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CBFA2T3 is a Novel Breast Cancer Tumour Suppressor Gene: Investigation as a Prognostic Marker and Development of Drug Targets.
Following an extensive research effort we have identified CBFA2T3 as a novel putative breast cancer tumor suppressor gene at human chromosome band 16q24.3. Our studies indicate that properties of CBFA2T3 are consistent with a breast cancer tumor suppressor and that this gene encodes a transcriptional repressor. We have recently identified a cDNA from a normal breast expression library encoding a protein that interacts with the CBFA2T3 protein. This is a previously uncharacterized zinc finger protein and we have termed the gene CBFAIP1. We believe this repressor complex provides a new avenue for the identification of drug targets in breast cancer.
The objective of the proposed research is to test the hypotheses that: 1) CBFA2T3 is a critical tumor suppressor gene in a significant proportion of breast cancers and its expression is related to patient prognosis; and 2) Potential breast cancer drug targets are genes that are transcriptionally repressed by the CBFA2T3/CBFAIP1 complex and that these can be rapidly identified for further validation studies.
The specific aims of the proposed research are: 1) To develop optimum conditions for the immunohistochemistry of breast biopsy paraffin sections with CBFA2T3 antibodies. The relative protein expression of CBFA2T3 will be determined in a cohort of breast tumors by analysis of tissue arrays. These data will then be analyzed to determine the association between the proportion of tumors that down-regulate the CBFA2T3 protein and tumor type, staging, prognosis and other known tumor markers. 2) To confirm that CBFAIP1 binds DNA and to identify target genes, repressed by the CBFA2T3/CBFAIP1 complex, that are potential drug targets.
The study design is to use polyclonal antibodies being raised in rabbits immunized with a short CBFA2T3 protein fragment. The affinity-purified antibodies will be optimized for immunohistochemistry to paraffin sections of breast tumors by testing different techniques for antigen retrieval and varying the first and second antibody concentrations. CBFA2T3 protein expression in tissue microarrays (derived from 194 breast tumors) will be determined using a computer-based image analysis system. The clinical data associated with the tissue microarrays will be analyzed to determine the proportion of tumors with reduced CBFA2T3 protein expression and its association with prognosis. It is important to emphasize that the absence of a correlation between CBFA2T3 expression and prognosis will not affect the potential for drug target development. The proportion of tumors showing down-regulation of the CBFA2T3 protein will, to some extent, determine the potential interest as a commercial drug target.
The down-stream targets of the CBFA2T3/CBFAIP1 repressor complex will be determined using an innovative melding of expression microarrays and SELEX (systematic evolution of ligands by exponential enrichment). In silico analysis of the identified genes will be used to select optimum drug targets for subsequent validation studies. The major outcome of these investigations is both to determine the significance of the CBFA2T3 protein in the development of breast cancer and to quickly identify suitable novel breast cancer drug targets to ensure rapid target validation.
There is only a limited understanding of details of the molecular changes that cause a normal breast cell to progress to a tumor. The initial changes involve genes that control normal cell growth. One of the types of genes involved in growth are negative regulators (termed tumor suppressor genes) as they normally function by stopping the activity of genes that promote cell growth. Following a large research effort, we have identified a novel putative breast cancer tumor suppressor gene called CBFA2T3. The objective of the proposed study is to determine the importance of this gene in breast cancer. We will determine the proportion of breast cancers where this gene has a critical role, whether this gene controls an early event in the development of cancers, and if it is related to the clinical outcome of patients. This will be achieved by developing a probe that can be applied to tumor samples. This retrospective study will assess small samples of each tumor arranged as an array on a glass slide enabling the simultaneous analysis of breast tumors from 194 patients. We will use a computer-based image analysis system to ensure that the recorded data is unbiased. Finally, we will undertake detailed statistical analysis to determine the proportion of breast tumors where CBFA2T3 is a critical factor and to see if this is related to patient outcomes.
The second part of the proposed study will focus on the identification of new targets for drug development. The development of new drugs based on an understanding of the molecular changes in breast cancer offer the hope of improved treatments with fewer side effects associated with traditional cytotoxic drugs. A common approach to the identification of molecular-based drugs is to screen large libraries of chemical compounds against a particular “target” molecule. However, the identified drugs often fail in subsequent clinical testing due to the limited original characterization and investigation of these targets. To ensure our targets are better validated, we have emphasized the selection of potential targets at an early stage of the proposed research project. We have already identified a novel protein that interacts with CBFA2T3 and the resulting protein complex is proposed to control a number of genes whose protein products will be promising drug targets. We propose to identify these targets by using a novel combination of technologies. Finally, we will approach companies as partners for developing new breast cancer drugs.