家系样本有无p53基因突变以及与瘢痕疙瘩发病的关系。方法:实验标本来自南方医科大学南方医院整形外科2005年收集的A和B两个瘢痕疙瘩家系。2005-01/05在上海基康公司采用聚合酶链反应及基因测序技术,分别以A家系两例患者的瘢痕疙瘩组织为研究对象,以其周围正常皮肤及外周静脉血作为自身对照。其配偶的外周静脉血作为正常对照,并以B家系中两例患者的外周静脉血作为不同家系之间的对照。检测10份样本中p53基因外显子1～11的基因序列。结果:①经基因序列分析发现所有被检测样本p53基因外显子1～10均未发现突变。②p53基因外显子11在所有的被检测样本中均发现突变,而且突变的位点及突变的类型均相同。其中119 bp,577 bp,604 bp,626 bp,627 bp,726 bp,727 bp,1148～1149 bp为点突变,440 bp为插入突变,397～400 bp,600 bp,602 bp,605～607 bp,609 bp为缺失突变。结论:瘢痕疙瘩家系样本p53基因突变提示p53基因的突变可能与瘢痕疙瘩的发病无关。     

结直肠癌p53基因点突变和蛋白表达的研究

目的了解中国人结直肠癌ｐ５３基因的突变谱，探讨聚合酶链反应－单链构象多态（ＰＣＲ－ＳＳＣＰ）银染技术用于研究结直肠癌中ｐ５３基因突变的可行性。方法应用ＰＣＲ－ＳＳＣＰ银染技术检测４１例结直肠癌ｐ５３基因突变，以ＡＢＣ免疫组织化学染色检测结直肠癌Ｐ５３蛋白的表达。结果３４％（１４／４１）的病例显示有ｐ５３基因突变，其中外显子５，６，７和８各有４，１，５和４例突变。２０例有Ｐ５３蛋白异常表达，阳性率为４９％。结论导致Ｐ５３蛋白异常堆积的ｐ５３基因突变是结直肠癌的一种常见的分子结构改变，可能在结直肠癌的发生和发展中起着重要的作用。ＰＣＲ－ＳＳＣＰ银染技术是一简便、快速、有效的检测基因点突变的方法     

Histone deacetylase inhibitor FK228 enhances adenovirus-mediated p53 family gene therapy in cancer models

Therapeutic replacement of the wild-type p53 gene has been pursued as a potential gene therapy strategy in a variety of cancer types; however, some cancer models are resistant to p53 in vivo and in vitro. Therefore, to improve p53 gene therapy, it is important to overcome the resistance to p53-mediated apoptosis. Histone deacetylase inhibitors are a novel class of chemotherapeutic agents that are able to reverse the malignant phenotype of transformed cells. A natural histone deacetylase inhibitor, FK228, is reported to enhance adenovirus infection due in part to the up-regulation of coxsackievirus adenovirus receptor expression. In this study, preclinical experiments were done to establish a mechanistic rationale for the combination of adenovirus-mediated p53 family gene transfer and FK228 pretreatment in future clinical trials. Pretreatment with FK228 enhanced apoptosis in human cancer cells through enhanced transduction of Ad-p53. FK228 also induced hyperacetylation of the p53 protein and specifically enhanced p53-mediated Noxa expression. Additionally, the combination of FK228 and Ad-p53 induced Bax translocation to the mitochondria. The double knockdown of Bax and Noxa expression by small interfering RNA antagonized the synergistic effect of Ad-p53 and FK228 on apoptosis induction. In human cancer xenograft models, FK228 significantly increased the therapeutic effectiveness of p53 as well as p63 gene therapy. These results provide a strong rationale for combining p53 gene therapy and FK228 pretreatment in cancer therapy.     

p53 gene mutation, tumor p53 protein overexpression, and serum p53 autoantibody generation in patients with breast cancer

OBJECTIVES: Autoantibodies against the p53 tumor suppressor protein have been detected in the serum of a proportion of patients with various cancers. The generation of such antibodies has been proposed to be due to either tumor p53 protein accumulation or to the type of p53 gene mutation. These hypotheses are examined in the present study. DESIGN AND METHODS: Using immunofluorometric assays, we studied 195 patients with primary breast cancer for the presence of p53 antibodies in serum and p53 protein accumulation in the corresponding tumor. Seventeen patients (9%) were p53 antibody-positive and 77 (40%) overexpressed p53. Ten of the 17 p53 antibody-positive patients had tumor p53 accumulation and 7 were negative for p53. Statistical analysis revealed a weak association between the presence of p53 antibodies and p53 protein accumulation (p = 0.05). Direct DNA sequencing of exons 1-11 of the p53 gene was performed for 16 p53 antibody-positive and 16 p53 antibody-negative patients. RESULTS: Five of the seropositive and eight of the seronegative patients had a p53 gene mutation. Four of the five mutations in the p53 antibody-positive patients affected a Tyr residue, whereas none of the gene abnormalities in the seronegative patients had such an effect. CONCLUSIONS: We conclude that p53 antibodies tend to develop in patients with tumor p53 accumulation, but p53 accumulation is neither sufficient nor necessary for the generation of the immune response. Further, p53 antibody-positive patients do not have higher frequency of p53 gene mutations than p53 antibody-negative patients, but the former patient group is associated with a Tyr substitution in the protein product.     

Adenoviral p53 gene therapy in squamous cell cancer of the head and neck region

Treatment options for recurrent head and neck squamous cell carcinoma are limited. Morbidity and mortality are largely related to local recurrence. Experimental investigation of adenoviral p53 gene therapy is being explored as a new therapeutic modality for patients with recurrent squamous cell carcinoma in the head and neck region. The function of the p53 gene is to maintain the genetic integrity of the cell and induce apoptosis when DNA damage is irreparable. Phase I trial results indicate that multiple injections of up to 2.5 x 10(12) viral particles per injection administered during a 3-week cycle are well tolerated and demonstrate evidence of local regional control. Dose-related activity correlating with transient survival advantage is demonstrated in a meta-analysis of phase II trials. This review will summarize phase I and II trial results. Conclusions drawn from these studies justify the currently ongoing phase III investigation.     

重组人p53腺病毒治疗脊柱转移瘤的近期疗效观察

目的探讨重组人p53腺病毒（recombinant human p53 adenovirus,rAd-p53）在脊柱转移瘤的治疗中的近期疗效。方法 2006年6月2009年8月,以经皮注射rAd-p53联合放疗及单独放疗方法治疗肺鳞状细胞癌来源脊柱转移瘤患者各18例,通过比较两组治疗前后的肿瘤体积变化评价疗效,观察两组治疗后肿瘤细胞坏死率情况,检测P53蛋白在癌组织中的表达以及血清中抗特异p53基因腺病毒抗体水平。结果联合治疗组疗效评定有效率为66.7%,高于单独放疗组的27.8%,差异有统计学意义（P〈0.05）。联合治疗组可见明显肿瘤细胞坏死、P53蛋白表达阳性及血清中抗特异p53基因腺病毒抗体水平强阳性。结论 rAd-p53基因治疗能抑制肿瘤生长,联合放疗可弥补单一放疗的不足,提高放疗的敏感性,有效治疗脊柱转移瘤。