- Email: [email protected]
Why did p53 gene therapy fail in ovarian cancer?
Review
p53 and ovarian cancer
Why did p53 gene therapy fail in ovarian cancer? Alain G Zeimet and Christian Marth
Promising preclinical and clinical data led to the initiation of an international randomised phase II/III trial of p53 genetherapy trial for first-line treatment of patients with ovarian cancer. In that trial, replication-deficient adenoviral vectors carrying wild-type p53 were given intraperitoneally in combination with standard chemotherapy to patients with ovarian cancers harbouring p53 mutations. The study was closed after the first interim analysis because an adequate therapeutic benefit was not shown. In this review, we discuss the possible reasons for failure of p53 gene therapy, which include the multiple genetic changes in cancer and epigenetic dysregulations leading to aberrant silencing of genes. These complex interactions lead us to conclude that repair of single genes might not be a suitable strategy for the treatment of cancer. Moreover, dominant negative cross talk between ectopic wild-type p53 and recently identified dominant p53 mutants and splice variants of p63 and p73— which are frequently overexpressed in ovarian cancers— could seriously compromise the effectiveness of p53 gene therapy. Other substantial problems in targeting tumour cells with adenoviral vectors are the heterogeneity or lack of expression of coxsackie-adenovirus receptors and integrin co-receptors in ovarian tumours and the presence of adenovirus-neutralising antibodies in ovarian cancerrelated ascites. Lancet Oncol 2003 4: 415–22
Correction of specific genetic defects responsible for the aberrant biological behaviour of cancer cells is a fascinating new approach for cancer treatment. The high frequency of p53 mutations in human cancers and the central role of p53 in regulating growth and apoptosis led researchers to believe that it would be an appealing target for gene replacement therapy. Ovarian cancer is regarded as a particularly good candidate for gene transfer using viral vectors because the disease generally remains confined to the abdominal cavity throughout its course, enabling intraperitoneal delivery of therapy (figure 1). This approach has the advantage of producing high viral loads close to the tumour and adverse events are controlled because there is a delay before the vector gets into the systemic circulation. Promising preclinical data on in-vitro transfection of wild-type p53 into ovarian cancer cells with p53 mutations and into xenograft models, coupled with evidence from phase I trials, led to the introduction of this innovative technique for front-line treatment of patients with advanced ovarian cancer.1–3 Thus, a large international p53 gene-therapy study in patients with primary stage III ovarian cancer with p53 mutations was set up. In this phase II/III trial, patients with no or residual disease (less than 2 cm in diameter) who had undergone surgical cytoreduction were THE LANCET Oncology Vol 4 July 2003
Figure 1. Intraperitoneal delivery of p53 gene therapy for ovarian cancer.
AGZ and CM are both professors at the Gynaecologic Oncology Unit of the Department of Obstetrics and Gynaecology, Innsbruck University Hospital, Austria. CM is the head of that department. Correspondence: Dr Alain G Zeimet, Department of Obstetrics and Gynaecology, Innsbruck University Hospital, Anichstrasse 35, 6020 Innsbruck, Austria. Tel: +43 (0)512 504 3050. Fax: +43 (0)512 504 3055. Email: [email protected]
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
415
Review randomly assigned to receive either standard therapy of six systemic cycles of carboplatin and paclitaxel or the same regimen plus five cycles of gene therapy. Each 5-day genetherapy cycle consisted of intraperitoneal administration of 1013 particles a day of replication-deficient wild-type p53 delivered in a serotype-5 adenovirus (figure 2). The first interim analysis of this study showed that addition of p53 gene therapy to standard treatment did not improve therapeutic effectiveness in patients with optimally debulked advanced ovarian cancer and actually increased treatment morbidity. This unexpected result led to the study being closed. Some questions need to be addressed concerning these disappointing results. The first is whether this failure means the end for this novel therapeutic approach in ovarian cancer. The answer is clearly no. On the contrary, it should be the starting point from which to make improvements. Thus, for future attempts at gene therapy the reasons why p53 gene therapy failed to work in patients with ovarian cancer are very important—the reasons for failure are likely to be multiple.
Multiple genetic and epigenetic changes Malignant transformation is known to be a multistep process involving changes in several genes. In addition to p53, aberrations of C-ERBB2, C-MYC, K-RAS, and several others have an important role in ovarian cancer.4 Protein products of these genes—although core molecules in cellular pathways such as apoptosis, differentiation, proliferation, and cell-cycle control—are generally not final effectors but intermediate steps in signalling cascades. p53 is directly inactivated in about half of human cancers through mutation or loss and, in most tumours characterised to date, disruption of the p53 tumour suppressor pathway, at one or more positions, is thought to contribute to the malignant phenotype. Consequently, therapeutic replacement of a gene such as p53 can only restore full functionality to the corresponding pathway if none of the downstream genes are affected. Indeed, a subset of ECV-304 tumour cells selected for resistance to p53-mediated apoptosis by repeated p53 wildtype transductions were shown to be deficient in upregulating downstream proline oxidase, which is involved in the proline/pyrroline-5-carboxylate redox cycle.5 Furthermore, about 77% of primary ovarian cancers have been found to have serious impairments in cytochrome-cdependent apoptosome activation because of their inability to activate either caspase 9 or caspase 3, which are important members of the p53-induced caspase cascade (figure 3).6 In addition to loss or mutation of genes, there is growing evidence that epigenetic phenomena, such as the state of methylation of CpG islands in promoter regions of genes, and the consequent extent of histone acetylation, have a substantial role in the evolution and progression of the malignant phenotype.7,8 Epigenetic dysregulation is believed to result in either aberrant gene silencing9 or possibly gene overexpression.10 This suggests that the presence of a defined gene in its wild-type state in the genome of malignant cells is no guarantee for its accurate expression and functionality. Recent data have shown that the p53-mediated death cascade can be subverted by DNA methylation. Soengas and
416
p53 and ovarian cancer
Randomised phase II/III study in primary FIGO stage III ovarian cancer Primary surgical debulking No residual tumour, or ⭐2 cm DNA sequencing Evidenced p53 null or p53 mutated status 1 cycle carboplatin/paclitaxel Randomisation
iv carboplatin/ paclitaxel (d1) plus ip p53 gene therapy (d1–5) cycle 2 to 6
Control group iv carboplatin/ paclitaxel (d1) cycle 2 to 6
Figure 2. Study design of the phase II/III randomised international trial of p53 gene therapy for the first-line treatment of patients with optimally debulked, advanced stage (FIGO stage III), ovarian cancer.
colleagues reported that one downstream component of the p53-dependent death cascade, apoptotic protease-activating factor 1 (APAF1), is not expressed in malignant melanomas, which can then escape apoptosis when treated with chemotherapy. Although the promoter region of APAF1 was not found to be hypermethylated, treatment with methylation-inhibiting agents restored expression of the gene. Thus, it is conceivable that methylation inhibitors reactivate a yet unspecified APAF1 regulator or demethylate another control region of the gene (figure 3).11 Similarly, defective APAF1 activity was also shown in a subset of ovarian cancer cell lines that were resistant to induction of p53-mediated apoptosis.12 Thus, aberrant methylation in genes controlling the expression of APAF1 that are associated with mutated or lost p53 could seriously compromise p53 replacement therapy. Overall, epigenetic phenomena add a completely new dimension to our basic understanding of the molecular biology of cancer and should be taken into account in future gene-therapy approaches. Ono and colleagues used cDNA microarrays to identify 55 genes that were commonly upregulated and 48 genes that were downregulated in ovarian carcinomas. The group also reported 115 genes that were differentially expressed in serous and mucinous adenocarcinomas of the ovary.13 Furthermore, the different cellular signalling pathways cannot be understood by focusing on each isolated path and it is crucial to consider the tangled networks into which these signalling pathways are integrated. It seems optimistic to believe that cancer can be cured by repairing one single aberrant gene and it is tempting to speculate that therapeutic strategies of multigene repair, focusing on a several key genes involved in the development and progression of cancer, might be more effective than singlegene therapy. THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
Review
p53 and ovarian cancer
Problems with the transgene
operation with DNA-binding protein MIZ1 to the p21WAF1 Was p53 the best transgene for the intended therapeutic promoter, blocking its induction by activators such as p53 and purpose? Or would another gene have been more suitable? switching the p53-dependent cellular response from cytostatic There were two convincing arguments in favour of to apoptotic.23,24 Despite rapid advances in the study of p53, therapeutic restoration of wild-type p53 in altered malignant the exact mechanisms governing this pivotal cellular decision cells. First, p53 is a master regulator of the apoptotic pathway have not been elucidated. Nonetheless, it seems that and co-ordinates programmed cell death at many levels malignant cells are generally more likely to undergo apoptosis though numerous mechanisms and apoptotic pathways are after p53 activation than normal cells which tend to undergo believed to contribute decisively to the cytotoxic action of cell-cycle arrest. Because of this difference, therapies based on several chemotherapeutic drugs. And second, loss or p53 reactivation have been developed to selectively promote mutation of p53 has been causally linked to chemoresistance apoptosis in tumour cells. However, some normal cell types in various tumours, including ovarian cancer.14–16 Therefore, a such as intestinal epithelium and haematopoietic cells are rational hypothesis for treatment of these cancers is to attempt sensitive to p53-induced death even in a non-transformed repair of the p53-dependent death programme in the affected state. Since vector targeting is far from being tumour-cell cells, and then attack them with cytotoxic agents. However, it specific, this discrepancy could account for side-effects of is important to note that not all chemotherapeutic drugs externally delivered p53 or for more severe toxic effects when require p53 for their apoptotic function and the overall gene transfer is combined with chemotherapy.25 However, contribution of apoptotic defects to clinical drug resistance is data about the real susceptibility of malignant cells to cellcycle arrest and DNA repair in response to DNA-damaging still debatable.17 Another question concerns the cellular response to p53 agents after restoration of wild-type p53 is inconclusive. activation. What factors determine whether p53 induces Although studies in p53-deficient SK-OV-3 ovarian-cancer apoptosis or the potentially reversible p21WAF1-mediated xenografts have shown that transfection of wild-type p53 cell-cycle arrest (figure 3)? Vousden proposed several before treatment with cisplatin induces apoptosis, but does molecular mechanisms for this decision pathway, of which not improve DNA repair,26 it is conceivable that therapeutic one of the most promising is based on the intracellular effects of concomitantly administered cytotoxic drugs can be concentration of p53. Low concentrations cause cell-cycle attenuated or even completely neutralised by DNA repair if arrest, which turns to apoptosis as p53 concentrations reactivated p53 pathways in tumour cells favour cell-cycle increase. The molecular basis for this hypothesis is the lower arrest over cell death. In some tumour types permanent loss of affinity of promoters of the apoptotic target genes for p53.18 p53 was found to increase cell death induced by doxorubicin More recent studies have shown how transcriptional cofactors or platinum drugs.27–29 However, these findings contrast with such as the ASPP apoptotic-enhancer proteins, the junction the outcome of phase I and II trials of non-small-cell lung mediating and regulatory protein JMY, and several others, are cancer in which intratumoural injections of an adenovirus able to modulate the affinity of p53 for its target promoters carrying p53 combined with cisplatin, carboplatin, and and thus are involved in the choice between cell-cycle arrest paclitaxel, or cisplatin and vinorelbine resulted in locoregional and apoptosis. The activity of ASPP proteins is of particular activity. Clinical proof of principle was clearly achieved in interest because their expression is required for p53-induced apoptosis and frequently they are downregulated in breast cancer; mutated this constitutes yet another mechanism BAX MYC JunD RAS by which tumours can avoid p5319 Furthermore, induced cell death. p53 several investigations have provided DAPK p14ARF HDM2 evidence that the cyclin-dependent Mitochondria kinase inhibitor p21WAF1 is a critical downstream checkpoint in the BCL2 differential response after p53 activation. E2 F1 Cytochrome c release In addition to its close association with cell-cycle control (in regulating transition from G1 to S phase), APAF1 p21WAF1 was also found to have The antiapoptotic properties.20–22 unknown Gene silencing through important role of p21WAF1 in decisionAPAF1 DNA methylation Activation of the activator making was further underscored in a in promotor region caspase cascade paper by Seoane and co-workers, which reported a pivotal role of mitogenic transcripton factor MYC in influencing Apoptosis The p53-mediated apoptotic pathway the outcome of p53 response to DNA damage in favour of cell death. The group revealed that MYC binds in co- Figure 3. The principle p53 pathways leading either to apoptosis or cell-cycle arrest. THE LANCET Oncology Vol 4 July 2003
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
417
Review
p53 and ovarian cancer
One important question is why the complexity of genetic and epigenetic changes in tumours and the limitations of the p53 transgene go a long way to explaining the failure of the clinical gene-therapy trial in ovarian cancer, when in preclinical ovarian cancer
418
Adenoviral vector
␣v3
␣v5
Preclinical research
␣v
3
␣v
5
␣v3
5 ␣v
these studies as shown by greater regression of injected lesions xenograft models intraperitoneal p53 replacement therapy compared with control lesions and by progression of distant showed promising activity. First, it is important to note that metastases.30–32 In the phase II trial of these combinations of inhibition of tumour growth was observed in most but not all drugs, locoregional response rates could not be verified with of these xenogeneic transplant models. In a nude mouse classic radiographical response criteria, although trends model, von Grueningen and co-workers showed effective toward greater tumour shrinkage of targeted lesions were growth inhibition but it was found not to be specifically reported by use of size criteria. Nevertheless, the findings led attributable to p53 gene transfer.41 Furthermore, a review of the authors to conclude that there was no clear evidence of available preclinical studies revealed that only a small number significant improvement with ectopic p53 replacement of different ovarian-cancer cell lines were used in engraftment studies. Preclinical data of the efficacy of p53 gene transfer in therapy in these settings.30,31 Another obstacle, which may compromise the efficacy of combination with chemotherapy in vivo are less clear than gene replacement of wild-type p53, could be the necessity of generally assumed. Kigawa and colleagues reported a p53 tetramerisation for transregulation of downstream genes. significant short-term survival advantage in SCID mice The dominant negative hypothesis relating to tetramers of treated intraperitoneally with p53 gene therapy in conjunction mutant and wild-type p53 has been refuted in several with cisplatin compared with single treatments. However, investigations,33–35 and wild-type p53 was considered to be when combined therapy was given repeatedly, the researchers phenotypically dominant over its mutant forms. However, could not identify significant long-term survival advantages.42 Monti and colleagues recently showed that roughly 30% of Thus, the main limitations of xenograft models in this context p53 mutants are dominant over wild-type p53 and also that might be the result of short intervals between engraftment and dominance is target-gene specific. Genes harbouring response treatment and short follow-up. In addition, in sporadic elements with a low affinity for wild-type p53 such as BAX tumours the reasons for treatment failure are not necessarily (figure 3) or PIG3 were much more sensitive to dominant- the same for each tumour. For instance, not all ovarian negative inhibition by p53 mutants than genes with high- cancers overexpress p73EX2DEL or are affected by a p53 affinity response elements such as p21WAF1.36 Furthermore, mutation which has negative dominance over wild-type the apoptotic pathway seems more likely to be affected by this p53.36,40 Also, it is questionable whether xenogeneic transplant dominant-negative cross talk than p53-induced cell-cycle models based on a limited number of established cell lines are arrest with subsequent DNA repair. Some splice variants of representative of all sporadic tumours and can mimic the p53-related genes, such as ⌬NP73, ⌬NP63, and p73-EX2DEL, heterogeneity and genetic instability that occurs in which lack the transactivation domain have recently been progression of sporadic ovarian cancers. identified. The intact oligomerisation domain of these proteins enables hetero-oligomer formation with wild-type Adenoviral vectors and intraperitoneal delivery p53, and they have been found to act as dominant-negative The adenoviral vector system and the intraperitoneal mode of inhibitors of transactivation and the apoptotic ability of vector delivery could be major causes of the failure of the p53.37–39 Overexpression of p73EX2DEL was shown in a large clinical trial. Entry of adenovirus into target cells is the rateproportion of primary ovarian cancers and ovarian cancer cell limiting step in ectopic gene transfer and determines lines.40 Therefore, such dominant-negative cross talk between therapeutic efficacy of adenovirus-based gene therapy. wild-type p53 and dominant p53 mutants or variants of p53- Attachment of adenoviruses to the cell surface is mediated by related proteins (especially when the latter are overexpressed in tumours) Internalisation Attachment could negatively affect p53 gene therapy. These aspects were not considered in the disappointing trial of p53 gene therapy in patients with Integrins ␣v3 ovarian cancer because when the Integrins protocol was devised the dominant␣v5 negative hypothesis on p53 mutants ␣v3 had been refuted and the other Nucleus Nucleus CAR members of the p53 family were not yet Adenoviral CAR known or their function was not fully vector ␣v ␣v5 3 understood. ␣v5
Target cell membrane
Target cell membrane
Figure 4. Entry of a serotype-5 adenovirus into a target cell.
THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
Review
p53 and ovarian cancer
the coxsackie-adenovirus receptor (CAR), and internalisation is achieved via interactions with integrins of the ␣v3 and ␣v5 classes (figure 4).43,44 Deficiency in one or both of these membranous molecule classes has been reported to confer relative resistance to adenoviral vectors.45–47 Marked differences in CAR expression were noted in primary cultured ovarian cancer cells,1 and in addition to total absence of CAR or both classes of integrins, a pronounced intratumour heterogeneity in the expression of these molecules was detected immunohistochemically in ovarian tumours, which could account for negative selection during adenovirus-based therapy. Ovarian cancers classified as undifferentiated were found to completely lack expression of ␣v3 integrins.48 Furthermore, Bruning and colleagues reported that loss of ␣v3 integrins and especially the 3 subunit compromises adenoviral transduction efficiency in established ovarian cancer cell lines.49 According to our immunohistochemical data, only 40% of the randomly investigated ovarian cancers would meet the criteria to be adequately and evenly targeted by adenoviral vectors.48 Whether or not other molecules of the cell membrane such as ␣v1 integrins are involved in virus uptake or can bypass the defective classic pathway of adenoviral infection remains to be clarified.50 However, to overcome limitations posed by wide variability in the expression of molecules regulating cell entry of adenoviruses, adenoviral vectors were retargeted to specifically enter cancer cells, either by the insertion of targeting moieties, such as the arginine-glycine-aspartic acid (RGD) peptide motif in the viral fibre knob protein,51,52 or by use of bispecific antibody conjugates consisting of the Fab-fragment of an antiadenovirus knob antibody and an antibody of choice directed against a defined molecule readily expressed in malignant cells. For instance, adenoviral targeting with bispecific antibodies via the tumour-associated glycoprotein TAG72—which is expressed on virtually all ovarian carcinomas but not on the surrounding normal mesothelial cells—resulted in 28 times better gene transfer to ovarian cancer cells.53 The use of a genetically modified adenoviral vector containing a TAG72-specific epitope in the viral fibre protein is currently being explored for gene therapy in ovarian cancer. Targeting via other molecules such as growth factor receptors—eg, epidermal growth-factor receptor or basic fibroblast growth-factor receptor—which are overexpressed in malignant disease has also been done.54,55 Overall, the generation of tropism-modified adenoviral vectors is a very promising approach and should provide CAR-independent vectors with a higher transduction efficiency and an improved tumour selectivity in the near future. Locoregional administration of adenoviral vectors has long been considered opportune, not only because of the high vector loads delivered directly to target cells but also as a means of avoiding vector neutralisation by systemic antibodies—those for adenoviruses are highly prevalent in the general population. However, significant in-vitro inhibition of adenovirus-mediated gene transfer has been shown in the presence of ascitic fluid from patients with ovarian cancer. Depletion of antibodies directed against the adenoviral fibre protein from the ascites resulted in a large but not total elimination of the inhibitory activity,56 indicating that THE LANCET Oncology Vol 4 July 2003
Pseudocyst
Figure 5. Computed tomography of a peritoneal pseudocyst after four courses of intraperitoneally administered adenovirus-based p53 gene therapy.
components of the ascites other than neutralising antibodies, such as fibronectin, fibrinogen, and vitronectin (which have high binding affinity for ␣v integrins), may contribute to the obstruction of vector internalisation. Moreover, splice variants of CAR which lack a transmembrane domain but have an intact adenovirus-binding domain have been identified.57 Thus, it is tempting to speculate that these splice variants, when shed into the ascitic fluid, may compromise adenoviral attachment to target cells by opsonising the fibre knobs of adenoviral vectors. Intraperitoneal drug delivery via an implanted catheter system, as described in the protocol of the p53 gene therapy trial, seems at first to be a comfortable means delivering anticancer treatment close to the tumour, especially in ovarian cancer which generally remains confined to the abdominal cavity. However, in practice the therapeutic window for this locoregional mode of drug administration was found to be small. Problems associated with intraperitoneal catheter delivery such as infections, bowel perforations, and obstructions were the limiting and most serious adverse events in phase I of the trial.2 Successful intraperitoneal treatment requires even distribution of the vector throughout the entire abdominal cavity. Because of peritoneal adhesion and pseudocyst formation after repeated administration, the chances of vector sequestration were found to be high; thus, large areas of the peritoneal cavity remained unaffected by the drug (figure 5). Normal human and murine mesothelial cells express the molecules required for adenoviral cell entry and infection of these cells was shown in vivo.58,59 However it remains to be established to what extent virus-triggered inflammation and apoptosis of peritoneal mesothelium account for the development of intraperitoneal adhesions and peritonitis-like conditions. Furthermore, earlier studies revealed that the effectiveness of intraperitoneal treatment with conventional cytotoxic agents is highly dependent on the diameter of the residual tumour nodules or plaques, with best responses in tumours less than 0·5 cm in diameter.60,61 The limitations of penetration of drug to the central part of the tumour are thought to explain this phenomenon. This situation should also be true
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
419
Review for adenoviral vectors with p53 when given intraperitoneally, and therefore the inclusion of patients with residual tumours up to 2 cm in diameter should be seriously questioned.
Host immune defence Neutralising antiadenoviral antibodies in the ascitic fluid of patients with ovarian cancer seem to be a major obstacle in tumour-cell targeting and are strongly suspected of adversely affecting the outcome of intraperitoneal adenoviral-based gene therapy.56 To what extent the activated complement system participates in the elimination of adenoviral vectors in the abdominal cavity either via enhanced phagocytosis or more importantly via immediate complement-directed virolysis remains to be determined although such interactions have been described for other virus types.62 However, several investigations have shown that adenoviral vectors trigger an MHC-class-Irestricted cytotoxic-T-lymphocyte response to viral antigens, which is sufficient to destroy virus-infected cells.63 This bystander effect of the cellular immune system could in fact increase therapeutic effectiveness of gene transfer by its beneficial contribution to tumour-cell kill. Non-specific p53 antitumour activity has been reported in breast and ovarian cancer xenografts in immunodeficient mice treated with empty adenoviral vectors and has been attributed to residual immunological functions.41,64 Moreover, the cytotoxic immune response directed against vector-infected cells should be larger than that triggered by genuine adenoviruses because recombinant replication-defective adenoviral vectors also used in the trial lack the genes encoding for early proteins E1 and E3. These proteins are vital elements in the molecular mechanisms leading to immune evasion of unmodified adenoviruses and work by blocking interferon-induced gene expression, preventing death-receptor-mediated apoptosis through FAS and TRAIL receptor removal from the cell surface and through interference with the cytolytic and proinflammatory activities of tumour necrosis factor, and by subverting MHC class I antigen presentation.65 Overall, these functions drastically limit lysis of cells infected by genuine adenoviruses, either by natural killer cells or cytotoxic T lymphocytes. However, because vector targeting is not tumour-cell specific, this amplified immune response caused by adenoviral vectors should be as harmful to normal tissue as to tumour cells and thus may contribute to intraperitoneal inflammatory conditions and other sideeffects, which could impair therapeutic outcome or even preclude continuation of treatment.2,3 Another phenomenon that can compromise the efficacy of gene therapy is the innate antiadenoviral immunity of cells. Infection with adenoviral vectors induces the production of cytokines, such as interferon ␥ and TNF␣, both of which have been shown to significantly inhibit the expression of transgenes directed by the human cytomegalovirus promoter.66 The E1/E3-deficient state of adenoviral vectors is considered largely responsible for this inhibitory effect on transgene expression. This cytokinerelated promoter attenuation may partially explain discrepancies in p53-transgene expression in various cell
420
p53 and ovarian cancer
Search strategy and selection criteria Data for this review were identified by searches of MEDLINE, Current Contents, and PubMed, with the search terms “gene therapy”, “ovarian cancer”, “p53”, “p21”, “apoptosis”, “adenoviral vector”, “coxsackie-adenovirus receptor”, and “epigenetic”. Reference lists of relevant articles were also searched. Only papers published in English or German between 1980 and 2003 were included.
lines, which could not be primarily ascribed to marked differences in the infectability of these cell lines.67
Future directions What are the prospects for future gene therapy approaches in ovarian cancer? The generation of more efficient vectors to improve tumour-cell targeting by reconsidering alternative types of viral and non-viral transfection systems, such as liposomes, seems to be mandatory. However, it is hard to conceive that non-viral systems will ever give transfection rates as effective as those produced by viral vectors. For millions of years the main job of viruses has been to penetrate cellular systems to ensure survival. Recent endeavours to develop new recombinant viral vectors with modified tropism and higher transduction rates, increased stability of transgene expression, and the ability to bypass the host immune response are the most encouraging and meaningful approaches for clinical translation in the near future.51,68,69 For instance, conditionally replicative viruses for cancer treatment constitute a rapidly evolving new strategy. A new generation of these replicative viral vectors, placing the expression of transgenes under the control of tumour-specific promoters or transcriptional control regions, such as the COX2 or MUC1/DF3 gene promoter in ovarian cancer cells, is of great interest. As shown in preclinical studies, this strategy confers tumour specificity59,70–73 which in turn could represent the rational basis for the use of either suicide genes, like HSVthymidine-kinase,71 or strong proapoptotic transgenes. These classes of transgenes have been shown to cause highly efficient cell killing in various tumour cell lines, but when delivered in unselective vectors they are as harmful to normal cells as to cancer cells and are expected to be associated with a broad range of side-effects in vivo. There is a large body of evidence that in gene therapy the battle has to be won before the treatment stage. Only identification of the genetic and epigenetic signature of the target tumour, including expression of cell-surface molecules governing the cellular uptake of the chosen vector, can provide the information needed to tailor a multigene or multivector treatment for each individual tumour. Although as yet tailoring of treatment remains complex and incomplete, and exact gene expression profiling requires delicate, complicated procedures, continued advances in biotechnology, such as genome-scale microarray analysis and proteomics should help to yield a manufactured but welltailored treatment in the near future. Conflict of interest
None declared.
THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
Review
p53 and ovarian cancer
References
1 Zeimet AG, Riha K, Berger J, et al. New insights into p53 regulation and gene therapy for cancer. Biochem Pharmacol 2000; 60: 1153–63. 2 Runnebaum IB. Intraperitoneale p53-gentherapie des ovarialkarzinoms in der klinischen prüfung: interimsanalyse eines vielversprechenden ansatzes zur ”kausalen” therapie. Arch Gynecol Obstet 1999; 263: 166–68. 3 Buller RE, Runnebaum IB, Karlan BY, et al. A phase I/II trial of rAD/p53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer Gene Therapy 2002; 9: 553–66. 4 Aunoble B, Sanches R, Didier E, Bignon YJ. Major oncogenes and tumor suppressor genes involved in epithelial ovarian cancer. Int J Oncol 2000; 16: 567–76. 5 Maxwell SA, Davis GE. Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis sensitive tumor cells. Proc Natl Acad Sci 2000; 97: 13009–14. 6 Liu JR, Opipari AW, Tan L, et al. Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance. Cancer Res 2002; 62: 924–31. 7 Jones PA. DNA methylation errors and cancer. Cancer Res 1996; 56: 2463–67. 8 Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16: 168–74. 9 Widschwendter M, Berger J, Müller HM, et al. Epigenetic downregulation of the retinoic acid receptor-β2 gene in breast cancer. J Mammar Gland Biol Neopl 2001; 6: 193–201. 10 Lu AP, Gupta A, Li C, et al. Molecular mechanisms for aberrant expression of the human breast cancer specific gene 1 in breast cancer cells: control of transcription by DNA methylation and intronic sequences. Oncogene 2001; 20: 5173–85. 11 Soengas MS, Capodieci P, Polsky D, et al. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 2001; 409: 207–11. 12 Wolf BB, Schuler M, Li W, et al. Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity. J Biol Chem 2001; 276: 34244–51. 13 Ono K, Tanaka T, Tsunoda T, et al. Identification by c-DNA microarray of genes involved in ovarian carcinogenesis. Cancer Res 2000; 60: 5007–11. 14 Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell 2002; 108: 153–64. 15 Kigawa J,Sato S, Shimada M, et al. p53 gene status and chemosensitivity in ovarian cancer. Hum Cell 2001; 14: 165–71. 16 Reles A, Wen WH, Schmider A, et al. Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer. Clin Cancer Res 2001; 7: 2984–97. 17 Brown JM, Wouters BG. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res 1999; 59: 1391–99. 18 Vousden KH. p53: death star. Cell 2000; 103: 691–94. 19 Vousden KH. Activation of the p53 tumor suppressor protein. Biochim Biophys Acta 2002; 1602: 47–59. 20 Lu YJ, Yamagishi N, Yagi T, Takebe H. Mutated p21WAF1/CIP1/SDI1 lacking CDK inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells. Oncogene 1998; 16: 705–12. 21 Gorospe M, Cirielli C, Wang XT, et al. p21WAF1/CIP1 protects against p53-mediated apoptosis of human melanoma cells. Oncogene 1997; 14: 929–35. 22 Wang Y, Blandino G, Givol D. Induced p21WAF1 expression in H1299 cell line promotes cell scenescence and protects against cytotoxic effects of radiation and doxorubicin. Oncogene 1999; 18: 2643–49 23 Seoane J, Le HV, Massagué J. Myc suppression of the p21Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 2002; 419: 729–34. 24 Vousden KH. Switching from life to death: The Miz-ing link between Myc and p53. Cancer Cell 2002; 2: 351–52. 25 Komarova EA, Gudkov AV. Suppression of p53: a new approach to overcome side effects of antitumor therapy. Biochemistry 2000; 65: 41–48. 26 Shimada M, Kigawa J, Kanamori Y, et al. Mechanism of the combination effect of wild-type TP53 gene transfection and cisplatin treatment for ovarian cancer xenografts. Eur J Cancer 2000; 36: 1869–75. 27 Bunz F, Hwang PM, Torrance C, et al. Disruption of p53 in human
THE LANCET Oncology Vol 4 July 2003
28 29 30
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
50 51
52 53
cancer cells alters the responses to therapeutic agents. J Clin Invest 1999; 104: 263–69 Zamble DB, Jacks T, Lippard SJ. p53-dependent and independent responses to cisplatin in mouse testicular teratocarcinoma cells. Proc Natl Acad Sci USA 1998; 95: 6163–68. Pestell KE, Hobbs SM, Titley JC, et al. Effect of p53 status on sensitivity to platinum complexes in a human ovarian cancer cell line. Mol Pharmacol 2000; 57: 503–11. Schuler M, Herrmann R, DeGreve JL, et al. Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemo-therapy for advanced non-small cell lung cancer: results of a multicenter phase II study. J Clin Oncol 2001; 19: 1750–58. Swisher SG, Roth JA. p53 Gene therapy for lung cancer. Curr Oncol Rep 2002; 4: 334–40. Swisher SG, Roth JA, Carbone DP. Genetic and immunologic therapies for lung cancer. Semin Oncol 2002; 29 (suppl): 95–101. Chen PL, Chen Y, Bookstein R, Lee WH. Genetic mechanisms of tumor suppression by the human p53 gene. Science 1990; 250: 1576–80. Yonish-Rouach E, Resnitzky D, Lotem J, et al. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin6. Nature 1991; 352: 345–47. Frebourg T, Sadelein M, Ng YS, et al. Equal transcription of wild-type and mutant p53 using bicistronic vectors results in the wild-type phenotype. Cancer Res 1994; 54: 878–81. Monti P, Campomenosi P, Ciribilli Y, et al. Tumour p53 mutations exhibit promoter selective dominance over wild type p53. Oncogene 2002; 21: 1641–48. Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998; 2: 305–16. Pozniak CD, Radinovic S, Yang A, et al. An anti-apoptotic role for the p53 family member, p73, during developmental neuron death. Science 2000; 289: 304–06 Yang A, Walker N, Bronson R, et al. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 2000; 404: 99–103. Ng SW, Yiu GK, Liu Y, et al. Analysis of p73 in human borderline and invasive ovarian tumors. Oncogene 2000; 19: 1885–90. Von Grueningen VE, Santoso JT, Coleman RL, et al. In vivo studies of adenovirus-based p53 gene therapy for ovarian cancer. Gynecol Oncol 1998; 69: 197–20. Kigawa J, Sato S, Shimada M, et al. Effect of p53 gene transfer and cisplatin in a peritonitis carcinomatosa model with p53-deficient ovarian cancer cells. Gynecol Oncol 2002; 84: 210–15. Stevenson SC, Rollence M, White B, et al. Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain. J Virol 1995; 69: 2850–57. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment. Cell 1993; 73: 309–19. Sewell DA, Li D, Duan L, et al. Optimizing suicide gene therapy for head and neck cancer. Laryngoscope 1997; 107: 1490–95. Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997; 275: 1320–23. Li Y, Pong RC, Bergelson JM, et al. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res 1999; 59: 325–30. Zeimet AG, Müller-Holzner E, Schuler A, et al. Determination of molecules regulating gene delivery using adenoviral vectors in ovarian carcinomas. Gene Ther 2002; 9: 1093–100. Bruning A, Kohler T, Quist S, et al. Adenoviral transduction efficiency of ovarian cancer cells can be limited by loss of integrin beta3 subunit expression and increased by reconstitution of integrin alphavbeta3. Hum Gene Ther 2001; 12: 391–99. Li E, Brown SL, Stupack DG, et al. Integrin αvβ1 is an adenovirus coreceptor. J Virol 2001; 75: 5405–09. Dmitriev I, Krasnykh V, MillerCR, et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998; 72: 9706–13. Hemminki A, Zinn KR, Liu B, et al. In vivo molecular chemotherapy and noninvasive imaging with an infectivity-enhanced adenovirus. J Natl Cancer Inst 2002; 94: 741–49. Kelly FJ, Miller CR, Buchsbaum DJ, et al. Selectivity of TAG-72targeted adenovirus gene transfer to primary ovarian carcinoma cells
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
421
Review
54
55 56 57 58 59
60
61
62
versus autologous mesothelial cells in vitro. Clin Cancer Res 2000; 6: 4323–33. Miller RM, Buchsbaum DJ, Reynolds PN, et al. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer. Cancer Res 1998; 58: 5738–48. Goldman CK, Rogers BE, Douglas JT, et al. Targeted gene delivery to Kaposi’s sarcoma cells via the fibroblast growth factor receptor. Cancer Res 1997; 57: 1447–51. Stallwood Y, Fisher KD, Gallimore PH, Mautner V. Neutralisation of adenovirus infectivity by ascitic fluid from ovarian cancer patients. Gene Ther 2000; 7: 637–43 Thoelen I, Magnusson C, Tagerud S, et al. Identification of alternative splice products encoded by the human coxsackie-adenovirus receptor gene. Biochem Biophys Res Comm 2001; 287: 216–22. Kim J, Hwang ES, Kim JS, et al. Intraperitoneal gene therapy with adenoviral-mediated p53 tumor suppressor gene for ovarian cancer model in nude mouse. Cancer Gene Ther 1999; 6: 172–78 Casado E, Gomez-Navarro J, Yamamoto M, et al. Strategies to accomplish targeted expression of transgenes in ovarian cancer for molecular therapeutic applications. Clin Cancer Res 2001; 7: 2496–504. Pujade-Lauraine E, Guastalla JP, Colombo N, et al. Intraperitoneal recombinant interferon gamma in ovarian cancer patients with residual disease at second look laparotomy. J Clin Oncol 1996; 14: 343–50. Alberts DS, Liu PY, Hannigan EV, et al. Intraperitoneal cisplatin plus intravenous cyclophosphamide versus intravenous cisplatin plus intravenous cyclophosphamide for stage III ovarian cancer. N Engl J Med 1996; 335: 1950–55. Stoiber H, Pinter C, Siccardi AG, et al. Efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective
422
p53 and ovarian cancer
63 64 65 66 67 68 69 70 71
72 73
action of complement factor H and decay accelerating factor (DAF, CD55). J Exp Med 1996; 183: 307–10. Yang, Y, Jooss KU, Su Q, et al. Immune response to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Ther 1996; 3: 137–44. Nielsen LL, Dell J, Maxwell E, et al. Efficacy of p53 adenovirusmediated gene therapy against human breast cancer xenografts. Cancer Gene Ther 1997; 4: 129–38. Burgert HG, Ruzsics Z, Obermeier S, et al. Subversion of host defense mechanisms by adenoviruses. Curr Top Microbiol Immunol 2002; 269: 273–318. Qin L, Ding Y, Pahud DR, et al. Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum Gene Ther 1997; 8: 2019–29. Hwang ES, Kim J, Kim JS, et al. The effects of the adenovirusmediated wild-type p53 delivery in human epithelial ovarian cancer cell line in vitro and in vivo. Int J Gynecol Cancer 1998; 8: 27–36. Kanerva A, Mikheeva GV, Krasnykh V, et al. Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res 2002; 8: 275–80. Breyer B, Jiang W, Cheng H et al. Adenoviral vector mediated gene transfer for human gene therapy. Curr Gene Ther 2001; 1: 149–62. Tai Y, Strobel T, Kufe D, Cannistra S. In vivo cytotoxicity of ovarian cancer cells through selective Bax gene. Cancer Res 1999; 59: 2121–26. Ring C, Blouin P, Martin L, et al. Use of transcriptional regulatory elements of the MUC1 and ERBB2 genes to drive tumor selective expression of a prodrug activating enzyme. Gene Ther 1997; 4: 1045–52. Ichige K, Perey L, Vogel C, et al. Expression of the DF3-P epitope in human ovarian carcinomas. Clin Cancer Res 1995; 1: 565–71. Casado E, Nettelbeck DM, Gomez-Navarro J, et al. Transcriptional targeting for ovarian cancer gene therapy. Gynecol Oncol 2001; 82: 229–37.
THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
p53 and ovarian cancer
Why did p53 gene therapy fail in ovarian cancer? Alain G Zeimet and Christian Marth
Promising preclinical and clinical data led to the initiation of an international randomised phase II/III trial of p53 genetherapy trial for first-line treatment of patients with ovarian cancer. In that trial, replication-deficient adenoviral vectors carrying wild-type p53 were given intraperitoneally in combination with standard chemotherapy to patients with ovarian cancers harbouring p53 mutations. The study was closed after the first interim analysis because an adequate therapeutic benefit was not shown. In this review, we discuss the possible reasons for failure of p53 gene therapy, which include the multiple genetic changes in cancer and epigenetic dysregulations leading to aberrant silencing of genes. These complex interactions lead us to conclude that repair of single genes might not be a suitable strategy for the treatment of cancer. Moreover, dominant negative cross talk between ectopic wild-type p53 and recently identified dominant p53 mutants and splice variants of p63 and p73— which are frequently overexpressed in ovarian cancers— could seriously compromise the effectiveness of p53 gene therapy. Other substantial problems in targeting tumour cells with adenoviral vectors are the heterogeneity or lack of expression of coxsackie-adenovirus receptors and integrin co-receptors in ovarian tumours and the presence of adenovirus-neutralising antibodies in ovarian cancerrelated ascites. Lancet Oncol 2003 4: 415–22
Correction of specific genetic defects responsible for the aberrant biological behaviour of cancer cells is a fascinating new approach for cancer treatment. The high frequency of p53 mutations in human cancers and the central role of p53 in regulating growth and apoptosis led researchers to believe that it would be an appealing target for gene replacement therapy. Ovarian cancer is regarded as a particularly good candidate for gene transfer using viral vectors because the disease generally remains confined to the abdominal cavity throughout its course, enabling intraperitoneal delivery of therapy (figure 1). This approach has the advantage of producing high viral loads close to the tumour and adverse events are controlled because there is a delay before the vector gets into the systemic circulation. Promising preclinical data on in-vitro transfection of wild-type p53 into ovarian cancer cells with p53 mutations and into xenograft models, coupled with evidence from phase I trials, led to the introduction of this innovative technique for front-line treatment of patients with advanced ovarian cancer.1–3 Thus, a large international p53 gene-therapy study in patients with primary stage III ovarian cancer with p53 mutations was set up. In this phase II/III trial, patients with no or residual disease (less than 2 cm in diameter) who had undergone surgical cytoreduction were THE LANCET Oncology Vol 4 July 2003
Figure 1. Intraperitoneal delivery of p53 gene therapy for ovarian cancer.
AGZ and CM are both professors at the Gynaecologic Oncology Unit of the Department of Obstetrics and Gynaecology, Innsbruck University Hospital, Austria. CM is the head of that department. Correspondence: Dr Alain G Zeimet, Department of Obstetrics and Gynaecology, Innsbruck University Hospital, Anichstrasse 35, 6020 Innsbruck, Austria. Tel: +43 (0)512 504 3050. Fax: +43 (0)512 504 3055. Email: [email protected]
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
415
Review randomly assigned to receive either standard therapy of six systemic cycles of carboplatin and paclitaxel or the same regimen plus five cycles of gene therapy. Each 5-day genetherapy cycle consisted of intraperitoneal administration of 1013 particles a day of replication-deficient wild-type p53 delivered in a serotype-5 adenovirus (figure 2). The first interim analysis of this study showed that addition of p53 gene therapy to standard treatment did not improve therapeutic effectiveness in patients with optimally debulked advanced ovarian cancer and actually increased treatment morbidity. This unexpected result led to the study being closed. Some questions need to be addressed concerning these disappointing results. The first is whether this failure means the end for this novel therapeutic approach in ovarian cancer. The answer is clearly no. On the contrary, it should be the starting point from which to make improvements. Thus, for future attempts at gene therapy the reasons why p53 gene therapy failed to work in patients with ovarian cancer are very important—the reasons for failure are likely to be multiple.
Multiple genetic and epigenetic changes Malignant transformation is known to be a multistep process involving changes in several genes. In addition to p53, aberrations of C-ERBB2, C-MYC, K-RAS, and several others have an important role in ovarian cancer.4 Protein products of these genes—although core molecules in cellular pathways such as apoptosis, differentiation, proliferation, and cell-cycle control—are generally not final effectors but intermediate steps in signalling cascades. p53 is directly inactivated in about half of human cancers through mutation or loss and, in most tumours characterised to date, disruption of the p53 tumour suppressor pathway, at one or more positions, is thought to contribute to the malignant phenotype. Consequently, therapeutic replacement of a gene such as p53 can only restore full functionality to the corresponding pathway if none of the downstream genes are affected. Indeed, a subset of ECV-304 tumour cells selected for resistance to p53-mediated apoptosis by repeated p53 wildtype transductions were shown to be deficient in upregulating downstream proline oxidase, which is involved in the proline/pyrroline-5-carboxylate redox cycle.5 Furthermore, about 77% of primary ovarian cancers have been found to have serious impairments in cytochrome-cdependent apoptosome activation because of their inability to activate either caspase 9 or caspase 3, which are important members of the p53-induced caspase cascade (figure 3).6 In addition to loss or mutation of genes, there is growing evidence that epigenetic phenomena, such as the state of methylation of CpG islands in promoter regions of genes, and the consequent extent of histone acetylation, have a substantial role in the evolution and progression of the malignant phenotype.7,8 Epigenetic dysregulation is believed to result in either aberrant gene silencing9 or possibly gene overexpression.10 This suggests that the presence of a defined gene in its wild-type state in the genome of malignant cells is no guarantee for its accurate expression and functionality. Recent data have shown that the p53-mediated death cascade can be subverted by DNA methylation. Soengas and
416
p53 and ovarian cancer
Randomised phase II/III study in primary FIGO stage III ovarian cancer Primary surgical debulking No residual tumour, or ⭐2 cm DNA sequencing Evidenced p53 null or p53 mutated status 1 cycle carboplatin/paclitaxel Randomisation
iv carboplatin/ paclitaxel (d1) plus ip p53 gene therapy (d1–5) cycle 2 to 6
Control group iv carboplatin/ paclitaxel (d1) cycle 2 to 6
Figure 2. Study design of the phase II/III randomised international trial of p53 gene therapy for the first-line treatment of patients with optimally debulked, advanced stage (FIGO stage III), ovarian cancer.
colleagues reported that one downstream component of the p53-dependent death cascade, apoptotic protease-activating factor 1 (APAF1), is not expressed in malignant melanomas, which can then escape apoptosis when treated with chemotherapy. Although the promoter region of APAF1 was not found to be hypermethylated, treatment with methylation-inhibiting agents restored expression of the gene. Thus, it is conceivable that methylation inhibitors reactivate a yet unspecified APAF1 regulator or demethylate another control region of the gene (figure 3).11 Similarly, defective APAF1 activity was also shown in a subset of ovarian cancer cell lines that were resistant to induction of p53-mediated apoptosis.12 Thus, aberrant methylation in genes controlling the expression of APAF1 that are associated with mutated or lost p53 could seriously compromise p53 replacement therapy. Overall, epigenetic phenomena add a completely new dimension to our basic understanding of the molecular biology of cancer and should be taken into account in future gene-therapy approaches. Ono and colleagues used cDNA microarrays to identify 55 genes that were commonly upregulated and 48 genes that were downregulated in ovarian carcinomas. The group also reported 115 genes that were differentially expressed in serous and mucinous adenocarcinomas of the ovary.13 Furthermore, the different cellular signalling pathways cannot be understood by focusing on each isolated path and it is crucial to consider the tangled networks into which these signalling pathways are integrated. It seems optimistic to believe that cancer can be cured by repairing one single aberrant gene and it is tempting to speculate that therapeutic strategies of multigene repair, focusing on a several key genes involved in the development and progression of cancer, might be more effective than singlegene therapy. THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
Review
p53 and ovarian cancer
Problems with the transgene
operation with DNA-binding protein MIZ1 to the p21WAF1 Was p53 the best transgene for the intended therapeutic promoter, blocking its induction by activators such as p53 and purpose? Or would another gene have been more suitable? switching the p53-dependent cellular response from cytostatic There were two convincing arguments in favour of to apoptotic.23,24 Despite rapid advances in the study of p53, therapeutic restoration of wild-type p53 in altered malignant the exact mechanisms governing this pivotal cellular decision cells. First, p53 is a master regulator of the apoptotic pathway have not been elucidated. Nonetheless, it seems that and co-ordinates programmed cell death at many levels malignant cells are generally more likely to undergo apoptosis though numerous mechanisms and apoptotic pathways are after p53 activation than normal cells which tend to undergo believed to contribute decisively to the cytotoxic action of cell-cycle arrest. Because of this difference, therapies based on several chemotherapeutic drugs. And second, loss or p53 reactivation have been developed to selectively promote mutation of p53 has been causally linked to chemoresistance apoptosis in tumour cells. However, some normal cell types in various tumours, including ovarian cancer.14–16 Therefore, a such as intestinal epithelium and haematopoietic cells are rational hypothesis for treatment of these cancers is to attempt sensitive to p53-induced death even in a non-transformed repair of the p53-dependent death programme in the affected state. Since vector targeting is far from being tumour-cell cells, and then attack them with cytotoxic agents. However, it specific, this discrepancy could account for side-effects of is important to note that not all chemotherapeutic drugs externally delivered p53 or for more severe toxic effects when require p53 for their apoptotic function and the overall gene transfer is combined with chemotherapy.25 However, contribution of apoptotic defects to clinical drug resistance is data about the real susceptibility of malignant cells to cellcycle arrest and DNA repair in response to DNA-damaging still debatable.17 Another question concerns the cellular response to p53 agents after restoration of wild-type p53 is inconclusive. activation. What factors determine whether p53 induces Although studies in p53-deficient SK-OV-3 ovarian-cancer apoptosis or the potentially reversible p21WAF1-mediated xenografts have shown that transfection of wild-type p53 cell-cycle arrest (figure 3)? Vousden proposed several before treatment with cisplatin induces apoptosis, but does molecular mechanisms for this decision pathway, of which not improve DNA repair,26 it is conceivable that therapeutic one of the most promising is based on the intracellular effects of concomitantly administered cytotoxic drugs can be concentration of p53. Low concentrations cause cell-cycle attenuated or even completely neutralised by DNA repair if arrest, which turns to apoptosis as p53 concentrations reactivated p53 pathways in tumour cells favour cell-cycle increase. The molecular basis for this hypothesis is the lower arrest over cell death. In some tumour types permanent loss of affinity of promoters of the apoptotic target genes for p53.18 p53 was found to increase cell death induced by doxorubicin More recent studies have shown how transcriptional cofactors or platinum drugs.27–29 However, these findings contrast with such as the ASPP apoptotic-enhancer proteins, the junction the outcome of phase I and II trials of non-small-cell lung mediating and regulatory protein JMY, and several others, are cancer in which intratumoural injections of an adenovirus able to modulate the affinity of p53 for its target promoters carrying p53 combined with cisplatin, carboplatin, and and thus are involved in the choice between cell-cycle arrest paclitaxel, or cisplatin and vinorelbine resulted in locoregional and apoptosis. The activity of ASPP proteins is of particular activity. Clinical proof of principle was clearly achieved in interest because their expression is required for p53-induced apoptosis and frequently they are downregulated in breast cancer; mutated this constitutes yet another mechanism BAX MYC JunD RAS by which tumours can avoid p5319 Furthermore, induced cell death. p53 several investigations have provided DAPK p14ARF HDM2 evidence that the cyclin-dependent Mitochondria kinase inhibitor p21WAF1 is a critical downstream checkpoint in the BCL2 differential response after p53 activation. E2 F1 Cytochrome c release In addition to its close association with cell-cycle control (in regulating transition from G1 to S phase), APAF1 p21WAF1 was also found to have The antiapoptotic properties.20–22 unknown Gene silencing through important role of p21WAF1 in decisionAPAF1 DNA methylation Activation of the activator making was further underscored in a in promotor region caspase cascade paper by Seoane and co-workers, which reported a pivotal role of mitogenic transcripton factor MYC in influencing Apoptosis The p53-mediated apoptotic pathway the outcome of p53 response to DNA damage in favour of cell death. The group revealed that MYC binds in co- Figure 3. The principle p53 pathways leading either to apoptosis or cell-cycle arrest. THE LANCET Oncology Vol 4 July 2003
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
417
Review
p53 and ovarian cancer
One important question is why the complexity of genetic and epigenetic changes in tumours and the limitations of the p53 transgene go a long way to explaining the failure of the clinical gene-therapy trial in ovarian cancer, when in preclinical ovarian cancer
418
Adenoviral vector
␣v3
␣v5
Preclinical research
␣v
3
␣v
5
␣v3
5 ␣v
these studies as shown by greater regression of injected lesions xenograft models intraperitoneal p53 replacement therapy compared with control lesions and by progression of distant showed promising activity. First, it is important to note that metastases.30–32 In the phase II trial of these combinations of inhibition of tumour growth was observed in most but not all drugs, locoregional response rates could not be verified with of these xenogeneic transplant models. In a nude mouse classic radiographical response criteria, although trends model, von Grueningen and co-workers showed effective toward greater tumour shrinkage of targeted lesions were growth inhibition but it was found not to be specifically reported by use of size criteria. Nevertheless, the findings led attributable to p53 gene transfer.41 Furthermore, a review of the authors to conclude that there was no clear evidence of available preclinical studies revealed that only a small number significant improvement with ectopic p53 replacement of different ovarian-cancer cell lines were used in engraftment studies. Preclinical data of the efficacy of p53 gene transfer in therapy in these settings.30,31 Another obstacle, which may compromise the efficacy of combination with chemotherapy in vivo are less clear than gene replacement of wild-type p53, could be the necessity of generally assumed. Kigawa and colleagues reported a p53 tetramerisation for transregulation of downstream genes. significant short-term survival advantage in SCID mice The dominant negative hypothesis relating to tetramers of treated intraperitoneally with p53 gene therapy in conjunction mutant and wild-type p53 has been refuted in several with cisplatin compared with single treatments. However, investigations,33–35 and wild-type p53 was considered to be when combined therapy was given repeatedly, the researchers phenotypically dominant over its mutant forms. However, could not identify significant long-term survival advantages.42 Monti and colleagues recently showed that roughly 30% of Thus, the main limitations of xenograft models in this context p53 mutants are dominant over wild-type p53 and also that might be the result of short intervals between engraftment and dominance is target-gene specific. Genes harbouring response treatment and short follow-up. In addition, in sporadic elements with a low affinity for wild-type p53 such as BAX tumours the reasons for treatment failure are not necessarily (figure 3) or PIG3 were much more sensitive to dominant- the same for each tumour. For instance, not all ovarian negative inhibition by p53 mutants than genes with high- cancers overexpress p73EX2DEL or are affected by a p53 affinity response elements such as p21WAF1.36 Furthermore, mutation which has negative dominance over wild-type the apoptotic pathway seems more likely to be affected by this p53.36,40 Also, it is questionable whether xenogeneic transplant dominant-negative cross talk than p53-induced cell-cycle models based on a limited number of established cell lines are arrest with subsequent DNA repair. Some splice variants of representative of all sporadic tumours and can mimic the p53-related genes, such as ⌬NP73, ⌬NP63, and p73-EX2DEL, heterogeneity and genetic instability that occurs in which lack the transactivation domain have recently been progression of sporadic ovarian cancers. identified. The intact oligomerisation domain of these proteins enables hetero-oligomer formation with wild-type Adenoviral vectors and intraperitoneal delivery p53, and they have been found to act as dominant-negative The adenoviral vector system and the intraperitoneal mode of inhibitors of transactivation and the apoptotic ability of vector delivery could be major causes of the failure of the p53.37–39 Overexpression of p73EX2DEL was shown in a large clinical trial. Entry of adenovirus into target cells is the rateproportion of primary ovarian cancers and ovarian cancer cell limiting step in ectopic gene transfer and determines lines.40 Therefore, such dominant-negative cross talk between therapeutic efficacy of adenovirus-based gene therapy. wild-type p53 and dominant p53 mutants or variants of p53- Attachment of adenoviruses to the cell surface is mediated by related proteins (especially when the latter are overexpressed in tumours) Internalisation Attachment could negatively affect p53 gene therapy. These aspects were not considered in the disappointing trial of p53 gene therapy in patients with Integrins ␣v3 ovarian cancer because when the Integrins protocol was devised the dominant␣v5 negative hypothesis on p53 mutants ␣v3 had been refuted and the other Nucleus Nucleus CAR members of the p53 family were not yet Adenoviral CAR known or their function was not fully vector ␣v ␣v5 3 understood. ␣v5
Target cell membrane
Target cell membrane
Figure 4. Entry of a serotype-5 adenovirus into a target cell.
THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
Review
p53 and ovarian cancer
the coxsackie-adenovirus receptor (CAR), and internalisation is achieved via interactions with integrins of the ␣v3 and ␣v5 classes (figure 4).43,44 Deficiency in one or both of these membranous molecule classes has been reported to confer relative resistance to adenoviral vectors.45–47 Marked differences in CAR expression were noted in primary cultured ovarian cancer cells,1 and in addition to total absence of CAR or both classes of integrins, a pronounced intratumour heterogeneity in the expression of these molecules was detected immunohistochemically in ovarian tumours, which could account for negative selection during adenovirus-based therapy. Ovarian cancers classified as undifferentiated were found to completely lack expression of ␣v3 integrins.48 Furthermore, Bruning and colleagues reported that loss of ␣v3 integrins and especially the 3 subunit compromises adenoviral transduction efficiency in established ovarian cancer cell lines.49 According to our immunohistochemical data, only 40% of the randomly investigated ovarian cancers would meet the criteria to be adequately and evenly targeted by adenoviral vectors.48 Whether or not other molecules of the cell membrane such as ␣v1 integrins are involved in virus uptake or can bypass the defective classic pathway of adenoviral infection remains to be clarified.50 However, to overcome limitations posed by wide variability in the expression of molecules regulating cell entry of adenoviruses, adenoviral vectors were retargeted to specifically enter cancer cells, either by the insertion of targeting moieties, such as the arginine-glycine-aspartic acid (RGD) peptide motif in the viral fibre knob protein,51,52 or by use of bispecific antibody conjugates consisting of the Fab-fragment of an antiadenovirus knob antibody and an antibody of choice directed against a defined molecule readily expressed in malignant cells. For instance, adenoviral targeting with bispecific antibodies via the tumour-associated glycoprotein TAG72—which is expressed on virtually all ovarian carcinomas but not on the surrounding normal mesothelial cells—resulted in 28 times better gene transfer to ovarian cancer cells.53 The use of a genetically modified adenoviral vector containing a TAG72-specific epitope in the viral fibre protein is currently being explored for gene therapy in ovarian cancer. Targeting via other molecules such as growth factor receptors—eg, epidermal growth-factor receptor or basic fibroblast growth-factor receptor—which are overexpressed in malignant disease has also been done.54,55 Overall, the generation of tropism-modified adenoviral vectors is a very promising approach and should provide CAR-independent vectors with a higher transduction efficiency and an improved tumour selectivity in the near future. Locoregional administration of adenoviral vectors has long been considered opportune, not only because of the high vector loads delivered directly to target cells but also as a means of avoiding vector neutralisation by systemic antibodies—those for adenoviruses are highly prevalent in the general population. However, significant in-vitro inhibition of adenovirus-mediated gene transfer has been shown in the presence of ascitic fluid from patients with ovarian cancer. Depletion of antibodies directed against the adenoviral fibre protein from the ascites resulted in a large but not total elimination of the inhibitory activity,56 indicating that THE LANCET Oncology Vol 4 July 2003
Pseudocyst
Figure 5. Computed tomography of a peritoneal pseudocyst after four courses of intraperitoneally administered adenovirus-based p53 gene therapy.
components of the ascites other than neutralising antibodies, such as fibronectin, fibrinogen, and vitronectin (which have high binding affinity for ␣v integrins), may contribute to the obstruction of vector internalisation. Moreover, splice variants of CAR which lack a transmembrane domain but have an intact adenovirus-binding domain have been identified.57 Thus, it is tempting to speculate that these splice variants, when shed into the ascitic fluid, may compromise adenoviral attachment to target cells by opsonising the fibre knobs of adenoviral vectors. Intraperitoneal drug delivery via an implanted catheter system, as described in the protocol of the p53 gene therapy trial, seems at first to be a comfortable means delivering anticancer treatment close to the tumour, especially in ovarian cancer which generally remains confined to the abdominal cavity. However, in practice the therapeutic window for this locoregional mode of drug administration was found to be small. Problems associated with intraperitoneal catheter delivery such as infections, bowel perforations, and obstructions were the limiting and most serious adverse events in phase I of the trial.2 Successful intraperitoneal treatment requires even distribution of the vector throughout the entire abdominal cavity. Because of peritoneal adhesion and pseudocyst formation after repeated administration, the chances of vector sequestration were found to be high; thus, large areas of the peritoneal cavity remained unaffected by the drug (figure 5). Normal human and murine mesothelial cells express the molecules required for adenoviral cell entry and infection of these cells was shown in vivo.58,59 However it remains to be established to what extent virus-triggered inflammation and apoptosis of peritoneal mesothelium account for the development of intraperitoneal adhesions and peritonitis-like conditions. Furthermore, earlier studies revealed that the effectiveness of intraperitoneal treatment with conventional cytotoxic agents is highly dependent on the diameter of the residual tumour nodules or plaques, with best responses in tumours less than 0·5 cm in diameter.60,61 The limitations of penetration of drug to the central part of the tumour are thought to explain this phenomenon. This situation should also be true
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
419
Review for adenoviral vectors with p53 when given intraperitoneally, and therefore the inclusion of patients with residual tumours up to 2 cm in diameter should be seriously questioned.
Host immune defence Neutralising antiadenoviral antibodies in the ascitic fluid of patients with ovarian cancer seem to be a major obstacle in tumour-cell targeting and are strongly suspected of adversely affecting the outcome of intraperitoneal adenoviral-based gene therapy.56 To what extent the activated complement system participates in the elimination of adenoviral vectors in the abdominal cavity either via enhanced phagocytosis or more importantly via immediate complement-directed virolysis remains to be determined although such interactions have been described for other virus types.62 However, several investigations have shown that adenoviral vectors trigger an MHC-class-Irestricted cytotoxic-T-lymphocyte response to viral antigens, which is sufficient to destroy virus-infected cells.63 This bystander effect of the cellular immune system could in fact increase therapeutic effectiveness of gene transfer by its beneficial contribution to tumour-cell kill. Non-specific p53 antitumour activity has been reported in breast and ovarian cancer xenografts in immunodeficient mice treated with empty adenoviral vectors and has been attributed to residual immunological functions.41,64 Moreover, the cytotoxic immune response directed against vector-infected cells should be larger than that triggered by genuine adenoviruses because recombinant replication-defective adenoviral vectors also used in the trial lack the genes encoding for early proteins E1 and E3. These proteins are vital elements in the molecular mechanisms leading to immune evasion of unmodified adenoviruses and work by blocking interferon-induced gene expression, preventing death-receptor-mediated apoptosis through FAS and TRAIL receptor removal from the cell surface and through interference with the cytolytic and proinflammatory activities of tumour necrosis factor, and by subverting MHC class I antigen presentation.65 Overall, these functions drastically limit lysis of cells infected by genuine adenoviruses, either by natural killer cells or cytotoxic T lymphocytes. However, because vector targeting is not tumour-cell specific, this amplified immune response caused by adenoviral vectors should be as harmful to normal tissue as to tumour cells and thus may contribute to intraperitoneal inflammatory conditions and other sideeffects, which could impair therapeutic outcome or even preclude continuation of treatment.2,3 Another phenomenon that can compromise the efficacy of gene therapy is the innate antiadenoviral immunity of cells. Infection with adenoviral vectors induces the production of cytokines, such as interferon ␥ and TNF␣, both of which have been shown to significantly inhibit the expression of transgenes directed by the human cytomegalovirus promoter.66 The E1/E3-deficient state of adenoviral vectors is considered largely responsible for this inhibitory effect on transgene expression. This cytokinerelated promoter attenuation may partially explain discrepancies in p53-transgene expression in various cell
420
p53 and ovarian cancer
Search strategy and selection criteria Data for this review were identified by searches of MEDLINE, Current Contents, and PubMed, with the search terms “gene therapy”, “ovarian cancer”, “p53”, “p21”, “apoptosis”, “adenoviral vector”, “coxsackie-adenovirus receptor”, and “epigenetic”. Reference lists of relevant articles were also searched. Only papers published in English or German between 1980 and 2003 were included.
lines, which could not be primarily ascribed to marked differences in the infectability of these cell lines.67
Future directions What are the prospects for future gene therapy approaches in ovarian cancer? The generation of more efficient vectors to improve tumour-cell targeting by reconsidering alternative types of viral and non-viral transfection systems, such as liposomes, seems to be mandatory. However, it is hard to conceive that non-viral systems will ever give transfection rates as effective as those produced by viral vectors. For millions of years the main job of viruses has been to penetrate cellular systems to ensure survival. Recent endeavours to develop new recombinant viral vectors with modified tropism and higher transduction rates, increased stability of transgene expression, and the ability to bypass the host immune response are the most encouraging and meaningful approaches for clinical translation in the near future.51,68,69 For instance, conditionally replicative viruses for cancer treatment constitute a rapidly evolving new strategy. A new generation of these replicative viral vectors, placing the expression of transgenes under the control of tumour-specific promoters or transcriptional control regions, such as the COX2 or MUC1/DF3 gene promoter in ovarian cancer cells, is of great interest. As shown in preclinical studies, this strategy confers tumour specificity59,70–73 which in turn could represent the rational basis for the use of either suicide genes, like HSVthymidine-kinase,71 or strong proapoptotic transgenes. These classes of transgenes have been shown to cause highly efficient cell killing in various tumour cell lines, but when delivered in unselective vectors they are as harmful to normal cells as to cancer cells and are expected to be associated with a broad range of side-effects in vivo. There is a large body of evidence that in gene therapy the battle has to be won before the treatment stage. Only identification of the genetic and epigenetic signature of the target tumour, including expression of cell-surface molecules governing the cellular uptake of the chosen vector, can provide the information needed to tailor a multigene or multivector treatment for each individual tumour. Although as yet tailoring of treatment remains complex and incomplete, and exact gene expression profiling requires delicate, complicated procedures, continued advances in biotechnology, such as genome-scale microarray analysis and proteomics should help to yield a manufactured but welltailored treatment in the near future. Conflict of interest
None declared.
THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com
Review
p53 and ovarian cancer
References
1 Zeimet AG, Riha K, Berger J, et al. New insights into p53 regulation and gene therapy for cancer. Biochem Pharmacol 2000; 60: 1153–63. 2 Runnebaum IB. Intraperitoneale p53-gentherapie des ovarialkarzinoms in der klinischen prüfung: interimsanalyse eines vielversprechenden ansatzes zur ”kausalen” therapie. Arch Gynecol Obstet 1999; 263: 166–68. 3 Buller RE, Runnebaum IB, Karlan BY, et al. A phase I/II trial of rAD/p53 (SCH 58500) gene replacement in recurrent ovarian cancer. Cancer Gene Therapy 2002; 9: 553–66. 4 Aunoble B, Sanches R, Didier E, Bignon YJ. Major oncogenes and tumor suppressor genes involved in epithelial ovarian cancer. Int J Oncol 2000; 16: 567–76. 5 Maxwell SA, Davis GE. Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis sensitive tumor cells. Proc Natl Acad Sci 2000; 97: 13009–14. 6 Liu JR, Opipari AW, Tan L, et al. Dysfunctional apoptosome activation in ovarian cancer: implications for chemoresistance. Cancer Res 2002; 62: 924–31. 7 Jones PA. DNA methylation errors and cancer. Cancer Res 1996; 56: 2463–67. 8 Baylin SB, Herman JG. DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet 2000; 16: 168–74. 9 Widschwendter M, Berger J, Müller HM, et al. Epigenetic downregulation of the retinoic acid receptor-β2 gene in breast cancer. J Mammar Gland Biol Neopl 2001; 6: 193–201. 10 Lu AP, Gupta A, Li C, et al. Molecular mechanisms for aberrant expression of the human breast cancer specific gene 1 in breast cancer cells: control of transcription by DNA methylation and intronic sequences. Oncogene 2001; 20: 5173–85. 11 Soengas MS, Capodieci P, Polsky D, et al. Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 2001; 409: 207–11. 12 Wolf BB, Schuler M, Li W, et al. Defective cytochrome c-dependent caspase activation in ovarian cancer cell lines due to diminished or absent apoptotic protease activating factor-1 activity. J Biol Chem 2001; 276: 34244–51. 13 Ono K, Tanaka T, Tsunoda T, et al. Identification by c-DNA microarray of genes involved in ovarian carcinogenesis. Cancer Res 2000; 60: 5007–11. 14 Johnstone RW, Ruefli AA, Lowe SW. Apoptosis: a link between cancer genetics and chemotherapy. Cell 2002; 108: 153–64. 15 Kigawa J,Sato S, Shimada M, et al. p53 gene status and chemosensitivity in ovarian cancer. Hum Cell 2001; 14: 165–71. 16 Reles A, Wen WH, Schmider A, et al. Correlation of p53 mutations with resistance to platinum-based chemotherapy and shortened survival in ovarian cancer. Clin Cancer Res 2001; 7: 2984–97. 17 Brown JM, Wouters BG. Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res 1999; 59: 1391–99. 18 Vousden KH. p53: death star. Cell 2000; 103: 691–94. 19 Vousden KH. Activation of the p53 tumor suppressor protein. Biochim Biophys Acta 2002; 1602: 47–59. 20 Lu YJ, Yamagishi N, Yagi T, Takebe H. Mutated p21WAF1/CIP1/SDI1 lacking CDK inhibitory activity fails to prevent apoptosis in human colorectal carcinoma cells. Oncogene 1998; 16: 705–12. 21 Gorospe M, Cirielli C, Wang XT, et al. p21WAF1/CIP1 protects against p53-mediated apoptosis of human melanoma cells. Oncogene 1997; 14: 929–35. 22 Wang Y, Blandino G, Givol D. Induced p21WAF1 expression in H1299 cell line promotes cell scenescence and protects against cytotoxic effects of radiation and doxorubicin. Oncogene 1999; 18: 2643–49 23 Seoane J, Le HV, Massagué J. Myc suppression of the p21Cip1 Cdk inhibitor influences the outcome of the p53 response to DNA damage. Nature 2002; 419: 729–34. 24 Vousden KH. Switching from life to death: The Miz-ing link between Myc and p53. Cancer Cell 2002; 2: 351–52. 25 Komarova EA, Gudkov AV. Suppression of p53: a new approach to overcome side effects of antitumor therapy. Biochemistry 2000; 65: 41–48. 26 Shimada M, Kigawa J, Kanamori Y, et al. Mechanism of the combination effect of wild-type TP53 gene transfection and cisplatin treatment for ovarian cancer xenografts. Eur J Cancer 2000; 36: 1869–75. 27 Bunz F, Hwang PM, Torrance C, et al. Disruption of p53 in human
THE LANCET Oncology Vol 4 July 2003
28 29 30
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
50 51
52 53
cancer cells alters the responses to therapeutic agents. J Clin Invest 1999; 104: 263–69 Zamble DB, Jacks T, Lippard SJ. p53-dependent and independent responses to cisplatin in mouse testicular teratocarcinoma cells. Proc Natl Acad Sci USA 1998; 95: 6163–68. Pestell KE, Hobbs SM, Titley JC, et al. Effect of p53 status on sensitivity to platinum complexes in a human ovarian cancer cell line. Mol Pharmacol 2000; 57: 503–11. Schuler M, Herrmann R, DeGreve JL, et al. Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemo-therapy for advanced non-small cell lung cancer: results of a multicenter phase II study. J Clin Oncol 2001; 19: 1750–58. Swisher SG, Roth JA. p53 Gene therapy for lung cancer. Curr Oncol Rep 2002; 4: 334–40. Swisher SG, Roth JA, Carbone DP. Genetic and immunologic therapies for lung cancer. Semin Oncol 2002; 29 (suppl): 95–101. Chen PL, Chen Y, Bookstein R, Lee WH. Genetic mechanisms of tumor suppression by the human p53 gene. Science 1990; 250: 1576–80. Yonish-Rouach E, Resnitzky D, Lotem J, et al. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin6. Nature 1991; 352: 345–47. Frebourg T, Sadelein M, Ng YS, et al. Equal transcription of wild-type and mutant p53 using bicistronic vectors results in the wild-type phenotype. Cancer Res 1994; 54: 878–81. Monti P, Campomenosi P, Ciribilli Y, et al. Tumour p53 mutations exhibit promoter selective dominance over wild type p53. Oncogene 2002; 21: 1641–48. Yang A, Kaghad M, Wang Y, et al. p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities. Mol Cell 1998; 2: 305–16. Pozniak CD, Radinovic S, Yang A, et al. An anti-apoptotic role for the p53 family member, p73, during developmental neuron death. Science 2000; 289: 304–06 Yang A, Walker N, Bronson R, et al. p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 2000; 404: 99–103. Ng SW, Yiu GK, Liu Y, et al. Analysis of p73 in human borderline and invasive ovarian tumors. Oncogene 2000; 19: 1885–90. Von Grueningen VE, Santoso JT, Coleman RL, et al. In vivo studies of adenovirus-based p53 gene therapy for ovarian cancer. Gynecol Oncol 1998; 69: 197–20. Kigawa J, Sato S, Shimada M, et al. Effect of p53 gene transfer and cisplatin in a peritonitis carcinomatosa model with p53-deficient ovarian cancer cells. Gynecol Oncol 2002; 84: 210–15. Stevenson SC, Rollence M, White B, et al. Human adenovirus serotypes 3 and 5 bind to two different cellular receptors via the fiber head domain. J Virol 1995; 69: 2850–57. Wickham TJ, Mathias P, Cheresh DA, Nemerow GR. Integrins αvβ3 and αvβ5 promote adenovirus internalization but not virus attachment. Cell 1993; 73: 309–19. Sewell DA, Li D, Duan L, et al. Optimizing suicide gene therapy for head and neck cancer. Laryngoscope 1997; 107: 1490–95. Bergelson JM, Cunningham JA, Droguett G, et al. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 1997; 275: 1320–23. Li Y, Pong RC, Bergelson JM, et al. Loss of adenoviral receptor expression in human bladder cancer cells: a potential impact on the efficacy of gene therapy. Cancer Res 1999; 59: 325–30. Zeimet AG, Müller-Holzner E, Schuler A, et al. Determination of molecules regulating gene delivery using adenoviral vectors in ovarian carcinomas. Gene Ther 2002; 9: 1093–100. Bruning A, Kohler T, Quist S, et al. Adenoviral transduction efficiency of ovarian cancer cells can be limited by loss of integrin beta3 subunit expression and increased by reconstitution of integrin alphavbeta3. Hum Gene Ther 2001; 12: 391–99. Li E, Brown SL, Stupack DG, et al. Integrin αvβ1 is an adenovirus coreceptor. J Virol 2001; 75: 5405–09. Dmitriev I, Krasnykh V, MillerCR, et al. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol 1998; 72: 9706–13. Hemminki A, Zinn KR, Liu B, et al. In vivo molecular chemotherapy and noninvasive imaging with an infectivity-enhanced adenovirus. J Natl Cancer Inst 2002; 94: 741–49. Kelly FJ, Miller CR, Buchsbaum DJ, et al. Selectivity of TAG-72targeted adenovirus gene transfer to primary ovarian carcinoma cells
http://oncology.thelancet.com
For personal use. Only reproduce with permission from The Lancet.
421
Review
54
55 56 57 58 59
60
61
62
versus autologous mesothelial cells in vitro. Clin Cancer Res 2000; 6: 4323–33. Miller RM, Buchsbaum DJ, Reynolds PN, et al. Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer. Cancer Res 1998; 58: 5738–48. Goldman CK, Rogers BE, Douglas JT, et al. Targeted gene delivery to Kaposi’s sarcoma cells via the fibroblast growth factor receptor. Cancer Res 1997; 57: 1447–51. Stallwood Y, Fisher KD, Gallimore PH, Mautner V. Neutralisation of adenovirus infectivity by ascitic fluid from ovarian cancer patients. Gene Ther 2000; 7: 637–43 Thoelen I, Magnusson C, Tagerud S, et al. Identification of alternative splice products encoded by the human coxsackie-adenovirus receptor gene. Biochem Biophys Res Comm 2001; 287: 216–22. Kim J, Hwang ES, Kim JS, et al. Intraperitoneal gene therapy with adenoviral-mediated p53 tumor suppressor gene for ovarian cancer model in nude mouse. Cancer Gene Ther 1999; 6: 172–78 Casado E, Gomez-Navarro J, Yamamoto M, et al. Strategies to accomplish targeted expression of transgenes in ovarian cancer for molecular therapeutic applications. Clin Cancer Res 2001; 7: 2496–504. Pujade-Lauraine E, Guastalla JP, Colombo N, et al. Intraperitoneal recombinant interferon gamma in ovarian cancer patients with residual disease at second look laparotomy. J Clin Oncol 1996; 14: 343–50. Alberts DS, Liu PY, Hannigan EV, et al. Intraperitoneal cisplatin plus intravenous cyclophosphamide versus intravenous cisplatin plus intravenous cyclophosphamide for stage III ovarian cancer. N Engl J Med 1996; 335: 1950–55. Stoiber H, Pinter C, Siccardi AG, et al. Efficient destruction of human immunodeficiency virus in human serum by inhibiting the protective
422
p53 and ovarian cancer
63 64 65 66 67 68 69 70 71
72 73
action of complement factor H and decay accelerating factor (DAF, CD55). J Exp Med 1996; 183: 307–10. Yang, Y, Jooss KU, Su Q, et al. Immune response to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Ther 1996; 3: 137–44. Nielsen LL, Dell J, Maxwell E, et al. Efficacy of p53 adenovirusmediated gene therapy against human breast cancer xenografts. Cancer Gene Ther 1997; 4: 129–38. Burgert HG, Ruzsics Z, Obermeier S, et al. Subversion of host defense mechanisms by adenoviruses. Curr Top Microbiol Immunol 2002; 269: 273–318. Qin L, Ding Y, Pahud DR, et al. Promoter attenuation in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum Gene Ther 1997; 8: 2019–29. Hwang ES, Kim J, Kim JS, et al. The effects of the adenovirusmediated wild-type p53 delivery in human epithelial ovarian cancer cell line in vitro and in vivo. Int J Gynecol Cancer 1998; 8: 27–36. Kanerva A, Mikheeva GV, Krasnykh V, et al. Targeting adenovirus to the serotype 3 receptor increases gene transfer efficiency to ovarian cancer cells. Clin Cancer Res 2002; 8: 275–80. Breyer B, Jiang W, Cheng H et al. Adenoviral vector mediated gene transfer for human gene therapy. Curr Gene Ther 2001; 1: 149–62. Tai Y, Strobel T, Kufe D, Cannistra S. In vivo cytotoxicity of ovarian cancer cells through selective Bax gene. Cancer Res 1999; 59: 2121–26. Ring C, Blouin P, Martin L, et al. Use of transcriptional regulatory elements of the MUC1 and ERBB2 genes to drive tumor selective expression of a prodrug activating enzyme. Gene Ther 1997; 4: 1045–52. Ichige K, Perey L, Vogel C, et al. Expression of the DF3-P epitope in human ovarian carcinomas. Clin Cancer Res 1995; 1: 565–71. Casado E, Nettelbeck DM, Gomez-Navarro J, et al. Transcriptional targeting for ovarian cancer gene therapy. Gynecol Oncol 2001; 82: 229–37.
THE LANCET Oncology Vol 4 July 2003
For personal use. Only reproduce with permission from The Lancet.
http://oncology.thelancet.com