The consequences of dysregulation of apoptosis on tumor pathophysiology are extremely diverse (see above and Fig. ). The core machinery of cell death, however, seems to be formed by a discreet number of protein families (Fig. ). Modulation of a limited number of targets by delivery of genes that encode apoptosis-related proteins would therefore have multiple beneficial effects on the malignant phenotype. Not only would tumor cell proliferation be properly balanced by increased cell death, but also barriers to the immune response would fall, tumor invasiveness would be crippled, and sensitivity to the cytotoxicity induced by drugs and radiation would be restored. Importantly, the need that tumor cells have for mechanisms that allow them to avoid apoptosis is universal, and shared by all tumor localizations in the body. This avoidance represents a 'window of homogeneity' that can be targeted in the context of the formidable tumor heterogeneity. With the increasing recognition of the molecular basis of the apoptotic pathway [1
], and the description of several of its components acting as oncogenes or tumor suppressor genes, gene therapy has thus emerged as a rational strategy for the modulation of apoptosis [20
]. The following is a brief summary of approaches that have already been clinically explored in the context of breast cancer. A discussion of the requirements for successful exploitation of the apoptotic machinery using gene transfer and some of the solutions being developed is then provided.
Clinically explored strategies
Preliminary attempts to explore the therapeutic modulation of apoptosis against cancer by gene transfer have been started, driven by encouraging preclinical data in animal models. Clinical trials are currently ongoing to evaluate the value of pro-apoptotic p53
and adenoviral E1A
, and a growing number of other candidate genes are being considered and tested preclinically (see [20
] and Table ).
Supplementation or restoration of p53
Direct induction of apoptosis has been attempted by replacing the tumor suppressor gene p53
. Several factors made this gene an attractive candidate, including its frequent inactivation in human tumors, the observed lack of toxicity of wild-type p53
itself, the control that it exerts in multiple other genes implicated in apoptosis and cell cycle regulation, and some experimental evidence for a bystander effect. Both in vitro
and in vivo
animal studies [84
] have indeed shown induction of apoptosis and suppression of tumorigenicity in human breast cancer models after p53
gene delivery via recombinant adenoviral vectors. Of interest, this result has also been observed in tumor cell sublines selected for resistance to drugs that are commonly used in the treatment of breast cancer [88
] and in tumor cells expressing wild-type p53
]. Furthermore, adenovirus-mediated delivery of p53
augments the cytotoxic effect of the chemotherapeutic drug paclitaxel [90
]. These preclinical studies were followed by two clinical trials in breast cancer in which p53
is currently being administered intralesionally, or incubated ex vivo
with bone marrow for purging of contaminating breast cancer cells [91
]. Potential obstacles for the successful clinical exploitation of p53
, however, have arisen with the observation that wild-type p53
can be inactivated in human breast cancer cells that express mutant p53
], and with the low efficiency in vivo
of current gene delivery vectors.
In addition to restoration of p53
, the other pro-apoptotic approach currently being clinically tested is based on liposome-mediated delivery of the adenoviral gene E1A
]. Gene transfer of E1A
inhibits transcription of the human HER-2/neu promoter, and suppresses the tumorigenicity and metastatic potential of the HER-2/neu oncogene in cells that overexpress this oncogene [94
]. An analogous effect of E1A
in anchorage-independent growth and tumorigenicity has been shown in a HER-2/neu-independent manner [95
]. Finally, E1A
sensitizes mammalian cells to immune-mediated apoptosis in a p53-
independent manner [96
]. Thus, delivery of E1A
could have an effect in vivo
that exceeds the limitations imposed by the heterogeneity in the HER-2/neu or p53 status within the tumor.
Requirements for pro-apoptotic gene therapy
A variety of additional interventions and targets have been proposed for the gene-based therapeutic induction of apoptosis (see Table , and the references therein). Here, comments are limited to theoretical aspects that need to be considered for designing successful therapeutic interventions for breast cancer based on the genetic modulation of apoptosis. Specifically, the gene transfer systems and knowledge of the genetic pathophysiology of the disease as the most critical aspects are considered.
High levels of gene transfer
Gene transfer vectors are needed that can trigger apopto-sis in most malignant cells in any given tumor. Most frequently, current vector systems have been employed for delivery of therapeutic nucleic acids to relevant target cells in loco-regional contexts. In general, a fundamental recognition in most of these studies has been the disparity noted between the in vitro
and in vivo
gene transfer efficiencies of these vectors, with a generally suboptimal tumor transduction [97
]. For disseminated disease, employment of available vectors is not presently feasible. This restriction is based on their limited capacity to accomplish efficient gene transfer to widely disseminated tumor targets in vivo
. Implicit in this limitation is the recognition of three requirements for any candidate vector system for this purpose: the ability to accomplish highly efficient and nontoxic gene delivery after direct in vivo
delivery via the intravascular route; the ability to accomplish targeted, specific gene delivery to a selected cellular subset; and the ability to escape the innate and adaptive immune responses.
With regard to the first requirement, only two presently available vector systems, cationic liposomes and recombinant adenoviral vectors, have been reported to transduce various end organs after in vivo
gene delivery [98
]. However, low levels of gene expression [102
], or promiscuous tropism and entrapment of the vector by the reticuloendothelial system, mostly in the liver [104
], respectively, have undermined the utility of the aforementioned vector systems for accomplishing transduction of normal breast tissue and disseminated breast cancer [105
]. This fundamental limit has hindered achieving the second requirement (ie the evaluation of tissue-specific or tumor-specific promoters for transcriptional targeting of therapeutic genes in the context of in vivo
models of breast cancer) [105
]. With regard to the effects of the immune response, viral vectors display foreign antigens that can induce a strong cellular and humoral immune response in humans [106
]. This can ultimately determine immune-mediated clearance of the vector and of the virally infected target cells [109
], and precludes efficient readministration of the vector.
Thus, important limitations of current approaches used for implementation of pro-apoptotic gene therapy for disseminated cancers, including breast cancer, have been noted. Although many potentially effective strategies exist to achieve the molecular treatment of breast cancer, gene delivery issues have limited definitive evaluation of these methods in clinically relevant models.
In this regard, understanding of the determinants of vector efficacy at a cellular level has recently allowed vectors to be designed that achieve cell-specific gene delivery in the loco-regional context. For instance, altering the tropism of adenoviral vectors by bifunctional, antibody-based conjugates and by genetic engineering of the binding sites of the virus dramatically enhances the infectivity of otherwise refractory tumor cells [110
]. This occurs by allowing the cellular entry of the virus through heterologous pathways, thus overcoming the paucity of its receptor, coxsackie and adenovirus receptor (CAR), which characterizes human primary tumors. Here the mandate for a high level of gene transfer may be stricter, given the lack of firm evidence for a useful bystander effect mediated by pro-apoptotic interventions, perhaps with the exception of replacement of p53
]. In this case, a better definition of the basis of bystander effects observed after gene delivery of p53
should lead to the exploitation of similar schemas by designing ad hoc
therapeutic payloads. Examples would be secretory molecules such as the IGF binding protein 3 [112
], which is induced by p53
and inhibits the powerful growth factor IGF-1; and translocating molecules, such as fusion proteins derived from the herpes simplex virus protein VP22 [113
] or from the Tat protein of human immunodeficiency virus type 1 [114
]. Further advances are also needed to understand the determinants of the biodistribution of viral vector after systemic intravascular administration. To this end, novel molecular and imaging technologies [118
] have been developed that allow the amount of vector in relevant tissues to be quantified [119
] and gene expression to be followed noninvasively in vivo
*], both of which should accelerate these fundamental studies.
High therapeutic index
Given the ubiquity of the numerous cellular proteins involved in apoptotic pathways, highly selective activation in cancer cells of lethal processes may also be a critical requirement of therapeutic maneuvers. The lower threshold for undergoing apoptosis that characterizes tumor cells [1
] could, however, offer an advantageous therapeutic window that makes this requirement less stringent. Certain pro-apoptotic interventions might still require a more stringent level of specificity. In such cases, vector targeting could provide the required restriction in the expression of the pro-apoptotic molecules.
Targeted gene therapy for breast cancer can be accomplished at different levels [121
]. In one approach, the tumor cell can be targeted at the level of transduction to achieve the selective delivery of the therapeutic gene. This involves the derivation of a vector that binds selectively to the target breast cancer cell. Alternatively, the therapeutic gene can be placed under the control of breast tumor-specific transcriptional regulatory sequences that are activated in tumor cells, but not in normal cells, and therefore target expression selectively to the tumor cell (for a review see [28
]). In addition, targeted gene therapy for cancer can exploit the unique physiology of solid tumors, such as hypoxia [122
]. To date, targeted gene therapy has been attempted by employing either transductional targeting or transcriptional targeting alone. Again, enhancement of the overall level of specificity by combining the complementary approaches of transductional and transcriptional targeting may be required, each of which might be imperfect or 'leaky' by itself [121
]. Perhaps more importantly, vector targeting may confer, in addition to selectivity, a significantly higher level of gene transfer, as mentioned above.
Multiple interventions and modalities
The signaling circuits that control apoptosis are complex and redundant, and tumors are formidably heterogeneous. Therefore, concomitant modulation of several components of the apoptotic pathways may be needed to provoke cell death in a robust and consistent manner. In this regard, the complex phenotype of the caspase knockout mice revealed that multiple and redundant mechanisms of caspase activation operate in parallel [123
]. Recent high-throughput studies (for instance employing complementary DNA microarrays and serial analysis of gene expression) allow identification of patterns of gene expression and their variation under appropriate stimuli, which will contribute to the dissection of the relevant pathways in breast cancer [124
]. Interventions downstream in the circuits might also be preferable for avoiding regulatory counterbalances that may dissipate the effect of the intervention. For instance, multiple genes act downstream of p53
that alter the response to p53
restoration. In contrast, Bax has a more downstream role in effecting apoptosis, which perhaps explains its more predictable cytotoxicity on tumor cells [126
*]. Finally, the magnitude of cell death after most pro-apoptotic interventions in vitro
is cell line-dependent, with some tumor cells typically showing refractoriness to the induction of apoptosis. This has prompted the evaluation of combining a variety of pro-apoptotic interventions, and their association with chemotherapy [4
**], radiotherapy [6
], and other biologicals [128
]. These multimodality treatments have consistently showed that higher levels of cell death are obtained when different pathways are modulated and modalities are combined, clearly indicating that the targeted pathways do not totally overlap.