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Research Grants Awarded
Discovery and Functional Analysis of Fusion Transcripts In Breast Cancer
Tumor Cell Biology V
End Sequence Profiling (ESP) is a sequence-based technique developed by us for the elucidation of the structural organization of tumor genomes while cloning genome breakpoints en masse . The large numbers of breakpoints identified in tumor genomes prompted modification of ESP to test the hypothesis that they result in fusion transcripts. To do this full length enriched and normalized cDNA libraries are constructed, arrayed, and end-sequenced. ESP mapping software is used to map cDNA paired ends revealing candidate fusion transcripts. Transcript ESP (tESP) was piloted on a primary tumor and cancer cell lines, and revealed fusion transcripts in each that were then validated. Our goal is to identify fusion transcripts in two breast cancer cell lines and determine if they are recurrent between tumors and encode biological functions. In Aims 1-3 tESP will be used to identify candidate fusion transcripts. Aims 4-6 will focus on validation of fusion transcripts and identification of open reading frames (ORF). The focus of Aims 7-9 is to determine if the validated fusion transcripts are recurrent and encode biological activity. Near full length normalized cDNA libraries will be constructed from all cell lines. 5000 clones from each will be arrayed and end-sequenced. Candidate fusions will be identified using ESP software and validated using RT-PCR. Validated cDNAs will be sequenced to completion to identify ORFs. We will take two approaches to determine if they are recurrent. Normal BAC clones flanking genomic breakpoints for all validated fusions will be selected and used for dual color fluorescent in situ hybridization (FISH) to a tissue microarray containing 120 breast tumors. Co-localized FISH signals will signify the presence of a corresponding genome breakpoint, and show if it is recurrent. We will interrogate a panel of 1 st strand cDNAs from breast cancer cell lines and primary tumors by RT-PCR. A subset of fusions will be silenced using siRNA in the relevant cell lines. Perturbations in proliferation, motility, apoptosis, morphology, and cell cycle distribution will be assessed using flow cytometry, high content imaging and confocal microscopy. This project is based on solid preliminary data showing that fusion transcripts are present in primary tumors and cell lines. If it is established that they have a biological function important to breast tumors, it may fundamentally alter approaches to the development of biomarkers and therapies for breast cancer.
The much heralded anti cancer drug Gleevec targets a fusion protein arising from a chromosome translocation in chronic myeloid leukemia (CML). The translocation fuses two chromosomes resulting in production of a fusion transcript that produces the fusion protein called BCR-ABL. The fusion is recurrent meaning it happens in the tumors of many CML patients. BCR-ABL only occurs in tumors having the translocation and is required for tumor survival. Gleevec inactivates it resulting in very high rates of CML remission and cures. We recently invented and patented a technology called End Sequence Profiling (ESP) for mapping and cloning translocations in solid tumors. We were surprised by the large numbers of translocations detected by ESP and sought to determine if they encode fusion transcripts like BCR-ABL. To do this we modified ESP for detection of fusion transcripts (tESP). Our pilot studies demonstrated that fusion transcripts are produced in a primary brain tumor and breast and prostate cancer cell lines. The detection and confirmation of multiple independent fusion transcripts in all three samples suggest that they are common to solid tumors. This project is designed to test the hypothesis that fusion transcripts are common in breast tumors, that they are recurrent, and that they encode functions important to the tumor such as survival. To do this we will perform tESP on two cell lines since they are model systems in which to evaluate function. Once we identify candidate fusion transcripts, we will validate them using standard molecular biology techniques and determine if they are likely to encode fusion proteins like BCR-ABL. The next step is to determine if they are recurrent. Recurrence is viewed as evidence that something is important for a subset of tumors as is the case with many aberrations involving cancer genes. Finally, we will ask if the fusion transcripts encode detectable functions important to cancer cells. To do this we will inactivate them in these cell lines and measure cellular parameters such as proliferation, programmed cell death, and motility. Establishing that fusion transcripts are present in primary breast tumors, are recurrent and encode biological functions will have enormous implications for development of biomarkers, targeted therapies and vaccines. The work should be viewed as “basic” however our long-term goals are translational and it is reasonable to expect translation of this work to occur within five years of funding.