Download Breast Cancer Gene Therapy

34 Breast Cancer Gene Therapy
Kun-Ming Rau, Chi-Ping Day, and Mien-Chie Hung
34.1 Introduction
Breast cancer is one of the major health threats for women all over the world. It is
the most commonly diagnosed cancer in women and the second leading cause of
cancer death in women in Western society (unpublished data from the Comprehensive Cancer Monitoring Program Meeting in Europe, 2003). In recent years, the
mortality rate of breast cancer has declined slightly [80], thanks to early detection
programs and the advances in therapy, and especially improvements in systemic
therapy, such as new chemotherapeutic agents and aromatase inhibitors. However,
even localized diseases will relapse locally or distantly in a significant proportion
of patients. When disease progresses to the metastatic stage it becomes essentially
incurable and the median survival time is about 2 years. Chemotherapy is the main
treatment at this stage. The response rate of combined chemotherapies ranges from
35% to 67%, and the median response duration is short, usually approximately
9 months [6]. Therefore, finding alternative therapies for patients whose disease is
refractory to chemotherapy or hormone therapy is critical. Gene therapy is one of
the alternative therapies.
Cancer is a genetic disease. Almost all types of cancers exhibit genomic instability [92, 121, 149]. Cancer may arise as a result of hereditary or acquired somatic
mutations. These genetic alternations may occur as changes in a single or a cluster of
nucleotides, microsatellite instability, loss or gain of whole genes, changes in chromosomal structures, or even complete loss of a chromosome. The consequences of
genetic changes leads to loss of tumor suppressive function, to oncogenicity, or to
activation or inactivation of those genes whose products regulate genomic stability.
Familial clustered breast cancers are found in only 10% of all breast cancers cases.
In this group, hereditary breast cancers occur in some syndromes. Although hereditary breast cancers are highly penetrative and are associated with definitive genetic changes, such as BRCA1, BRCA2, and p53, the estimated incidence of these
breast cancers is only about 20% in patients with familial clustered breast cancer.
The majority of other cases of familial clustered breast cancer and sporadic cases
result from the accumulation of multiple somatic genetic changes interacting with
environmental factors.
Once a key mutated gene or a dysregulated signaling pathway is identified in
cancers, conceptually we can transfer a therapeutic gene to cancer cells, reversing
the malignant phenotype by correcting its genetic defects, blocking the abnormal
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signaling, or inducing the death of cancer cells specifically. Such treatment, known
as gene therapy, is the most straightforward, effective treatment for cancer. In recent
years, advances in the technology of gene transfer have made gene therapy feasible
for cancer treatment. The gene therapy system is composed of DNA that contains
the therapeutic gene and a biological or synthetic vector that can be complexed with
DNA and carries the DNA to cancer cells, releasing it inside the cells. In this chapter,
we will discuss the principles of gene therapy, including delivery systems, therapeutic targets, in vitro and preclinical experiments, and ongoing clinical trials of breast
cancer gene therapy.
34.2 DNA Delivery System in Breast Cancer Gene Therapy
Cancer gene therapy is a treatment that transfers DNA to cancer cells, resulting in
the suppression of breast tumor growth or death of cancer cells. For this purpose,
gene carrier agents (i.e., vectors) are used in vitro or in vivo to deliver DNA to cells.
Many types of vectors have been developed for gene therapy, and each vector has its
own advantages and limitations. In general, the most efficient gene delivery agents
are viral vectors, but their immunogenicity or genomic integration usually limits
their use in local or ex vivo treatment. Chemical vectors are less efficient of gene delivery agents than viruses; however, their low immunogenicity and easy preparation
make them attractive in breast cancer gene therapy.
34.2.1 Viral Vectors
34.2.1.1 Adenoviral Vectors
Adenoviruses can carry and transduce the inserted gene to the host cell efficiently,
so they are used widely in gene therapy. Adenoviruses consist of nonenveloped icosahedral protein capsids and inner DNA/protein cores. Serotypes 2 and 5 (Ad2 and
Ad5, respectively) are the most commonly used in the development of gene delivery
vectors. The infection is initiated by the interaction of adenoviral fiber protein with
the cellular Coxsackievirus and adenovirus receptor (CAR), and subsequently the
virus enters the cell through internalization. The lytic cycles of Ad2 and Ad5 are so
efficient that it is easy to produce a high titer of virus particles in infected cells. The
whole replicative process does not require integration of the viral DNA into the host
cell genome [72].
The characteristics of adenoviruses make them excellent vectors for use in cancer
gene therapy. For example, the infection spectrum of adenoviruses is wide with
respect to different types of tissues and cells, and the virus is readily prepared in
high titers with high transducing efficiency [214]. Viral DNA mediates transient and
effective gene expression ectopically without becoming integrated into the cellular
chromosomes and inducing gene mutation of the host cell. More importantly,
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because adenoviral vectors have been used in preclinical and clinical trials of gene
therapy for many years, cost-effective methods for the propagation and purification
of clinical trial-scale quantities of the virus, as well as assays to determine its
identity, purity, integrity, viability, infectivity, potency, stability, safety, toxicity, and
therapeutic efficacy, are well established [214]. In addition, adenoviral vectors can
be easily engineered to generate desired functions, such as tumor targeting [196]
and conditional replication [214]. Despite the many advantages and feasibility of
gene delivery, the immune response induced by virus treatment limits the spread of
the virus, reducing its therapeutic efficacy. Moreover, there are always risks of toxic
immunological reactions to the virus that may induce high fever or organ failure
[214].
Adenoviral vectors have been used in several studies to deliver therapeutic genes
to breast tumor cells. Therapeutic genes include early-region 1A (E1A) [24], proapoptotic genes such as p53 [132], Bcl-xL [170], and Fas [143], intracellular singlechain antibodies such as anti-ErbB2 [194], and dominant-negative estrogen receptor [99]. Several types of oncolytic adenovirus, which replicates specifically in breast
tumor cells, have been reported [70, 192]. In conclusion, adenovirus is an effective
and feasible gene delivery vector, but the immune response and long-term effect
may limit its use in clinical gene therapy for breast cancer.
34.2.1.2 Other Viral Vectors
Retroviruses are RNA-containing viruses that reproduce their genome through reverse transcription. On entering a cell, the retrovirus RNA is reverse transcribed
into DNA, which then randomly becomes integrated into the host genome. Retroviral vectors have been used to deliver genes to hepatic metastatic breast cancer
in nude mice [162]. However, the ability of DNA to integrate into the host genome
becomes the drawback for cancer gene therapy, because the integration of therapeutic genes into normal cell genomes may cause long-term damage to the host gene.
Thus, retroviruses are not recommended for breast cancer gene therapy. Other viral
vectors, such as adenoassociated virus and herpes simplex virus (HSV), are primarily used to transduce genes ex vivo in cancer gene therapy, but we will not elaborate
on these vectors in this chapter.
34.2.2 Chemical and Biochemical Vectors
Many types of lipids and polymers can bind with DNA to form aggregates. These
aggregates can either interact with receptors on the cell surface or even fuse with
the plasma membrane, delivering DNA into cells. These chemicals, therefore, can
be used as gene therapy vectors. Recently, amphiphilic or modified peptides were
developed as gene carriers. These chemical and biochemical vectors are described
below.
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34.2.2.1 Cationic Liposomes
A cationic liposome consists of a cationic lipid and a neutral helper lipid. Cationic
liposomes are the most extensively investigated nonviral vectors. In the past decades, a large number of cationic lipids, such as 3-β-[N-(N’,N’-dimethylaminoethane)-carbamoyl] cholesterol plus dioleoylphosphatidyl-ethanolamine plus plasmid
DNA (together known as DC-Chol liposome), DC-Chol liposome/polymer/DNA
(known as LPD-1), stabilized nonviral cationic liposome (SN), and extruded 1,2-dioleoyl-3-trimethyl-ammoniumpropane cholesterol (DOTAP:chol), were developed
for cancer gene therapy. These positively charged lipids form electrostatic complexes
with the negatively charged plasmid DNA, forming lipoplexes [74]. Cells take up
these complexes and transport them into the nucleus. Compared with viral vectors,
lipoplexes are easy to produce and relatively nonimmunogenic. The transfection efficiency of lipoplexes is lower than that of viruses, however the low immunogenicity
and toxicity of lipoplexes allows repetitive administration to patients, thus increasing the overall efficiency of gene delivery. Lipoplexes have been used successfully
in preclinical models and clinical trials of cancer gene therapy. Different versions
of lipoplexes have been developed to enhance the efficiency of systemic delivery.
Steric stabilization of liposomes can increase their biocompatibility and stability in
vivo, delaying their clearance by the reticuloendothelial system. Lipid molecules can
also be linked with protein factors to promote specific interactions with tumor cells.
These improvements are expected to greatly enhance the application of liposomes in
gene therapy. Several liposomes are being evaluated in preclinical and clinical trials
of breast cancer gene therapy.
DC-chol Liposome
DC-chol liposome consists of 3-β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]
cholesterol (serving as cationic lipid), dioleoylphosphatidyl-ethanolamine (DOPE;
serving as helper lipid), and plasmid DNA [74, 120]. This cationic liposome reagent
facilitates efficient DNA-mediated transfection [58]. DC-chol/DOPE liposome has
been used as gene therapy in clinical trials for breast cancer [73], head and neck
cancer [205], cystic fibrosis [22], and melanoma [128]. In these trials, the lipoplex is
administered by intrapleural injection, as aerosolized spray to the nasal epithelium,
or by intratumoral injection.
LPD-1
The introduction of cationic polymers in the appropriate charge ratio to DC-chol/
DOPE liposome may change its overall structure and transfection capacity. This
modified formulation, a DC-chol liposome/polymer/DNA lipoplex, is named LPD1. An LPD-1 formulation, which is composed of 1,2-dioleoyl-3-trimethyl- ammoniumpropane (DOTAP)/cholesterol, protamine, and DNA, has been developed for
intravenous (i.v.) administration. Early studies of gene delivery with LPD-1 com-
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plexes have demonstrated that i.v. administration through the tail vein of nude mice
facilitated the delivery of the DNA to distant organs such as the lung or liver [105,
106]. Thus, the LPD-1 system is an attractive candidate for the systemic delivery of
therapeutic genes to treat advanced or metastatic cancer. In a recent report, LPD-1
was used to deliver the E1A gene to tumor xenograft models of breast cancer and
head and neck cancer [178]. The results demonstrated that LPD-1 led to effective
delivery and subsequent expression of E1A at the tumor site, resulting in tumor suppression and increased survival in animals treated with LPD-E1A, either alone or in
combination with paclitaxel.
Stabilized Nonviral Cationic Liposome
Another efficient in vivo gene delivery system, stabilized non-viral cationic liposome
(SN), has recently been developed. Like LPD-1, SN is also a polymer-modified liposome, which consists of 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-ethylphosphoethanolamine-N-polyethylene glycol-5000,
and polyethyleneimine (PEI). The transfection efficiency of SN is 5–10 times higher
than that of the common nonviral agents FuGENE6 transfection reagent (Roche,
Indianapolis, USA) and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in the
presence of serum [221]. The proapoptotic gene Bik delivered by SN induced significant apoptosis in breast cancer cell lines in vitro as well as in orthotopic tumor
tissues in nude mice. The systemically administered SN-Bik complex significantly
inhibited the growth and metastasis of human breast cancer cells implanted in nude
mice and prolonged their life span [108, 221].
Extruded DOTAP:chol liposome
A modified procedure to prepare cholesterol-based liposomes was developed for
in vivo gene delivery [171]. Liposomes consisting of cationic lipid, 1,2-dioleoyl-3trimethyl-ammoniumpropane (DOTAP), and neutral lipid, cholesterol (Chol), are
prepared using mild sonication, heating, and extrusion, and are finally dissolved
in water containing 5% dextrose. These extruded DOTAP:chol cationic liposomes
have been shown to deliver reporter genes efficiently to various tissues via i.v. administration, the maximal gene delivery being present in the lung. The extrusion
step during synthesis was critical for the very high gene-transfer efficiency of the
liposomes. The use of extruded DOTAP:chol liposomes in lung cancer gene therapy
has been approved (NIH Human Gene Transfer Protocol #0201-513).
34.2.2.2 Polycationic Polymers
Cationic polymers, such as poly-L-lysine (PLL) derivatives, PEI, polyamidoamine,
and polymethacrylate dendrimers, form electrostatic complexes with the negatively
charged DNA (i.e., polyplex) [185]. Like lipoplex, these complexes are taken up by
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cells via endocytosis, and subsequent endosomal escape; they are then transported
to the nucleus. The transfection efficiency of the cationic polymer-DNA complexes
is superior to that of the liposome-DNA complex. The properties of cationic polymers make them competitive nonviral vectors in gene therapy. Cationic polymers
provide high transfection efficiency for cell culture systems. However, when used
in vivo they face several obstacles, such as nonspecific electrostatic interaction with
tissues and membrane toxicity of the cationic polymers. Recently, there have been
a lot of efforts to modify the cationic polymers for in vivo gene delivery. Despite
their promise, cationic polymer vectors have not yet been approved for clinical trials
[90].
34.3 Strategies of Breast Cancer Gene Therapy
The most distinguishing feature of cancer gene therapy is the versatility of its therapeutic mechanisms. Unlike conventional chemotherapy and radiotherapy, which
can only differentiate rapidly proliferating cells from other cells, gene therapy can
target tumor cells directly and suppress their growth in multiple ways. Modified
vectors or tumor-specific control sequences can be used to target tumor cells. Delivery of therapeutic genes to tumor cells can block oncogenic signaling, block tumorinduced angiogenesis, kill tumor cells directly, or even enhance their immunogenicity. In the following sections we describe the strategies of breast cancer gene therapy
in tumor targeting and suppression. With multiple combinations of mechanisms to
choose from, gene therapy provides specificity and effectiveness for cancer treatment, and can even be tailored for individual patient or disease type to optimize the
therapeutic effect.
34.3.1 Strategies in Tumor Targeting
One of the essential developments in breast cancer gene therapy is its tumor specificity. Because of the altered gene expression of tumors, tumor cells can be distinguished from normal cells by their overexpression of specific membrane receptors,
defective tumor suppression pathways, dysregulated cell cycles, and active oncogenic signaling. In cancer gene therapy, vectors and the genes they carry can be
modified to target the altered genes or cell-signaling pathway, resulting in tumorspecific therapy.
34.3.1.1 Tumor-Targeting Vectors
Adenoviral Vectors
Entry of adenoviral vectors into cells is initiated by binding of the viral fiber knob
domain to the CAR on the cell surface. This process is facilitated by integrin and
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heparan sulfate glycosaminoglycans (HSGs) on the cell surface. To target tumor
cells specifically, viral binding to CAR, integrin, and HSG binding must be abolished and replaced with a tumor-specific interaction. This can be achieved by two
ways. First, a bispecific molecule can be used to block the virus–CAR interaction
and redirect the virus to the tumor-specific receptor. The bispecific molecule should
contain two components: one is the fiber-knob-binding domain and the other is the
tumor-specific ligand. The neutralizing antifiber antibody conjugated to an antiepithelial cellular adhesion molecule (anti-EpCAM) antibody has been used to direct
the adenovirus to the EpCAM antigen present on breast cancer cells [64]. Second,
the adenovirus can be engineered genetically to replace the sequence responsible for
binding the native cellular receptor to the sequence of tumor-specific ligand in the
fiber knob and penton base. For example, the genetically engineered adenoviruses,
into which an arg-gly-asp integrin-binding motif was inserted in the HI loop of the
adenovirus fiber knob, can target breast tumors in the xenograft mouse model [21].
Nonviral Vectors
The presence of ligand on a liposome facilitates the entry of DNA into cells through
initial ligand-receptor binding on the cell surface. The specificity of tumor targeting
can also be enhanced by conjugating cationic polymers with tumor-specific ligands
[185]. For example, the transferrin receptor level is upregulated in various types of
cancer cells, including breast cancer, and is correlated with the aggressiveness of tumor cells. The transfection efficiency of transferring-complexed cationic liposomes
is, as a result, 10–15 times higher than that of cationic liposomes without ligands in
cancer cells [174, 204].
The addition of epidermal growth factor (EGF) and insulin to liposomes has also
been shown to greatly increase transfection efficiency in lung cancer cell lines [204].
Specific antibodies can also be attached to the surface of liposomes, directing them
to antigens on the surface of tumor cells. For example, liposomes conjugated with
the Fab fragment of an anti-erbB2 monoclonal antibody can specifically bind to
erbB2-overexpressing breast cancer cell lines [107, 135]. Recently, Xu et al. reported
that liposomes conjugated with a single-chain Fv fragment (scFv) of an antitransferrin receptor monoclonal antibody delivered genes into breast cancer cells in an
animal model [200].
Polycationic polymer vectors can also be chemically modified and linked with
the tumor-specific ligand to obtain tumor specificity. For example, coating the surface of polyplex with polyethylene glycol (PEG) can block nonspecific interactions,
thus increasing the ratio of polyplex reaching tumors.
34.3.1.2 Tumor-Specific Control Elements
Many genes are specifically dysregulated in tumor cells relative to normal cells. Once
a gene is found to be dysregulated in tumor cells, its regulatory sequences for tran-
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scriptional, posttranscriptional, and translational controls can be isolated to examine which one is responsible for such tumor-specific expression. The identified regulatory elements have great potential to control gene expression in a tumor-specific
manner, and thus can be used to target tumor cells in gene therapy. These elements
include promoters, and the 5’- and 3’-untranslated regions (UTRs) of mRNA.
Tumor-specific promoters (TSPs) can be used to control the transcription of
genes delivered by vectors. The high promoter activity in tumor cells results in tumor-specific gene expression. Many promoters have been explored for gene therapy
in a variety of cancer cell types [68]. For example, the α-fetoprotein promoter in
hepatoma cells [77], tyrosinase promoter in melanoma cells [213], prostate-specific
antigen and probasin promoter in prostate cancer cells [98, 188], beta-catenin/Tcfresponse promoter in colon cancer cells [95], and carcinoembryonic antigen (CEA)
promoter in adenocarcinoma cells [145]. Many promoters have been used to drive
breast-cancer-specific gene expression in cell lines or animal models [68]. These include the hypoxia responsive element, glyceraldehyde-3-phosphate dehydrogenase
promoter [117], human alpha-lactalbumin promoter, ovine beta-lactoglobulin promoter [3], type II hexokinase promoter [87], HER2 promoter, and hTERT promoter
[111]. These studies have demonstrated the feasibility of using TSPs for targeting
tumor cells in animal models of gene therapy. However, the activity of most current
TSPs is pretty weak as compared with that of the cytomegalovirus (CMV) promoter
widely used in gene therapy. Efforts have been made to enhance the promoter activity, such as linking TSPs with strong enhancer sequences and amplifying the activity
using an artificial transcription factor [112].
Many genes are upregulated in tumor cells at the posttranscriptional or translational level, thus their control elements can also be used to target tumors in gene
therapy. For example, the 5’-UTR of mRNA can mediate inhibition of translation
initiation, and the 3’-UTR can mediate mRNA stabilization. The DNA sequence of
5’-UTR may also be involved in transcriptional control. Studies have now shown
that tumor-specific signaling can control gene expression through these elements.
34.3.1.3 Conditionally Replicating Vectors
The replication competence of viral vectors, such as oncolytic viruses, can be made
in a tumor-specific manner. Technically, key virus proteins can be deleted so that
the virus replicates only in tumor cells with specific dysregulated signaling pathways [61, 183]. The key virus proteins can also be placed under the control of TSPs,
thereby restricting virus production to the targeted tumor cells [68, 213]. Conceptually, the replication of viruses depends on alteration of the signaling pathway in the
host cells; thus most types of viruses can be engineered to become oncolytic viruses.
However, taking into account chromosome integration, tumor specificity, and replication efficiency, oncolytic adenoviruses are the most feasible agents.
Currently, three types of oncolytic adenoviruses are used in gene therapy. The
first type is the early region 1 B (E1B)-deficient adenovirus. The E1B gene of a nor-
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mal adenovirus binds to and inactivates the p53 gene, allowing viral replication in
normal cells. Deletion or mutation of the E1B region in the adenoviral genome results in the restriction of viral replication only in p53-defective context, which occurs frequently in tumor cells. The second type is the E1A-defective adenovirus [8].
The expression of E1A can force cells to enter the S phase from the G0/G1 phase,
allowing adenoviral replication to start. The E1A-defective adenovirus can replicate
only in tumor cells with a dysregulated G1-S transition background, such as Rb mutation [62]. This type of oncolytic virus has been tested in animal models and several
clinical trials [118]. Finally, TSPs such as hTERT and L-plastin have been inserted
into the viral genome to control E1A expression, resulting in the selective lysis of
breast tumors in animal models [70, 213].
34.3.2 Strategies in Tumor Suppression
34.3.2.1 Blocking the Activity of Oncogenes
Specific genes that contribute to tumor progression can be seen as targets for therapy. During tumorigenesis, genetic changes such as point mutations, amplification,
chromosome translocation, and rearrangement, can modify protooncogenes to
confer the transforming ability on them. Alternatively, in tumor cells, the signaling
pathways can be altered to increase the activity or expression level of key genes in
the cell cycle or survival (Table 34.1). Downregulation of abnormal oncogene expression has been shown to reverse the malignant phenotype of various types of
tumor cells, or suppress tumor growth. Approaches to block oncogene activity are
discussed below.
Table 34.1 Common genetic defects in breast cancers. OV Overexpression, UN underexpression,
M mutated, EFGR epidermal growth factor receptor, FGFRs fibroblast growth factor receptors,
IGF1R insulin-like growth factor receptor 1, MMPs metalloproteinases, VEGF vascular endothelial
growth factor, PA plasminogen activator, uPA urokinase-type plasminogen activator
Genes
Chromosomal
location
Expression
Reference
EGFR
7p13
OV
[146]
[103]
Erb-2/Her-2
17q21
OV
[140]
[218]
OV
[9]
[43]
OV
[49]
[216]
Growth factor receptors
FGFRs
IGF1R
15q25-26
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Table 34.1 continued
Genes
Chromosomal
location
Expression
Reference
11q22-23
UN
[168]
[39]
BAX
19q13.3
UN
[212]
Bcl-2
18q21.3
OV
[93]
Bcl-xL
20q11.2
OV
[93]
Bik
22q13
UN
[23]
[221]
CCND1
11q13
OV
[187]
[66]
c-Myc
8q24
OV
[139]
[130]
MDM2
12q14.3
OV
[129]
[218]
p16/INK4
9p21
UN
[175]
p21/CIP1
6p21.2
UN
[175]
[45]
p27/KIP1
12p13
UN
[175]
[160]
PTEN
10q23
UN(M)
[17]
[138]
Rb-1
13q14.1
UN(M)
[35]
[48]
TP53
17q13
OV(M)
[71]
[88]
Cell death regulators
ATM
Angiogenesis/metastasis
β-catenin
3p21-22
OV
[40]
[111]
[82]
CXCR4
2q21
OV
[127]
[126]
E-cadherin
16q22.1
UN
[32]
[69]
OV
[47]
[176]
MMPs
uPA/PAI1, PAI2
10q24
OV
[46]
[52]
VEGF
6p12
OV
[12]
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Table 34.1 continued
Genes
Chromosomal
location
Expression
Reference
BRCA1
17q21
UN(M)
[193]
[123]
BRCA2
13q12-13
UN(M)
[193]
[48]
[192]
Chromosomal stability
Inhibiting Oncogene Transcription
The transcription of oncogenes can be blocked by transcription repressors (TRs)
or dominant-negative (DN) mutants of transcription factors [36]. TRs can be molecules that bind to oncogene promoters, and thereby block the transcription factor
function. For example, a consensus binding sequence of PEA3, an ets transcription
factor family member, is present 26 nucleotides upstream from the transcriptional
starting site on the HER2/neu promoter. When overexpressed, PEA3 binds directly
to this consensus motif and prevents HER2/neu gene expression by suppressing
promoter function. Downregulation of HER2/neu expression inhibits cell growth
in vitro and blocks the development of HER2/neu-overexpressing cancer cells in
mouse models, thereby prolonging the survival of treated animals. Therefore, PEA3
gene therapy is a promising way to target HER2/neu transcription [198].
Alternatively, TRs can block transcription indirectly by binding to transcriptional
coactivators. For example, expression of the adenoviral E1A protein inhibits HER2/
neu promoter activity by targeting p300, the coactivator of HER2/neu transcription.
This idea is further supported by the finding that the p300-binding domain of E1A
is required for repression of HER2/neu transcription [25]. E1A shares structurally
homologous regions with the large T antigen of the simian virus 40 (SV40) and the
c-Myc protooncogene. Like E1A, the SV40 large T antigen [122] and c-Myc protooncogene [167] were also able to repress HER2/neu promoter activity in transient
transfection assays.
mRNA Sequestering
There are three strategies for sequestering mRNA [36]. First, the function of oncogene mRNA can be blocked by its antisense sequence. For example, expression
of the antisense RNA of HER2/neu reverses the transforming phenotype of breast
cancer cells [7]. The second approach is to induce the homology-dependent degradation of cognate mRNA, a process known as RNA interference by using siRNA,
a short RNA with 21–23 nucleotides. For example, cells infected with retroviruses
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expressing anti-HER2/neu siRNA exhibit slower proliferation, increased apoptosis,
increased G0/G1 arrest, and decreased tumor growth [29]. Third, ribozymes can be
used to recognize and cleave specified RNA sequences. For example, infection with
adenovirus encoding the surviving ribozymes results in a significant reduction of
surviving mRNA and protein, and hence in the sensitization of tumor cells to apoptosis induced by etoposide or serum starvation [26].
Inhibiting the Function of Oncogenic Protein
There are two approaches to preventing oncogene activity from interfering with the
function of their protein products [36]. First, the DN mutant, which serves as a
nonfunctional competitor of the wild-type protein, can be used to antagonize the
function of the oncogene products. Especially attractive targets for this approach are
the overexpressed receptor tyrosine kinases, such as EGF receptor (EGFR), ErbB-2,
and ErbB-3, in breast cancer cells. The dimerization of these receptors can be disrupted by expression of the DN proteins, resulting in the inhibition of downstream
signaling. Such mutant of EGFR has been reported to reduce the binding of EGF,
receptor endocytosis, and downstream signaling [86]. Alternatively, growth factor
(GF) binding to the receptor can be prevented by the expression of a mutant GF, or
a GF-binding protein to sequester it [33]. The second approach is the expression of
single-chain antibodies to sequester the oncogene product in the cell. For example,
the delivery of a gene encoding anti-ErbB2 single-chain variable fragment antibody
(sFv) to ErbB2-overexpressing ovarian and breast cancer cells resulted in the downregulation of cell-surface ErbB2 expression and growth suppression. The ability to
selectively abrogate oncogenes through the use of intracellular sFvs provides a feasible breast cancer gene therapy [60].
34.3.2.2 Restoring the Function of Tumor-Suppressor Genes
Tumor suppressor genes encode proteins that act as the braking system of cell
growth. During tumorigenesis and tumor progression, these genes are inactivated
by multiple mechanisms, including gene mutation, deletion, silencing, and expression downregulation. Conceptually, reinstating the wild-type tumor suppressor
gene can restore its function and reverse the malignancy of tumor cells.
The p53 tumor suppressor gene mutates in many inherited and sporadic cancers,
such as colon cancer, small cell lung cancer, ovarian cancer, bladder cancer, breast
cancer, and acute lymphocytic leukemia. In addition, the Li-Fraumeni syndrome is
associated with germ-line p53 mutations [71]. Animal and in vitro studies suggest
that delivery of the wild-type p53 gene to tumor cells will be useful as an adjuvant
therapy following conventional treatment. Clinical trials of p53 gene therapy for
breast cancer are in progress [79].
Another example of a tumor suppressor gene is BRAC1. BRAC1 is a gene that is
involved in DNA repair [182]. Although it is mutated in only a small percentage of
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717
breast or ovarian cancers, the BRAC1 level is downregulated in most sporadic breast
and ovarian cancers [81, 123, 172]. Expression of BRAC1 in breast cancer cell lines
can induce their apoptosis [152]. Likewise, the delivery of a functional BRAC1 gene
to an ovarian or breast cancer xenograft in nude mice resulted in tumor suppression.
34.3.2.3 Inducing Apoptosis or Suicide of Tumor Cells
The delivery of a growth suppression or suicide gene to the tumor cell is the most
straightforward mechanism for cancer gene therapy. Currently, three types of genes
have been used for this purpose: proapoptotic genes, cytotoxic suicide genes, and
signaling alteration genes.
Proapoptotic Genes
Overexpression of proapoptotic genes, including those that encode members of
the proapoptotic Bcl-2 family members, caspases, and death signal receptors, can
induce the apoptosis of cancer cells or sensitize them to therapeutic agents. Many
apoptosis-inducing genes, including interferon (INF)-β [16], p202 [44], and mapsin
[157], have also been used in cancer gene therapy.
Bax
Bax is a Bcl2-homologous protein that contains BH-1, BH-2, and BH-3 domains.
In response to cytotoxic signals, Bax undergoes a conformational change and forms
membrane-associated homo-oligomers [34], activating the mitochondrial apoptotic
pathway. Overexpression of Bax in human lung carcinoma cells [84], prostate cancer
cells [114], and cervical cancer [75] results in apoptosis, caspase activation, and cell
growth suppression. Expression of Bax can sensitize tumor cells to chemotherapy
and radiotherapy.
p202
p202 is a mouse IFN-inducible, chromatin-associated protein [83] that can interact
with several important transcriptional regulators, including E2Fs, Rb family pocket
proteins, Fos/Jun, c-Myc, NF-B, and p53BP-1, resulting in the transcriptional repression of genes induced by these transcriptional regulators [27]. Enforced expression
of p202 in stable murine fibroblasts and human cancer cell lines leads to retardation of cell growth and suppression of the transformation phenotype [28, 189, [203].
Furthermore, breast cancer cells stably transfected with p202 are sensitized to tumor
necrosis factor α (TNF-α)-induced apoptosis [189]. p202 gene therapy suppressed
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tumor growth in human pancreatic cancer [190] and breast cancer xenografts in
mouse models [44].
Bik
Bik, also known as nbk, is a proapoptotic gene that contains the BH-3 domain only,
and was recognized recently to be an essential initiator of apoptosis [13, 65]. Subsequently, Bik has been implicated in the development of human breast and colorectal
cancers by the finding that informative alleles are lost on chromosome 22q, where
the Bik gene is located [23]. The 18-kda Bik protein forms heterodimers with various antiapoptotic proteins, such as Bcl-2 and Bcl-XL, and thus inhibits their antiapoptotic function [65]. Bik can also sensitize tumor cells to the apoptosis induced
by certain chemotherapeutic agents [37, 133]. Bik lipoplex delivered by i.v. injection
has been shown to inhibit the growth and metastasis of human breast cancer xenografts in a nude mouse model, and prolong the life span of the treated animals [221].
Recently, it was found that the mutation of threonine 33 and serine 35 in Bik to aspartic acid enhanced the association of Bik with Bcl-2 and Bcl-XL. Moreover, these
mutants exhibited greater apoptotic activity in vitro and greater antitumor activity
in an animal model than wild-type Bik. This Bik mutant is also more potent than
wild-type Bik in a gene therapy application [108].
Cytotoxic Suicide Genes
Plants, fungi, bacteria, and viruses often utilize unique metabolic pathways that are
not present in mammalian cells. Some enzymes involved in these pathways can convert nontoxic prodrugs to metabolites toxic to mammalian cells. Therefore, when
the genes of such enzymes are expressed in tumor cells, a systematically administered nontoxic prodrug can be converted into toxic metabolites inside the cells
to kill them. This process is also selective, because the prodrug is nontoxic to untransfected cells [67, 125]. For example, HSV thymidine kinase (HSV-TK), the most
widely used prodrug activation gene in cancer gene therapy, converts the nontoxic
ganciclovir and acyclovir to their monophosphate forms, which in turn are metabolized into triphosphate forms by cellular enzymes [50]. The triphosphate forms inhibit DNA polymerase and thus DNA replication. Other prodrug activation genes
include cytosine deaminase (CD), which converts 5-fluorocytosine to 5-fluorouracil
[14], and nitroreductase, which converts nontoxic precursors of alkylating agents to
their functional forms [42]. Cytochrome P-450 activates cyclophosphamide [119]
and ifosfamide [113].
The tumor-suppression effect of this system can be further enhanced by the bystander effect, in which an active drug of low molecular weight passes from one
cell to another through gap junctions, inducing the death of the neighboring cells.
Alternatively, the death of transfected cells by prodrug treatment may induce T-celldependent antitumor immunity, resulting in the death of distant tumor cells. This
phenomenon is called the distant bystander effect.
34.3 Strategies of Breast Cancer Gene Therapy
719
Signaling Alteration Gene: Type 5 Adenoviral E1A Proteins
The E1A gene of human adenovirus type 5 (Ad5) is the first gene used in clinical trials of breast cancer gene therapy. E1A encodes the proteins that activate viral transcription and reprogram cellular gene expression in infected cells, permitting viral
replication [51]. Expression of E1A alone is sufficient to immortalize primary rodent
cells, and fully transforms them in cooperation with a second oncogene [59, 150].
However, the transformation of human cells by Ad5 E1A has never been achieved.
On the contrary, E1A reverses the transformed phenotype of human tumor cells
and suppresses primary human tumor growth. This anticancer activity of E1A was
first reported in 1988, when transfection of the type 2 adenoviral E1A gene reduced
the metastatic potential of ras-transformed rat embryo cells [141, 142, 163]. It was
then observed that Ad5 E1A inhibited metastasis in human tumor cell lines [57].
E1A was also shown to inhibit HER2/neu expression [208] and then suppressed
HER2/neu-induced tumorigenicity in an animal model [210]. Stable expression of
the E1A gene in human cancer cell lines was later shown to reduced tumorigenicity
[54]. Since then, E1A has been associated with anticancer activities through many
different mechanisms [41, 55, 56, 202].
Ad5 E1A exerts its anticancer activity through its interactions with multiple
target proteins, altering the balance between the normal state and transformation
signaling [56]. In particular, E1A binds to key cellular proteins that control gene expression and cell growth, including transcriptional coactivators, corepressors, cellcycle-regulatory proteins, components of the general and specific transcriptional
machinery, and numerous transcription factors (such as ATF-2, c-Jun). The wide
spectrum of E1A binding components provides feasible mechanisms for tumor suppression. As mentioned in a previous section (34.3.2.1), E1A suppresses HER2/neu
transcription, which transforms cells [211] and decreases metastatic potential on
tumor cells [206, 209]. In addition, E1A is able to promote the apoptosis induced
by serum deprivation, TNF-α, irradiation, and anticancer drugs. E1A also inhibits
the activation of NF-κB by suppressing IκB kinase activity and IκB phosphorylation,
rendering cells more sensitive to environmental stresses such as TNF-α [153] and γ
irradiation [54, 55]. E1A also negatively regulates the expression of the transforming receptor tyrosine kinase Axl at the transcriptional level, which is essential for the
proliferation of tumor cells [101].
Furthermore, E1A sensitizes tumor cells to anticancer drugs such as paclitaxel
[180], gemcitabine [102], and topoisomerase IIα inhibitors [219]. Several molecules
have been proposed to be involved in such E1A-induced chemosensitization, such
as Bax and caspase 9. Transcriptional upregulation of procaspases (such as procaspases 3, 7, 8, and 9) through E1A-mediated disruption of the pRB function and
subsequent release of free E2F-1 was reported to contribute to both p53-dependent
and p53-independent drug sensitization by E1A [177]. Recently, it was found that
E1A can activate p38 and inactivate Akt. This pathway may provide a general cellular mechanism for E1A to increase sensitization to different categories of anticancer
drugs [109].
E1A gene therapies with adenoviral and cationic liposome vectors have been
tested in an orthotopic HER-2/neu-overexpressing breast cancer animal model [24,
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Breast Cancer Gene Therapy
179]. In mice bearing an HER-2/neu-overexpressing breast cancer cell line, E1A significantly inhibited tumor growth and prolonged mouse survival compared with
the control group [24, 186, 199]. Additional preclinical and clinical developments of
E1A lipoplex were warranted for the treatment of advanced or metastatic cancer.
34.3.2.4 Indirect Strategies
The strategies in the previous sections require that the delivered genes act on tumor cells directly. An alternative strategy is to disrupt the environment for tumor
survival. For example, inhibiting the formation of new blood vessels (angiogenesis)
can block the supply of nutrients to tumors. In addition, enhancing the response of
immune cells enables them to eradicate tumor cells. To achieve this end, therapeutic
genes can be delivered to either the tumor cells themselves or to their host cells,
disrupting the environment for tumor survival.
Inhibiting Tumor-Induced Angiogenesis
Angiogenesis is required for solid tumors to grow and metastasize. Inhibiting angiogenesis can suppress tumor growth in animal models. There are three ways that gene
therapy blocks angiogenesis in tumors. First, expression of the angiogenic factor
genes in tumor cells can be suppressed by transferring antisense sequences or ribozymes. For example, antisense vascular endothelial growth factor cDNA delivered
by an adenoviral vector significantly suppressed the growth of breast tumor xenograft in a mouse model [104]. Second, expression of the secretory angiogenic inhibitor can block angiogenesis, resulting in the suppression of tumor growth. It has been
reported that delivery of antiangiogenic factor genes, such as endostatin, to breast
cancer cells can also suppress tumor growth in nude mice [20, 78, 110]. Finally, E1A
has been shown to exhibit a bystander effect on inhibition of tumor growth by suppressing angiogenesis and inducing apoptosis in tumors [156]. E1A gene therapy
also significantly reduced blood vessel density and induced cell apoptosis in mouse
tumors [220].
Enhancing the Immune Response to the Tumor
There are several mechanisms that allow cancer cells to escape from the immune
system and induce tolerance [15]. But tumor cells still can be eradicated by enhancing the immune response of the body.
The strategies of immunotherapy for cancer include nonspecific conditioning of
the immune system, with cytokines such as interleukin (IL)-12, IL-2, and INF-α, activation of the immune system by vaccination, adoptive transfer of antigen-specific
cytotoxic T lymphocytes or lymphokine as activated killer cells, and monoclonal or
polyclonal antibodies. Gene therapy, as modulating immunotherapy, can be used to
34.4 Clinical Trials of Breast Cancer Gene Therapy
721
transfer the genes of tumor-specific antigens to antigen-presenting cells as vaccines
[197], and to produce cytokine-expressing viruses [2] or cells [63] to enhance the
immune response.
34.4 Clinical Trials of Breast Cancer Gene Therapy
34.4.1 E1A Gene Therapy
Breast cancer cells overexpressing HER-2/neu show enhanced tumorigenicity, metastasis, and chemoresistance [5, 76, 207]. Patients with HER-2/neu-overexpressing breast cancers also have a poor prognosis [38, 158, 159]. The Ad5 E1A gene
has been found to inhibit HER-2/neu expression in both rodent and human cancer
cells through transcriptional repression of the HER-2/neu promoter [25, 201, 208].
It was found that transfecting the E1A gene into HER-2/neu-overexpressing cancer
cells could abolish the tumorigenic and metastatic potential of these cells [206, 207,
211].
On the basis of these results, a series of clinical trials of E1A gene therapy were
conducted. The first phase I trial of E1A gene therapy was completed by the end of
1997 [73]. This is the first gene therapy trial focused on HER-2/neu-overexpressing
cancers. In this trial, the E1A gene was encapsulated by a specific liposome, 3-β-[N(N’,N’-dimethyl-aminoethane)-carbamoyl]cholesterol/dioleoylphosphatidyl-ethanolamine, to form a DNA–liposome complex called tgDCC-E1A. This tgDCC-E1A
complex was injected into the thoracic cavity of breast cancer patients with pleural
effusion, or into the peritoneal cavity of ovarian cancer patients with ascites. The
goals of this trial were: (1) to determine the maximum-tolerated dose (MTD) of
the complex that was given intracavitarily, (2) to determine whether the E1A gene
could be delivered into tumor cells by this cationic liposome, and (3) to evaluate the
level of HER-2/neu expression as a possible marker of E1A-specific biologic activity.
In addition, the ability of this tgDCC-E1A complex to induce apoptosis of tumor
cells and cytokine expression levels that might contribute to the antitumor activity
of the E1A gene was evaluated. Six patients with breast cancer and 12 patients with
ovarian cancer were enrolled in this trial, all breast cancers and 6 ovarian cancers
overexpressed HER-2/neu. The starting dose was 1.8 mg/m2; 3-weekly injections
were followed by 1 week of rest. This dose was then escalated in 100% increments. A
median of six total injections (range, one to eight) was given over two cycles, and the
median cumulative dose of E1A plasmid was 10.8 mg/m2 (range, 5.4–32.4 mg/m2).
In most patients (77.8%), self-limited fever (temperature up to 103°F) developed 3–
48 h after injection of the tgDCC-E1A complex, regardless of dose. All five patients
who received the highest dose of E1A plasmid (7.2 mg/m2) developed moderate to
severe nausea, vomiting, and discomfort (pain or burning) at the sites of injection.
As a result, the MTD of the tgDCC-E1A complex was fixed at 3.6 mg/m2.
Although clinical response was not an endpoint of this trial, three breast cancer
patients achieved stable disease at the injected sites and improved performance status after two cycles of E1A gene therapy. Furthermore, transient decreases in levels
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Breast Cancer Gene Therapy
of tumor markers (CEA, CA 27–29, or CA-125) were noted in five of the patients.
Cancer cells were collected from pleural effusions before and after treatment in five
breast cancer patients. The signal intensity of HER-2/neu decreased after treatment
with the tqDCC-E1A complex (Fig. 34.1). Tumor clumps in pleural effusions and
Ki-67 expression in the cancer cells of all six breast cancer patients were also noted
(Fig. 34.2). In patients whose apoptotic cell percentage increased the most, TNFα levels were also increased. Since HER-2/neu overexpression can block TNF-αinduced apoptosis via the Akt/NF-κB pathway [217], and E1A can downregulate
HER-2/neu, the increase in apoptotic cells may be due to sensitization to TNF-αinduced apoptosis by the suppressive effect of E1A on HER-2/neu.
Another phase I trial was conducted in 1997 [205], the purpose of which was to
determine the MTD and maximum biologically active dose tgDCC-E1A given by
intratumoral injection. Nine patients with breast cancer and nine patients with head
and neck cancer were enrolled. One tumor nodule in each patient was injected with
tgDCC-E1A. No dose-limiting toxicity was noted in the four dose groups (15, 30,
Fig. 34.1 HER-2/neu downregulation after administration of tgDCC-E1A complex. a Pretreatment (day 1), 3+ HER-2/neu signal intensity. b After one injection of tgDCC-E1A complex (day
15), 2+ HER-2/neu signal intensity. c After four injections (day 38), HER-2/neu signal intensity.
d After six injections, 0 negative (day 57). Reprinted with the permission of Journal of Clinical
Oncology [73]
34.4 Clinical Trials of Breast Cancer Gene Therapy
723
Fig. 34.2 a Decreased number of tumor clumps after two cycles of E1A gene therapy. b Increased
percentage of apoptotic tumor cells after administration tqDCC-E1A complex. c, d Increased percentage of apoptotic tumor cells in patient (Pt) no. 2 (c, pretreatment; d, posttreatment). e tqDCCE1A complex suppressed Ki-67 expression of tumor cells in six breast cancer patients. Reprinted
with the permission of Journal of Clinical Oncology [73]
724
Breast Cancer Gene Therapy
60, and 120 µg DNA/cm of tumor). All patients tolerated the procedure well. The
MTD was reached in this study. E1A gene transfer could be detected in 14 out of
15 samples, and caused a subsequent downregulation of HER-2/neu expression and
tumor response. In one breast cancer patient, no pathological evidence of tumor
was found on biopsy of the treated tumor site at week 12. Of 16 patients who were
evaluable for tumor response, 2 had minor responses, 8 had stable disease, and 6
had progressive disease. This trial proved that gene therapy with an E1A gene:liposome complex is safe and warrants further testing.
In these clinical trials, E1A exerted its antitumor effects through a variety of
mechanisms, including downregulation of HER-2/neu, induction of apoptosis, inhibition of metastasis-related enzymes, and activation of the host immunosurveillance system.
34.4.2 p53 Gene Therapy
p53, the most well-known tumor suppressor gene, is normally responsible for detecting damaged DNA, causes the cell cycle to arrest at the G1 or G2 phase, and
either directs repair or commits cells to apoptosis [184]. p53 is mutated or otherwise
altered in more than 50% of human cancers [71]. Tumor cells expressing mutated
p53 or loss of p53 are resistant to chemotherapy [115, 195] and radiotherapy [116],
so mutated p53 has been associated with a poor prognosis in many types of cancers,
including breast cancer [173].
There are currently two p53-based therapeutic genes undergoing clinical trials.
One is SCH58500 (Schering-Plough), another one is INGN 201 (Introgen Therapeutics). Both constructs use the CMV promoter to drive the wild-type p53 gene on
different adenovirus gene transfer vectors with different modifications [191, 215].
SCH58500 is undergoing phase I/II trials of non-small cell lung cancer (NSCLC)
[131] and ovarian cancer [19], but until now, there are no clinical trials including
breast cancer.
In preclinical experiments, INGN 201 was shown to be effective against several
different cancer types, including breast cancer [136], lung cancer [169], glioma
[91], colorectal cancer [161], head and neck cancer [30], and ovarian cancers [124].
INGN 201 also has synergistic effects with chemotherapy [100] and radiotherapy
[97]. In the following phase I trials, NSCLC [131, 148, 169], glioma [96], and head
and neck cancer [31] were treated by intratumoral injection of an adenovirus-delivered p53 gene with minimal toxicity. Based on these data, there is an ongoing phase
II trial combining systemic chemotherapy docetaxel and doxorubicin, and intratumor injection of INGN 201 for stage III or IV breast cancer. Patients receive an
intratumoral injection of INGN 201 on days 1 and 2. Patients also receive i.v. doxorubicin over 15 min followed 1 h later by i.v. docetaxel over 1 h on day 1. Treatment
is repeated every 3 weeks for up to six courses. After completion of chemotherapy,
patients with a reasonable response undergo surgical resection (see: http://www.
clinicaltrials.gov/ct/show/NCT00044993? order=1).
34.4 Clinical Trials of Breast Cancer Gene Therapy
725
34.4.3 Gene-Directed Enzyme Prodrug Therapy
Gene-directed enzyme prodrug therapy (GDEPT) involves the delivery of a cytotoxic suicide gene that encodes a prodrug-activating enzyme, as mentioned in section 34.3.2. Such suicide genes, including HSV-TK, CD, and cytochrome P450, have
been studied extensively for their use in cancer gene therapy. The preclinical results
have demonstrated significant antitumor effects of all three of these genes, so they
are now undergoing phase I/II clinical trials in patients with different kinds of cancer, including breast cancer [134, 144, 151]. Almost all suicide genes in these trials
are delivered locally by virus, followed by systemic administration of prodrugs. In
a phase I trial for breast cancer, naked DNA of the CD gene driven by an HER-2/
neu promoter was directly injected into selected metastatic nodules of 12 patients
[134]. High CD gene expression was present at the injected site, the nodules of two
patients regressing after prodrug administration, and those of another two patients
regressing without prodrug treatment. The bystander effect is important for the tumoricidal response because the transfection rate is low [53].
Theoretically, the doses of systemically administrated prodrugs in GDEPT can
be decreased, so the toxicity that originates from the metabolites of these drugs can
be lessened. The dosages of chemotherapeutic agents in these clinical trials are the
same as those used alone, so we cannot see the benefit for toxicity from these trials.
More clinical trials are needed to confirm the synergistic effect of suicide genes and
prodrugs, and to modify these genes to enhance their tumor-specific expression.
34.4.4 Genetic Immunotherapy
As mentioned in the previous section, gene therapy can be used to transfer tumorspecific antigens to antigen-presenting cells, and to produce cytokine-expressing
viruses or cells to enhance immune responses. Several clinical trials of these two
therapeutic strategies are ongoing.
34.4.4.1 Dendritic Cells
Dendritic cells (DCs) originate from bone marrow and are distributed to almost
all organs by the bloodstream. They are the most potent antigen-presenting cells to
initiate and maintain primary immune responses when pulsed with immunogenic
antigens [4, 164], such as peptides, proteins, and cell lysates. In order to get the
most effective response, these antigens should be highly immunogenic and tumor
specific. One way to accomplish this is to transfect DCs with the cDNA or RNA
of a tumor-associated antigen, which is able to induce a strong, specific immune
response [11, 181].
Mucin-1 (MUC-1) is a high-molecular-weight glycoprotein that is overexpressed
in breast cancer [94]. Transfecting the MUC-1 gene into DCs as a breast cancer vac-
726
Breast Cancer Gene Therapy
cine was tested in a phase I/II clinical trial [137]. One million gene-transfected DCs
were injected subcutaneously into the upper limb of breast cancer patients on days
1, 21, and 42. Vaccination did not produce significant side effects. However, only
three out of ten patients had an immunological response, one patient developed
stable disease, and the others still showed signs of disease progression.
Clinical trials of DC-based therapy are under way, and there are still many issues
to be addressed, including methods of delivery, the maturation status of DCs, the
optimal loading of DCs, the possible roles of cytokines in enhancing the immune
response and the bystander effect, and methods of monitoring immune responses.
One of the greatest challenges is to find a tumor-specific antigen that is highly immunogenic, of clinical importance, and expressed on most tumor cells but not in
normal tissue, so that most of cancer cells can be destroyed.
34.4.4.2 Cytokines
Cytokines are transmitters that influence the induction of the immune response. Insufficient production of cytokines is one of the reasons that the immune system fails
to recognize and eradicate tumor cells. Systemic cytokine therapy usually results in
low systemic concentrations but unacceptable toxicity [18, 147, 166]. Locoregional
administration of cytokines can create higher concentrations at the target site, resulting in better clinical outcome and decreasing systemic toxicity. Many studies
now are focusing on using gene therapy to transfer the cDNAs of cytokines into
viruses or cells as delivery systems, followed by intratumoral injection, so that the
expressed cytokines can induce a locally immunological response against the tumor
instead of systemic toxicity.
The injection of adenovirus-expressing human IL-2 into tumors has been shown
to be effective in the resolution of tumors both locally and distantly [1, 2, 63]. In a
phase I study focusing on metastatic breast cancer and melanoma [165], different
doses of the adenovirus expressing human IL-2 were injected subcutaneously into
nodules of skin carcinomatosis in 23 patients, including 8 breast cancer patients.
Local inflammation was the most common toxicity; at higher doses, some patients
had fever. Pathological examination revealed T-lymphocyte infiltration and tumor
necrosis, enzyme-linked immunosorbent assay could detect IL-2 at tumor biopsy
48 h after injection. This phase I trial demonstrated that such a delivery method is
safe and relatively nontoxic.
In another dose-escalation phase I trial, IL-12-transduced autologous fibroblasts
were injected peritumorally to nine patients with disseminated cancer, including
five breast cancer patients [85]. Primary dermal fibroblasts from patients were
transduced with retroviral vectors carrying the human IL-12 gene. The locoregional
effects mediated by TNF-α and CD8+ T cells were observed with tumor regression.
No clinically significant toxicities were observed, only mild to moderate pain at the
injection site. A transient reduction in tumor size was observed at the injected sites
in four out of nine cases, including two breast cancer patients, and at noninjected
sites in one melanoma patient.
References 727
Although there are many cytokine-related gene therapies, most of the trials are
being conducted in patients with melanoma. To our knowledge, there is no clinical
trial of cytokines specifically focusing on breast cancer patients.
34.5 Conclusion
Since R.M. Blaese initiated the first gene therapy clinical trial in September 1990,
countless in vitro and in vivo experiments and gene therapy trials have been initiated, focusing on nearly all aspects of human diseases, including cancers. Most cancer gene therapy trials are at the phase I or II stage, and the results of many of these
trials confirm that cancer gene therapy is safe. Although there are technical issues
that need to be overcome, gene therapy for breast cancer should provide a promising avenue for development of new anticancer agents.
A highly efficient and specific delivery system is one of the key points to a successful gene therapy. Although the viral delivery systems can reach higher transfection efficiency than nonviral delivery system, most of these delivery systems are not
tumor specific. To prevent systemic toxicity and increase the local concentration, all
clinical trials of breast cancer gene therapies involve local or regional therapy. However, breast cancer should be viewed as a systemic disease, so one of the most important considerations is to modify the current delivery systems for systemic treatment.
Some preclinical experiments used ligands to coat these delivery vectors, so that
they could target cancer cells that overexpressed their receptors [10, 89]. Using TSPs
to limit gene expression within cells with high promoter activity can also enhance
the specificity [68, 134]. The most basic goal now is to extend our knowledge about
the molecular biology of breast cancer, and to understand the genetic changes during tumorigenesis so that we can target the specific gene, enhance or decrease its
expression, then control or kill cancer cells efficiently.
The genetic changes of breast cancer are polymorphic, and tumorigenesis usually
is the consequence of the interaction between genetic changes and environmental
factors. Trying to use a single gene therapy to treat breast cancer may therefore not
be sufficient. However, when it is combined with a multimodality therapy consisting
of radiotherapy, chemotherapy, small molecular therapy, and immunotherapy, the
effects may be synergistic. Gene therapy will indeed play an important role in the
treatment of breast cancer in the future.
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