The Role of Germline Mutations in DNA repair genes during Cancer Stem Cell Development and Renewal
Larecia Thomas, Aubrianna Jordan and Victoria Mgbemena Biology Department, Brailsford College of Arts and Sciences, Prairie View A&M University Methodology
Introduction A number of DNA repair pathway components, including BRCA1/2, are required for hematopoiesis, stem cell maintenance, and cancer suppression. BRCA1-deficient mice develop hematological disorders by four weeks, which include pancytopenia (1, 2) and display a pronounced inability to reconstitute stem cells. Although aplastic anemia does not develop, epithelial tumors are observed at a greater incidence in BRCA2 null mice and these mice also demonstrate a marked failure to reconstitute in competitive transplantation assays (3). In the U.S. approximately 63000 cases of uterine cancer develop each year (6). In a 2017 study, the number of patients developing uterine cancer with a BRCA2 mutation was reported to be double the expected number (7). Although clinical reports have statistically explored cases relating BRCA mutations to uterine cancer, the mechanisms that play a role in the pathogenesis of the disease is not clear. The Partner and localizer of BRCA2 (PALB2) encodes for a protein that may function as a tumor suppressor (8). It binds to BRCA2 and is assumed to facilitate their accumulation during homologous recombination repair of DNA breaks (9). While other investigators have reported an increased risk of developing cervical cancer for women with a familial history of BRCA1/2-related disease(s) or other high-risk factors (10, 11, 12, 13), the mechanism for oncogenesis triggered by germline mutations in other DNA repair genes for cervical cancer and other cancers remains unclear. .
Suspend cells
Count and dilute cells
Potential Significance
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PALB2 mutations will be designed onto plasmid vectors. We hypothesize that that the investigator will observe effects on gene expression, cell proliferation and proteincomplex interactions in the presence of mutations which have been inherited.
Seed cells in culture flask for expansion Transfection/ Transduction VUS PALB2, RAD51
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References
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Supernatant
Cells
Expression assay PALB2, RAD51
Proteomic interaction assay VUS PALB2, VUS RAD51
Transduction efficacy check
Cytokine analysis
Goals and Objectives The goal of this study is to determine the role of germline mutations on cervical cancer cell gene expression and invasion potential. The objectives are to express a clinical variant of PALB2 in cervical cancer cells and examine protein expression. As most cancers are epithelial in origin and can invade other tissues, we seek to investigate 1) the factors that may mediate development, renewal and metastasis of the epithelial cancer cell along with 2) how the cancer cells respond to exogenous factors.
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POSTERS AND REPORTS
The functional importance of germline mutations of DNA repair genes in cervical cancer remains unclear. Studies on the inherent characteristics of both cancer and stem cells demonstrate the involvement of factors needed for tumor suppression and cell cycle regulation. Martin et al. identified a group of genes important for cell-cycle regulation which were differentially expressed in cervical cancer cells (14). Additionally, a characterization of cervical cancer stem cells has revealed a phenotype identifying HPV-associated surface receptors, AII and CD49f, as well as transcription factor p63 and epithelial stem cell marker CK17 (14, 15, 16). The overall goal will be to determine what effect germline mutations have on cervical cancer pathogenesis and treatability. Understanding mechanisms of cancer cell expansion will broadly impact efforts to study the role of genes and the environment in chronic disease. This may have an effect on preventive care and prophylactic treatments, which may affect quality of life for a population of people.
HeLa cervical cancer line will be grown in-vitro using cell growth medium and factors. The cervical cancer component mutations associated with each line will be noted mapped accordingly. Cells will be transfected with plasmid vectors which encode for PALB2 variants of unknown significance, or transduced stably. Products will later be assayed for expression of wild type PALB2. In tandem co-immunoprecipitations will reveal resultant binding partners of ∆PALB2. Supernatants will be collected from cells and analyzed for secreted cytokines and tested for transduction targeting and efficiency.
Acknowledgements R&I’s Office of Undergraduate Research (OUR and Biology Department,, Prairie View A&M University.
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