Prostate cancer is one of the commonest causes ofillness and death from cancer. Radical prostatectomy,radiotherapy, and hormonal therapy are the mainconventional treatments. However, gene therapy isemerging as a promising adjuvant to conventionalstrategies, and several clinical trials are in progress.Here, we outline several approaches to gene therapy forprostate cancer that have been investigated. Methods ofgene delivery are described, particularly those that havecommonly been used in research on prostate cancer. Wediscuss efforts to achieve tissue-specific gene delivery,focusing on the use of tissue-specific gene promoters.Finally, the present use of gene therapy for prostatecancer is evaluated. The ability to deliver gene-therapyvectors directly to prostate tissue, and to regulate geneexpression in a tissue-specific manner, offers promisefor the use of gene therapy in prostate cancer.
Lancet Oncol 2004; 5: 469–79
Prostate cancer accounts for one in ten cases of cancer inmen.1 It is treated conventionally by radical prostatectomy(for localised disease), external-beam radiotherapy orbrachytherapy, or various types of androgen-withdrawalregimens. At present, conventional treatments control thedisease only for a short time in some patients, and genetherapy continues to emerge as a promising adjuvant totreatment. Several approaches to gene therapy for prostatecancer have been developed, and clinical trials are nowunder way.
Many patients, although they initially show afavourable response to hormone-withdrawal therapy,develop resistance to this treatment regimen over time.Androgen-independent prostate tumours are alsorefractory to other therapies and are an important problemfor the treatment of this disease. Thus, there is a need todevelop strategies for gene therapy that are effective inadvanced prostate cancer.
Successful gene therapy requires an appropriatetherapeutic gene that is delivered efficiently, is expressedtherapeutically for a sufficient amount of time with littleeffect on tissues other than the target tissue, and has fewtoxic effects. Many strategies have been developed,including several for cancer. A wide variety of both viral(figure 1) and non-viral methods of delivery are nowavailable. Attempts to make the therapeutic gene act in atissue-specific way might help ensure selective killing ofcancer cells.
Strategies for gene therapy in prostate cancer A range of approaches to gene therapy for prostate cancerhave been investigated preclinically, and several have beencarried through to phase I clinical studies. Figure 2summarises gene-based therapeutic methods that can beused in prostate cancer.
A first strategy uses vector-based gene augmentation tointroduce genes that encode tumour-suppressor proteins,proapoptotic proteins, cytokines, tumour-specific proteinsas vaccines, antiangiogenic proteins, or prodrug-activationenzymes, to block the growth of prostate-cancer cells orinduce prostate-cancer-specific apoptosis. A second strategyaims to exploit gene expression as a therapeutic target.Large-scale studies of the molecular events associated withdevelopment of prostate cancer have suggested many newpotential therapeutic targets.2,3 Genes that increase
RF is a postdoctoral researcher in the Department of Haematologyand Oncology and ML is Associate Professor of ExperimentalHaematology; both at the Institute of Molecular Medicine, StJames’ Hospital and Trinity College, Dublin, Ireland. DH isProfessor of Clinical Oncology at Trinity College, Dublin, Ireland.
Correspondence: Prof Donal Hollywood, Department ofHaematology and Oncology, Institute of Molecular Medicine, StJames’ Hospital, Dublin 8, Ireland. Tel: +353 1 4065000. Fax: +353 1 4967019. Email: [email protected]
Gene-based therapy in prostate cancer
Ruth Foley, Mark Lawler, and Donal Hollywood
Figure 1. Genes in a recombinant virus are spliced into the DNA of atarget cell.
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Rights were notgranted toinclude this
image inelectronic media.Please refer to
the printedjournal.
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expression with the development of prostate cancer providepotential targets for antisense or antigene methods oftherapy.
Cell-cycle control and apoptosisSelective killing of prostate-cancer cells can be achieved bymanipulation of cell-cycle control or apoptosis. Thisapproach might involve delivery of genes that activateprodrugs, tumour-suppressor genes, proapoptotic genes, orantiangiogenic genes.
The strategy of prodrug activation (also known as thesuicide-gene strategy or genetic prodrug-activation therapy,figure 3) introduces into target cells a gene that codes for adrug-metabolising enzyme: target cells can convert asystemically administered non-toxic prodrug into its toxic
form. Thus, prostate-cancer cells arekilled by the administration of prodrugonly when the appropriate gene codingfor a drug-metabolising enzyme ispresent. Examples of prodrugactivation include the herpes simplexvirus gene for thymidine kinasecombined with ganciclovir prodrug,4
and the cytosine deaminase system that converts flucytosine into 5-fluorouracil.5 The strategy of prodrugactivation has the advantage of beingless dependent on efficient genetransfer than other approaches, owingto the bystander effect of transduced ortransfected cells on neighbouring cellsthat do not express the transgene.Tumour regression was achieved by use of cytosine deaminase and flucytosine in human colorectal-cancercells, in which only 4% of tumour cellscontained the cytosine deaminasegene.6 The combination of the herpessimplex virus gene for thymidinekinase and the prodrug ganciclovir (orvalaciclovir) has been used widely inprostate cancer and other cancers, andis the subject of six clinical trials onprostate cancer.4,7,8–11
Other genes can also be used tocontrol cell proliferation, either directlyor indirectly. Tumour-suppressorgenes such as P53,12 and the proapop-totic genes BAX13 and CASP9,14 inhibittumour growth if overexpressed fromvectors introduced into prostate-cancercells. Introduction of genes that codefor toxins such as diphtheria toxin A15
into prostate-cancer cells might also beeffective if specific delivery to cancercells can be achieved. Viral genes thatelicit toxic effects have also beenexpressed in therapeutic vectors. Forexample, adenoviral vectors have been
designed in which adenoviral genes needed for replicationare expressed in a prostate-specific manner, resulting intissue-specific adenoviral replication and cell lysis.16 Lastly,there has been much interest in the role of angiogenesis intumour growth. For example, genes that encodeantiangiogenic proteins such as thrombospondin 1indirectly inhibit the growth of prostate tumours inimmunodeficient mice.17
Activation of the immune systemManipulation of the immune system to recognise tumourcells as foreign antigens has been a long-term goal of manyimmunotherapeutic approaches to cancer. Activation of animmune response against tumour cells might be achieved byexpression of a tumour protein (eg, prostate-specific
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Oligonucleotidedelivered to tumourtargets specificcellular genes toprevent proteinsynthesis
Delivery of vectorencoding therapeuticgene to tumourcauses transcription and translation oftherapeutic protein
Figure 2. Strategies for prostate-cancer gene therapy. The ability to transfer genes or oligonucleo-tides into prostate tumours, as well as an improved understanding of the molecular biology oftumours, allows for the use of an increasingly wide range of mechanisms to counter tumourgrowth.
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antigen),18 or by transfection of tumour cells with cytokines(eg, interleukin 2 and granulocyte-macrophage colony-stimulating factor).19,20 Shah and co-workers21 used atherapeutic gene to improve indirectly the ability of theimmune system to respond to challenges such as prostatecancer. Mice were treated with a gene that encoded a mutantform of the receptor for transforming growth factor �,which rendered bone-marrow cells insensitive to theimmunosuppressive effects of the growth factor andinhibited metastasis of prostate-cancer xenografts.21
Gene expressionIn vitro, several targeted approaches have been used forprostate cancer, and less commonly in animals or phase Iclinical studies. The androgen receptor is crucial because itstimulates proliferation of prostate-cancer cells by activatingtranscription of target genes. This transcriptional activationhas been competitively inhibited by transfection of double-stranded DNA decoy fragments that include the androgen-receptor binding site.22 Androgen-receptor mRNA has alsobeen targeted by use of an antisense strategy,23 as have theproto-oncogenes BCL224 and MYC.25 Antisense oligo-nucleotides inhibit gene expression by binding to a specifictarget mRNA that has a complementary nucleotidesequence, resulting in degradation and inhibition oftranslation of the target gene.
RNA can also be targeted by use of small interferingRNA—short RNA duplexes that mediate sequence-specificmRNA degradation and have a role in gene silencing ineukaryotes.26 Small interfering RNAs targeted against EZH2inhibited cell proliferation in prostate-cancer cell lines.27
EZH2, a homologue of a Drosophila melanogaster gene thatregulates gene transcription, was selected because micro-array studies identified the gene as significantly upregulatedin metastatic prostate cancer compared with localisedprostate cancer.27
Triplex-forming oligonucleotides offer another mecha-nism for sequence-specific silencing of genes, but they havefewer potential target sequences than antisense strategies orsmall interfering RNAs. Triplex-forming oligonucleotides
bind to double-stranded DNA to form a triple-helix structureand inhibit transcription of genes such as the transcriptionfactor ETS2, which has been implicated in the development ofprostate cancer.28
All of the strategies discussed above require the ability toidentify genes that are upregulated in prostate cancer (or inprogression of prostate cancer) and use gene targeting tointerfere with, or specifically ablate, expression of targetgenes.
Vectors for delivery of therapeutic genesCrucial to any strategy that relies on the introduction offoreign genetic material to cells is the ability to deliver genes tothe appropriate cell or tissue in sufficient numbers to achievea therapeutic effect. Viral vectors are designed to harness theattributes of viruses as delivery agents; the design involvesremoval of many virally encoded genes (generating so-calledgutless vectors) and introduction of the therapeutic gene.Vectors derived from retroviruses, adenoviruses, vacciniavirus, adeno-associated virus, and herpes simplex virus areused extensively.29 Non-viral methods of gene delivery includeuse of liposomes, cationic polymers, and disruption of the cellmembrane by physical methods (eg, electroporation andultrasonography).30 Although bacteria have not been inves-tigated extensively as vehicles for gene delivery, Salmonellatyphimurium has been modified to express prodrug-activationgenes, with resultant antitumour effects.31,32
Viral vectorsThe biological properties of the most widely used viralvectors have been reviewed,29 and are summarised in table 1.Retroviruses29 integrate into the cellular genome after reversetranscription, allowing long-term stable gene expression.Clearly, this integration is beneficial for gene replacement indisorders such as severe combined immunodeficiency,33 butit is less important in most strategies of gene therapy forcancer, in which the intention is to kill the target cells.Integration can also result in serious adverse events ifinsertional mutagenesis activates a proto-oncogene. Adverseevents have been reported in a clinical trial on retroviral genetherapy, in which two cases of uncontrolled proliferation ofT cells were described among ten patients receiving genetherapy for severe combined immunodeficiency (ie, replacement of IL2RG).33 These events have raisedserious concerns about ensuring the safety of gene therapy.
With the exception of lentiviruses, retroviruses cantransduce only dividing cells—an advantage for the targetingof malignant, rather than benign, cells. Although care istaken to ensure that retroviral vectors are replicationdeficient, the possibility of recombination in vivo resultingin a replication-competent vector is an additional safetyconcern for lentiviral vectors derived from HIV.34 Theusefulness of retroviruses is also limited by their smallgenome size (8–9 kb), and the difficulty in obtaining highviral titres during production.34 Despite these obstacles,protocols for research on use of retroviruses as vectorsaccount for many trials on gene therapy for cancer, andretroviruses have been used in two clinical trials on prostatecancer.20,35
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Prodrug activation gene vector
Inactive prodrug Active prodrug
Enzymes
Intratumoural
Intravenous
Figure 3. The strategy of prodrug activation aims to kill tumour cells bycombined treatment with a prodrug and a therapeutic gene. A prodrug isadministered intravenously, but requires a specific prodrug-activationenzyme to convert it to an active cytotoxic derivative. The prodrug-activation enzyme is produced in the tumour after intratumoural injectionof a prodrug-activation gene.
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Adenovirus vectors29 have also been investigatedextensively; adenovirus type 5 is the most commonly usedvector in clinical trials on gene therapy for prostate cancer.These parvoviruses are maintained transiently becausethey do not integrate into the genome and therefore poseno risk of insertional mutagenesis. Furthermore,adenoviruses transduce both dividing and non-dividingcells efficiently,29 and high titres can readily be prepared,4,9
thus offering several advantages over retroviruses.Adenovirus vectors can accommodate inserts of up to 30 kb, but their most important drawback is undoubtedlytheir immunogenicity.
Immune resistance to adenoviruses has causedsubstantial problems for repeated injections of the vector.29
Because transgene expression is transient after a singleinjection,7,11 adenovirus vectors are limited to applications inwhich short-term gene expression is sufficient. Toxic effectsin patients because of inflammatory responses to adenovirusare also an issue. Indeed, the only death of a patientundergoing gene therapy occurred after an immune reactionwhen an adenovirus vector was used to administer theornithine carbamoyltransferase gene (OTC) to a patient witha deficiency in this enzyme36 (although the trial protocol hadnot been followed).37 Adenovirus vectors have been modifiedextensively to decrease their immunogenicity by deletion ofthe E1 gene and other genes, including E2 and E3.4,10
Potentially, all adenovirus genes can be deleted from thevector genome and expressed during production of vectorsby helper viruses, plasmids, or cellular genes.38 Bothreplication-deficient adenoviruses4,7,8,10,39 and attenuatedreplication-competent9,11,16 adenoviruses have been used ingene therapy for prostate cancer.
Vaccinia virus and other poxviruses have emerged aspromising vectors for gene therapy. Unusually for DNAviruses, poxviruses replicate in the cytoplasm of the host cellrather than the nucleus and can carry substantially largerinserts (over 30 kb) than retroviruses.29 Vectors derived fromvaccinia virus elicit a rapid and sustained humoral immuneresponse.40 However, this response has not been associatedwith negative side-effects as much as that to adenoviruses,possibly because of modulation by genes encoded byvaccinia virus.7,10,41,42 Indeed, this property of fewer side-effects has been harnessed to deliver antigens as vaccines.Vaccinia viruses have been used in gene therapy for prostatecancer to deliver a vaccine of prostate-specific antigen(PSA).18,41,42
Adeno-associated viruses integrate in a non-homologous way into chromosomal DNA and can bemaintained there over long periods.43 The ability of thevirus to integrate is beneficial (especially for diseases that
require sustained treatment), but integration also posesrisks, as for retroviruses. Although no adverse eventsrelated to integration of adeno-associated virus have beenreported in clinical trials on gene therapy, it is neverthelessa potential concern. Adeno-associated viruses possiblyhave the best safety profile among the viruses used widelyto date because they have low immunogenicity, few toxiceffects, and no known association with any humandisease.29 However, these advantages are offset by the upperlimit of 4·5 kb on transgene size,29 which prevents theinsertion of long regulatory sequences, multiple genes, oreven a single large gene into vectors based on adeno-associated virus.
Because of the natural tropism of herpes simplex virusfor nerve cells, vectors based on herpes simplex virus type Ihave been used mainly to deliver genes to the CNS.44,45
Herpes simple virus type 1 virions are maintained inneurons as latent episomes, and stable expression for 2 months has been obtained under the control of wildtypepromoters and modified latency-associated promoters.44
Herpes simplex virus vectors have been modified by deletionof viral genes such ICP27, ICP4, and ICP34.5, in efforts toreduce their toxic effects.45 Although the large genome size ofthis virus makes deletion a more complex task than inadenoviruses, the process also offers the useful possibility ofinserting transgenes up to 30 kb in length.29
Non-viral vectorsLiposomes are cationic lipids, or more usually acombination of cationic and neutral lipids, which have beenused for the cellular delivery of drugs46 and to deliverplasmids carrying therapeutic genes. The positive charge ofliposomes facilitates the formation of complexes with DNA,and their lipid structure allows them to cross cellmembranes. Generally, liposomes have been less efficientthan viral vectors, although improved formulations47 andformation of complexes with targeting molecules48 have beendeveloped to improve uptake and decrease the problem ofthe sequestration of vectors in endosomes.49
Similarly, cationic polymers bind to nucleic acids tofacilitate intracellular delivery. In vitro, the dendrimerpolyethyleneimine has been used for transfection ofprostate-cancer cells.50 Plasmids have also been delivered byuse of electroporation, in which cell membranes are madepermeable temporarily by exposure to a strong electrostaticfield. Although used mainly in cell-culture systems,electroporation has also been used to deliver transgenes tosubcutaneous prostate tumours in mice.51
Liposomes, cationic polymers, and similar complexes—eg, poly(D,L-lactide-co-glycolide) and poly(lactic acid)—are
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Table 1. Properties of viral vectors for gene therapy
Vector type Nucleic acid Insert size (kb) Integrates into chromosome? Infects quiescent cells?
Retrovirus Double-stranded RNA 8·0 Yes No (except lentiviruses)
Adenovirus Double-stranded DNA 30·0 No Yes
Poxvirus Double-stranded DNA 30·0 or greater No Yes
Adeno-associated virus Double-stranded DNA 4·5 Yes Yes
Herpes simplex virus Single-stranded DNA 30·0 No Yes
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easy to prepare, cause few toxic effects, and elicit lessimmunogenicity than most viral vectors.30 Indeed, in somecircumstances, DNA can be delivered successfully withoutany carrier molecule. For example, a plasmid that encoded aPSA vaccine elicited a strong immune response in micewhen injected into muscle.52 Methods of chemical deliveryare also suitable for very large DNA molecules. Mammalianartificial chromosomes have been delivered by use ofliposomes and maintained in 70% of cells in vitro for 2 months without the application of selection.53
Thus, non-viral methods of gene delivery, althoughgenerally less efficient than techniques that use viruses,might prove very useful if barriers to their efficacy areovercome. Further research on the stability in vivo of vectorsnot based on viruses, the targeting of specific cell types, entryof vectors into cells, subsequent delivery of DNA to cellnuclei, and avoidance of vector degradation in endosomes,might lead to artificial vectors that have similar efficiencybut a better safety profile than vectors based on viruses.Liposomes have already been used frequently to delivertherapeutic genes in preclinical studies on prostate cancer54
and in several clinical trials, including one trial on prostatecancer.19
Routes of vector administrationThe anatomical route by which a vector is delivered is clearlyan important consideration for safety and efficacy. Vectorsfor gene therapy in clinical trials have been delivered byintratumoural, subcutaneous, intravenous, intramuscular,or intradermal injection, as well as other methods such asbone-marrow transplantation and aerosol application to theairway. Several techniques are suitable strategies for specificdiseases, such as intratumoural injection for cancers,intranasal delivery for cystic fibrosis, and bone-marrowtransplantation for severe combined immunodeficiency orother disorders of haemopoietic cells. Vectors can bedelivered to the skin, vasculature, or muscle for a wide rangeof applications.
Selection of an appropriate method of vectoradministration depends on both the disease in question andthe therapeutic gene to be used. For example, a vectordesigned to cause apoptosis should be restricted to the diseasesite as much as possible, even if this involves a more invasivemethod of delivery. Most clinical trials on gene therapy forpatients with prostate cancer have therefore administeredvectors into the tumour.8,9,16 Injection of vectors into prostatetumours has been facilitated by the routine use of similartechniques during brachytherapy. In a clinical trial10 ofprostate cancer, an adenovirus vector has been injecteddirectly into metastatic lesions of bone and lymph nodes.
If a vector encodes an antigen for the purpose ofimmunisation (eg, PSA), specific delivery to the disease siteis not needed, and the intradermal, subcutaneous, andintramuscular routes can be used.18,41,42,52 In a dose-escalationclinical trial42 on PSA vaccination, lower doses of vector weregiven intradermally and higher doses were givensubcutaneously because subcutaneous administrationdelivers greater volumes of vector. These approaches, as wellas that of intraprostatic injection, restrict gene delivery to the
area immediately surrounding the injection site; however, asmall amount of vector might be absorbed into the systemiccirculation and delivered to other organs, especially the liver.High concentrations of adenovirus vectors have beenreported in the liver after intravenous injection in mice.55
Intravenous administration is also limited by the presence ofserum proteins that inhibit non-virus transfection reagents,49
and this method also might not be suitable for transgenesthat, when expressed, can cause systemic toxic effects. In aphase I clinical trial24 of prostate cancer, intravenous admin-istration was used to deliver antisense oligonucleotidesagainst BCL2. Grade 4 toxic effects occurred, but could havebeen due to the use of docetaxel in the study.24
The risk of inadvertent transduction of non-target cellscan be eliminated completely by gene delivery ex vivo. Thecells are obtained from a patient, cultured and transduced invitro, and returned to the patient. However, technicaldifficulties (especially the culture conditions) have hinderedthe use of gene delivery ex vivo.20
Tissue-specific gene expressionSeveral approaches have aimed to achieve tissue-specificexpression of the therapeutic gene. In prostate cancer,transduction ex vivo20 and intraprostatic injection4,7–9,16 havebeen used for this purpose. However, transduction outside thebody is not always suitable practically, and intraprostaticinjection in animal studies causes some vector disseminationto other tissues.56 Alternatively, tissue specificity could beachieved during the process of expression of the therapeuticgene.
In addition to sequences that code for proteins, eukaryoticgenes include complex regulatory sequences that modify genetranscription and translation. These regulatory sequencesinclude promoters located adjacent to transcription start sites.As well as the basic function of allowing transcription to beginat appropriate sites, promoters can include sequences thatrestrict transcription to specific cell types by makingtranscription dependent on the presence of different factors,or by regulation of transcription through other methods.
Tissue-specific gene promoters, although rarelycompletely specific, can be a very useful way of restrictingeffective gene expression to the target tissue. Much researchhas focused on the development of tissue-specific genepromoters for use in prostate-cancer gene therapy, includingthe challenge of achieving high promoter activity withoutlosing tissue specificity. The ability of a promoter todistinguish the target tissue from liver and bladder is especiallyimportant: high concentrations of vectors have been noted inthe liver after intravenous delivery,55 and in the bladder afterintraprostatic injection,56 in studies in mice and dogs.
Several promoters have been used, or have potential use,in gene therapy for prostate cancer, including therapy formetastatic and androgen-independent disease (table 2).Most promoters have been selected because they havepreferential activity in prostate tissue; some promoters alsodistinguish between benign and malignant prostate tissue.An OTC gene promoter has also been developed to targetboth the epithelium of the prostate tumour and stromal cellsin bone.68
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Promoters such as the promoter of the human PSA gene(KLK3) have a role in improving the efficacy and safety ofvectors that express anticancer genes (figure 4). Commonly,promoters have been modified to increase their expressionwhile maintaining tissue specificity. Activity of the KLK3promoter has been improved by deletion of unnecessarysequences and by the engineering of multiple copies offunctional regions.55 The KLK3 promoter contains 15-bpregions called androgen response elements, which bind theandrogen receptor and are important for the activation ofgene transcription. We have generated recombinant KLK3promoters with sequence variations in androgen responseelement I at sites likely to improveactivity to mimic two mutations inbreast tumours that overexpressedPSA (figure 4), and are undertakingtests in human prostate-cancer celllines.69,70 A hybrid promoter has beengenerated from regions of the KLK3promoter and the promoter of theprostate-specific membrane antigengene FOLH1; the hybrid seems tomaintain the tissue specificity of thePSA sequences while being lessandrogen dependent.71 Modificationof the KLK3 promoter to become lessandrogen dependent might make thepromoter more efficient in somepatients with low androgenconcentrations as a result of hormone-withdrawal therapy.
Yoshimura and colleagues5
overcame the androgen dependence ofthe KLK3 promoter by use of the Crerecombinase system (figure 5). Theresearchers introduced two thera-peutic genes into a mouse withprostate cancer, including a genederived from bacteria that codes forthe enzyme Cre recombinase, underthe control of the KLK3 promoter. Inthe absence of androgen, there wasprostate-specific, but low, expressionof the enzyme. The Cre recombinaseenzyme bound specifically to two loxP
target sites in a second gene, resulting in a recombinationevent and subsequent deletion of a negative regulatoryelement between the two sites. After this modification by Crerecombinase, the second gene caused high expression of thetherapeutic gene cytosine deaminase under the control of ahighly efficient (but not tissue-specific) cytomegaloviruspromoter. Because one Cre recombinase molecule can causerecombination in many vectors, the system was designed toincrease the low expression of tissue-specific transgenes.5
The Cre recombinase system was active even in the absenceof androgen, as was the KLK3 promoter alone afterstimulation by androgens.5
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Gene expressedCell death
Gene expressedCell death
Neighbouringnon-prostate
tissues
A
B C D
Prostatetissue
PSA promoter Proapoptic gene
Vector
Figure 4. Development of prostate-specific gene promoters. For vectors expressing proapoptoticgenes, tissue-specific promoters offer greater safety and specificity to that provided by intratumoralinjection (A). Sequence of the PSA promoter (KLK3) before (B), and after (C, D), mutagenesis.
Table 2. Gene promoters with potential use in prostate-cancer gene therapy
Gene Expression profile Effectiveness in mice Refs
Rat probasin gene Generally restricted to prostate tissue in transgenic mice, but detected at Tumour regression 14, 57low amounts in testes
Kallikrein 3 (KLK3, prostate Used as clinical disease marker Tumour regression 59–63-specific antigen)* Low or absent expression in most non-prostate tissues
Higher expression in benign tissue than malignant tissue Folate hydrolase (FOLH1, Higher expression in malignant tissue than benign tissue Decreased tumour volume 64–67prostate-specific membrane Upregulated expression in the absence of androgensantigen) Variable tissue specificity in vitro Tumour regression 68Mouse osteocalcin gene* Expressed in osteoblasts, calcified smooth muscle, and prostate tumours
*The human kallikrein 3 promoter and mouse osteocalcin promoter have been used in clinical trials.
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The search for prostate cancer-specific expression oftransgenes has also led to several other strategies. Peng andco-workers15 have exploited the overexpression of theproto-oncogene BCL2 in prostate cancer by use of BCL2promoter sequences to direct expression of the gene fordiphtheria toxin A in a study in mice with prostate cancer(table 2). This transgene was engineered further so thatexpression depended on the presence of tamoxifen (figure6).15 Two genes, a fusion gene and a therapeutic gene, weredelivered to prostate tumours in vivo. The fusion gene wasregulated by a BCL2 promoter and encoded two proteins—a bacterial recombinase enzyme and an oestrogen receptormodified to bind tamoxifen—that were joined, but witheach retaining its own function. After the binding oftamoxifen to the oestrogen receptor, the conformation ofthe fusion protein changed so that the recombinase wasactive. The activated recombinase then deleted aninhibitory sequence from the therapeutic-gene construct,resulting in expression of the therapeutic gene under thecontrol of a non-specific but efficient promoter. Therefore,expression of the therapeutic gene occurred only in cellsthat expressed BCL2 (in other cells, the BCL2 promoter wasnot active) and in the presence of tamoxifen (in other cells,the recombinase was not active and the inhibitory sequenceremained in the therapeutic gene).15
Several approaches that used tissue-specific promotershave shown effectiveness against prostate cancer in vivo, andthe OTC10 and KLK316 promoters have both been used inphase I clinical trials on prostate cancer.
Clinical trials and promisingpreclinical studies Suicide-gene therapyGene therapy for prostate cancer basedon prodrug activation, most commonlyby use of the thymidine kinase system,has been investigated in several clinicaltrials. In a phase I trial,4 treatment ofrecurring non-metastatic disease withthymidine kinase and subsequent prodrugadministration caused sustained decreasesin serum concentration of PSA thatranged from 6 weeks to longer than 1 yearin three of 18 patients (although onepatient developed grade 4 thrombo-cytopenia at the highest dose of prodrug).In a phase I/II trial,8 gene therapy withthymidine kinase in an adenovirus vectorwas given in combination with radio-therapy. In another phase I/II trial7 ofthymidine kinase therapy, 78% of patientswith local recurrence of prostate cancerafter radiotherapy achieved partialdecreases in PSA. Most interestingly, asimilar cohort of patients were treated in aphase I trial9 with a replication-competentadenovirus that included a fusion gene fora cytosine deaminase and thymidinekinase. No dose-limiting toxic effects wereseen, and two of 18 patients showed no
evidence of having prostate cancer 1 year after treatment.9
A phase I clinical trial10 of 11 patients with metastatic or locallyrecurrent prostate cancer assessed the thymidine kinase genecombined with the prodrug valaciclovir. An adenovirusvector, which carried the thymidine kinase gene controlled byan OTC promoter, was injected into individual lesions and ledto temporary stabilisation of disease in one patient, and grade1–3 toxic effects.10 Use of cytosine deaminase combined withthe prodrug flucytosine has also shown effectiveness againstprostate cancer in mice.5
By use of mouse xenografts, Freytag and colleagues72 haveassessed trimodal therapy for prostate cancer, consisting of areplication-competent adenovirus vector, a cytosine-deaminase thymidine-kinase fusion gene and prodrugsactivated by both enzymes encoded by the fusion gene, andradiation. The researchers found that trimodal therapyimproved the cure rate and decreased the frequency ofmetastases. However, they also noted that use of viruscombined with external-beam radiotherapy caused synergistictoxic effects. In a phase I clinical trial11 of 15 patients withnewly diagnosed non-metastatic prostate cancer, use oftrimodal therapy did not lead to any dose-limiting toxiceffects, although four cases of grade 3 adverse events werereported that might have been a result of treatment. There wasno evidence of circulating infectious adenovirus, and toxiceffects in the liver were limited to grade 1 or 2, suggesting thattrimodal therapy is safe in patients.11 Patients in this trial whoreceived longer courses of prodrug therapy showed a morerapid decline in serum concentrations of PSA.11
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Non-specificpromoter
Recombinasetarget site
PSApromoter
Recombinasegene
Non-specificpromoter
Therapeuticgene
Inhibitorysequence
Recombinasetarget site
Therapeuticgene
A
Non-specificpromoter
Recombinasetarget site
Inhibitorysequence
Recombinasetarget site
Therapeuticgene
B
C
Not expressed
Therapeuticprotein
Figure 5. The Cre recombinase system amplifies low expression from a prostate-restrictedpromoter.15 Two transgenes were delivered into mouse prostate tumours (A). Recombinaseenzyme was expressed in prostate cells, and deleted an inhibitory sequence in the secondtransgene to activate the gene (B). A therapeutic gene was then highly expressed, indirectlyregulated by the PSA promoter KLK3 (C).
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ImmunotherapyImmunotherapy has also been investigated in thetreatment of prostate cancer. In a phase I trial,19 six of 24 patients with locally advanced or recurrent prostatecancer had a mean decrease in serum concentration of PSAof 39% 10 weeks after treatment with a plasmid thatexpressed interleukin 2 delivered by use of liposomes. Theresearchers reported an increase in the infiltration oflymphocytes into tumours, and no toxic effects higher thangrade 2 were seen.19 An adenovirus vector that expressedinterleukin 2 was administered by intraprostatic injectionto 12 patients with prostate cancer who had clinicallylocalised high-risk disease.39 In this phase I trial, treatmentincreased the presence of cytokines at the vaccine site.Grade 2 or lower toxic effects were seen (apart from in onepatient, who had grade 3 lymphopenia). Transduction ofprostate-cancer cells with granulocyte-monocyte colony-stimulating factor ex vivo followed by intradermalinjection of the transduced cells caused T-cell and B-cellimmune responses against prostate-cancer cells in a phase Iclinical trial.20
A PSA vaccine delivered by amodified vaccinia virus has shown lowtoxic effects and variable efficacy in threephase I trials of prostate cancer,18,41,42 afterstudies in animals showed that a similarstrategy elicited a strong and sustainedimmune response.52 In one of six patientswith recurrent prostate cancer treated byPSA vaccination, disease progression wasdelayed for longer than 8 months afterinterruption of hormonal therapy.18 Atrial41 of PSA vaccination in 33 patientswith advanced prostate cancer showedthat vaccination stabilised disease for11–25 months in nine patients. A PSA-specific T-cell response was seen in fiveof seven patients tested, all of whom alsoreceived granulocyte-monocyte colony-stimulating factor.41 A clinical trial42 ofPSA vaccination involving 42 patientswith metastatic androgen-independentprostate cancer showed a PSA-specificimmune response in four of six patientstested. In studies on mice with prostatecancer, a gene that encoded a modifiedtransforming growth factor � receptorimproved survival by overcoming sup-pression of the immune function of bonemarrow.21
Manipulation of the cell cycle andapoptosisThe tumour-suppressor gene BRCA1 hasbeen administered to patients withadvanced prostate cancer in a phase Iclinical trial35 by intraprostatic injection ofretrovirus without substantial toxic effects.The therapeutic potential of other
tumour-suppressor genes has also been investigated.Restoration of P53 decreased the growth and tumorigenicityof androgen-independent human prostate-cancer cells invivo.73 However, this restoration did not affect a P53-nullmouse prostate-cancer cell line, which was inhibited in vivoby transfection of the P21 gene (another cell-cycle regulator).74
A prostate-specific attenuated replication-competentadenovirus has led to a dose-dependent decrease in serumconcentration of PSA in a phase I trial.16 Tumour regressionhas been seen in mice with prostate tumours xenografted ontothe skeleton after treatment with an attenuated replication-
Review Gene therapy for prostate cancer
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Search strategy and selection criteriaData for this review were identified from PubMed andreferences of relevant articles. Searches used combinations ofthe search terms “prostate cancer”, “gene therapy”, “gene”,“therapy”, “treatment”, “delivery”, “vector”, “tissue-specific”,“prostate-specific”, and “promoter”. Abstracts of importantdevelopments for which no full-text articles were available, andof results from our own laboratory, were included. Only paperspublished in English between 1990 and 2004 were included.
BCL2promoter
Non-specificpromoter
Therapeuticgene
Recombinase ModifiedER
A
B
Non-specificpromoter
Recombinasetarget site
Inhibitorysequence
Therapeuticgene
C
D
Fusion protein
Fusion gene
Recombinaseactivated
Therapeuticprotein
Fusion protein
Tamoxifen
Recombinasetarget site
Figure 6. BCL2-dependent and tamoxifen-dependent expression of transgenes deliveredtogether into mouse prostate tumours. Expression of a recombinase-oestrogen receptor (ER)fusion gene (A). Activation of recombinase by the binding of ER to tamoxifen (B). Recombinasedeletes an inhibitory sequence from a therapeutic gene (C). Expression of therapeutic geneunder indirect control of the BCL2 promoter (D).
477
competent adenovirus controlled by an OTC promoter thattargeted the tumour and bone stroma.68 Survival of micexenografted with LNCaP human prostate-cancer cell lines hasbeen lengthened by treatment with a prostate-specific,inducible caspase 9 gene.14 The genes for diphtheria toxin A15
and FAS ligand75 have caused inhibition of tumour growth inmice with prostate-cancer xenografts; these genes meritfurther study as therapeutic agents.
Antisense approachesA phase I/II clinical trial is under way to ascertain thecombined efficacy and safety of BCL2 antisenseoligonucleotide and docetaxel in progressive metastaticprostate cancer. Four of 12 patients showed decreases inserum concentration of PSA, although one patient had dose-limiting grade 4 toxic effects.24 In studies in mice, antisenseoligonucleotides to MYC caused regression of prostatetumours;25 antisense targeted to the androgen receptor hasalso inhibited tumour growth.23
ConclusionSeveral gene-based approaches have been devised to treatprostate cancer, either by direct control of cancer-cellproliferation, by activation of an immune response against thetumour, or by inhibition of angiogenesis. Adenoviruses,retroviruses, and liposomes are the vectors used mostcommonly to deliver genes to prostate-cancer cells. Prostate-specific gene promoters have been developed to improve thesafety profile of therapeutic vectors. To date, at least 15 phase Ior phase I/II clinical trials on gene therapy for prostate cancerhave been started; in several trials, decreases in concentrationof serum PSA have been achieved. In one trial,9 two patientswith local recurrence of prostate cancer after radiotherapyremained in clinical remission 1 year after receiving genetherapy based on prodrug activation. In another trial,41 25% ofpatients with advanced prostate cancer given a PSA vaccineshowed stable disease for 11–25 months. Other approacheshave led to tumour regression or lengthened survival instudies in animals. With increasing knowledge of themolecular pathology of prostate cancer, new therapeutictargets might also emerge in the near future.
Conflict of interestNone declared.
AcknowledgmentsWe received funding from the Health Research Board, Ireland, and StLuke’s Institute for Cancer Research, Ireland.
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Clinical picture
A 69-year-old woman with nephrolithiasis and end-stagerenal disease presented at our clinic with recurrent, centralabdominal pain, which had persisted for 3 months. She hadbeen on haemodialysis for the past 10 years and had beendiagnosed with bilateral carpal tunnel syndrome.Colonoscopy showed a circumferential tumour at thetransverse colon. After a preoperative diagnosis ofobstructive carcinoma of the colon, the patient had anexploratory laparotomy. A bulky tumour was identified inthe proximal transverse colon adhering to a segment of themid-ileum, which needed an extended right hemicolectomyand an en bloc resection of the ileum. Histological analysisof the resected tissue (figure A) did not show any carcinoma
cells; instead, a pink amorphous deposit at the vessel wall inthe submucosa was noted. The amorphous substance stainedpositively with Congo red and showed green birefringenceunder polarised light.
3 years later, the patient was referred to a gynaecologicaloncologist after she detected a 2-cm mass in her right vulva;she underwent a Tru-cut biopsy. Histological analysis of thevulval tissue showed extensive deposition of a fragmentedpink amorphous substance within fibrous tissue. A diagnosis of dialysis-associated amyloidosis, whichmanifested as a tumour-like subcutaneous vulval mass, wasmade. Positive staining with Congo red (figure B)supported this diagnosis.
Amyloidosis presenting as malignant diseaseK M Chow, P C L Choi, and C C Szeto
Correspondence: Dr Kai Ming Chow, Department of Medicine and Therapeutics, Prince of Wales Hospital, Chinese University of HongKong, Shatin, Hong Kong, China. Tel: +852 2632 3131. Fax: +852 2637 5396. Email: [email protected]
