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    Awarded Grants
    Development of a Conditionally Replicative Adenovirus Targeted to HER-2/Neu-overexpressing Breast Cancer Through an Antisense Iron-responsive Element

    Scientific Abstract:
    The overexpression of HER-2/neu proto-oncogene is found in a variety of human cancers. In particular, amplification and/or overexpression of the HER-2/neu gene are found in 20-30% of patients with breast cancer. These patients usually have decreased survival, increased relapse rates, and are more resistant to chemotherapy. Thus, HER-2/neu overexpression has been an indicator of poor prognosis for breast cancer patients. Therefore, novel and effective therapies are urgently needed to treat this group of breast cancer patients. To develop an expression system that targets the HER-2/neu-overexpressing breast cancer cells, we have described a novel approach that combines the antisense principle and the biochemical property of a translational regulator, an iron-responsive element (IRE). IRE, when placed 5’ to a gene, functions as a negative translational regulator in that IRE interacts with iron-regulatory proteins (IRPs) and this protein-RNA complex blocks translation. One way to alleviate this translational block is to prevent the IRE/IRP interaction by disrupting the IRE stem-loop structure via a sense-antisense hybrid. We have tested this idea and showed a HER-2/neu antisense IRE (AS-IRE), that possesses the IRE consensus sequence, not only functions like a canonical IRE but also directs the expression of a reporter or a therapeutic gene preferentially in breast cancer cells that overexpress HER-2/neu mRNA, indicating that the AS-IRE-mediated translation inhibition can be overcome by the overexpression of HER-2/neu mRNA. Thus, this novel antisense IRE-mediated gene expression system can be used for targeting HER-2/neu-overexpressing cancer cells, and may be potentially used to target other cell types that uniquely express or overexpress a known gene as well. However, the treatment efficacy of an AS-IRE-mediated gene therapy has not been demonstrated in vivo. To accomplish this goal, the current plasmid-based gene expression system may have limited therapeutic effect because of low transfection efficiency. To overcome this obstacle and to enhance the specific therapeutic effect on HER-2/neu-overexpressing breast cancer, we propose to develop a conditionally replicative adenovirus in which E1A gene is under the control of E1A promoter and an optimized AS-IRE, i.e., AS-IRE4, with RGD-4C peptide motif added to the virus to facilitate viral internalization into the target cells. We hypothesize that this virus can efficiently infect and selectively replicate in breast tumor cells that overexpress HER-2/neu mRNA, and amplify and spread the killing effect from cell to cell within the breast tumor, but not the surrounding normal tissues. To test this hypothesis, three aims are proposed: 1. To generate a conditionally replicative adenovirus (ASI-RGD) in which E1A gene is under the control of E1A promoter and AS-IRE4, and an RGD-4C peptide motif inserted into the adenoviral fiber; 2. To determine the HER-2/neu overexpression-specific cell killing by ASI-RGD in vitro; and 3. To determine the HER-2/neu overexpression-specific therapeutic efficacy of ASI-RGD treatment in breast cancer xenograft models. The success of the proposed experiments will provide a novel therapeutic oncolytic adenovirus to specifically target HER-2/neu-overexpressing breast cancer.

    Lay Abstract:
    It is estimated that one in eight women will develop breast cancer in their lifetime. Many therapeutic strategies have been devised to combat this disease by targeting a variety of breast tumor-specific molecules. Among them, HER-2/neu has emerged as a major therapeutic target for breast cancer treatment due to the important finding that 20-30% of breast tumors have abnormally high HER-2/neu expression mainly caused by abnormal amplification of the gene. The dire consequence of HER-2/neu overproduction is the fact that these patients have decreased survival, increased relapse rates, and are relatively insensitive to chemotherapy treatment. Therefore, novel and effective therapies are urgently needed to treat this group of breast cancer patients. With this purpose in mind, we have developed a novel strategy that specifically target HER-2/neu overproducing breast cancer cells. This strategy combines an antisense principle, namely, two complementary RNA molecules can form A:U and G:C base pairs, and the biochemical property of a translational regulator, an iron-responsive element (IRE). The goal is to allow a therapeutic gene to be specifically translated in breast cancer cells with overproduction of HER-2/neu mRNA. The reason why this specific gene expression can be accomplished is the following: an intact IRE stem-loop (looks like a hairpin) structure, when placed in front of a therapeutic gene, can interact with proteins called IRPs, and the formation of IRE/IRPs complex can block the process of protein synthesis, called translation, of the gene. However, if an IRE was engineered in a way that is complementary (or antisensed) to a region of HER-2/neu mRNA, this IRE structure will be disrupted by the presence of overproduced HER-2/neu mRNA by forming a sense-antisense hybrid. As a result, the translational block is removed and thus allows the mRNA of the gene to be translated in HER-2/neu-overproducing breast cancer cells. We showed that this strategy worked in tissue culture. However, the effectiveness of this strategy to treat HER-2/neu-overproducing breast tumor has not yet been demonstrated in animal model. To do so, we will engineer a virus in which its replication mechanism is controlled by our IRE system. This engineered virus is expected to be efficiently internalized by breast cancer cells and to specifically replicate itself in breast cancer cells that overproduce HER-2/neu resulting in host cell destruction. Importantly, the newly made viruses bursting out of the host cell can spread to the neighboring cancer cells, replicate, and kill. This process in theory should continue within the breast tumor, but not in the surrounding normal cells because they have very low levels of HER-2/neu. In this proposal, we will test this idea by engineering the aforementioned virus. We will test the HER-2/neu-specific killing with this virus in tissue culture and determine the efficacy of treatment with this virus in animal models. The successful outcome of the proposed experiments will provide a novel and effective therapeutic agent in treating breast cancer that overproduces HER-2/neu.