The role of SEPT9_v1 in mammary tumorigenesis

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DESCRIPTION (provided by applicant): A novel breast cancer associated gene, SEPT9, was mapped by positional cloning to 17q25.2. SEPT9 was characterized as a member of a highly conserved family of cytokinesis genes encoding eukaryotic GTP- binding proteins named septins. The septin family has been implicated in several cellular processes including cytokinesis, membrane dynamics, cell polarity, apoptosis, cell-cycle regulation and most recently in oncogenesis. SEPT9 was found to be amplified and over-expressed in 50% of breast cancer sample available. Further analysis of SEPT9 locus demonstrated that it encodes different alternative transcripts, of which high SEPT9_v1 expression was observed in breast cancer cells by semi-quantitative RT-PCR and Western blot analysis. In addition, immortalized human mammary epithelial cells (IHMECs) over-expressing a SEPT9_v1 retroviral construct adquired malignant phenotypes, which includes an epithelial to mesenchymal transition (EMT), increased invasiveness, disrupted normal aipha-tubulin associated filaments and increased aneuploidy. Most interesting is the evidence of a direct interaction of SEPT9_v1 and alpha- tubulin, suggesting a mechanism by which overexpression of SEPT9_v1 promotes aneuploidy in IHMECs. The goal of this research is to study how increased expression of SEPT9_v1 contributes to genomic instability and to explore if the over-expression of SEPT9_v1 accelerate tumor formation and oncogenesis. To achieve this goal, SEPT9_v1 dynamic interaction with tubulin proteins during mitosis and cytokinesis will be studied using live-cell microscopy and Fluorescent Resonance Energy Transfer (FRET). In addition, the effect of SEPT9_v1 over-expression in chromosome dynamics and mitotic spindle function will be assayed by live-cell imaging and immunofluorescence. Cytogenetics analyses, including Spectral Karyotyping, will be performed to look for specific chromosomes or structural aberrations that may contribute to malignant progression in mammary epithelium when expression of SEPT9_v1 is altered. Finallly, IHMECs over- expressing SEPT9_v1 will be injected in mammary fat pads of female SCID mice to investigate SEPT9_v1's contribution to tumorigenesis and aneuploidy in an in vivo model. The goal of this project is to determine the importance of SEPT9_v1 in oncogenesis and its relevance to the early detection and clinical management of breast cancer. Breast cancer is a major public-health issue worldwide and is the second leading cause of death among women in US. To improve the genetic risk assessment, diagnosis and treatment for this common life threatening disease, the understanding of the functional role of novel potential oncogenes, such as SEPT9 v1 is essential.