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Observing the Variable Effects of Microbial Treatments in Different Cervical Cancer Cells
The Role of Germline Mutations in DNA repair genes during Cancer Stem Cell Development and Renewal Larecia Thomas, Aubrianna Jordan and Victoria Mgbemena
Introduction
A number of DNA repair pathway components, including BRCA 1/2, are required for hematopoiesis, stem cell maintenance, and cancer suppression. BRCA 1-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 BRCA 2 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 BRCA 2 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 BRCA 2 (PALB2) encodes for a protein that may function as a tumor suppressor (8). It binds to BRCA 2 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 BRCA 1/2-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. .
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 PALB 2 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.
Methodology
Suspend cells
Count and dilute cells
Seed cells in culture flask for expansion Seed cells in well plate
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.
Transfection/ Transduction VUS PALB2, RAD51
+/- treatment +/- treatment
Check morphology / confluency
Cells
Expression assay PALB2, RAD51
Proteomic interaction assay VUS PALB2, VUS RAD51
Supernatant
Transduction efficacy check Cytokine analysis
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 PALB 2 variants of unknown significance, or transduced stably. Products will later be assayed for expression of wild type PALB 2. 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.
Potential Significance
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 CD 49f, 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.
References
1. Mgbemena VE; et al. Cell Reports. 2017, 18, 947-960. 2. Vasanthakumar, A. et al. Blood. 2016, 127, 310- 313. 3. Navarro, S. et al. Molecular Therapy. 2006, 14, 525-535. 4. Alter, B. P. et al. Clinical Haematology. 2014, 27, 214–221. 5. Rebbeck, T. R. et al. JAMA. 2015, 313, 1347–1361. 6. The American Cancer Society. 2018 7. Lee, Y.C. et al. European Journal ofCancer. 2017, 114, 120-125. 8. Guzmán-Ramírez, Völler M et al. Prostate. 2009, 69, 1683-93. 9. Bing X., et al. Molecular Cell. 2006, 22, 719-729, 1097-2765. 10. Sellors J.W. et al. CMAJ. 2000, 163, 503-508. 11. Howlader N et al. SEER Cancer Statistics Review. 2017, 1975- 2014. 12. International Collaboration of Epidemiological Studies of Cervical Cancer. Int J Cancer. 2006, 119,1108-1124. 13. Kataja V et al. Am J Epidemiol. 1993, 138,735-745. 14. Martin, C.M. et. al. Methods in Molecular Biology. 2009, 511,333-59. 15. Li J, Zhou BP. BMCCancer. 2011, 11, 49 16. Chen C et al. Journal of Hematology & Oncology. 2018, 11,6.
OBSERVING THE VARIABLE EFFECTS OF MICROBIAL TREATMENTS IN DIFFERENT CERVICAL CANCER CELLS
Jocelyn Mejia and Victoria Mgbemena | Department if Biology, Brailsford College of Arts and Sciences, Prairie View A&M University
Introduction
Cervical cancer is among one of the fourth most common cancer among woman with approximately 570,000 cases worldwide and 311,000 deaths reported in 2018. [1] Symptoms in the early stages are undetectable unless getting proper screening, leading to a misdiagnosis such as premenstrual symptoms. However, many gynecologists have helped increase the early detection of cervical cancer with persistent recommendations of pap-test exams during regular checkups. [2] The screening of cells from a pap-test is examined by a pathologist and is checked for abnormalities. Another test is done for further examination for the cervical cells, including colposcopy, loop electrosurgical excision procedure (LEEP), and cone biopsy, to determine the stage and aggression of the carcinoma. [3] With early detection and proper diagnosis and treatment, help reduce the risk of cancer-related death. The vaginal microbiome is made of many different types of cells and bacteria, including Lactobacillus acidophilus (BSL1). Research has shown that changes in the vaginal microbiome contribute to the formation of cervical cancer. In a healthy vagina, the balance of the microbiome is an important factor that protects against sexually transmitted illnesses, such as Human papillomavirus (HPV) and multiple gynecological diseases, including cervical carcinoma. [4] A disturbance in the vaginal pH in an acidic range can lower one chance of fighting an infection such as HPV, which contributes to 70% of cervical cancer.[5] BSL1 is a significant influence in Cell lines used to further understand the change and development in cervical cancer cells in our study. It will include C33-A, SiHa, DoTc2-4510, and Ca-Ski obtained from the ATCC repository.
Hypothesis
We hypothesize that we will observe cytokine expression changes in cells treated with Lactobacillus Acidophilus. Specific Aim 1: Investigate the effect of Lactobacillus Acidophilus on gene expression in cervical cancer cell lines. Specific Aim 2: Investigate the effect of Lactobacillus Acidophilus in the presence of different germline and somatic PALB2 mutations in cervical cell lines and cervical cancer cell lines. Specific Aim 3: Assess expression changes in a cell co-culture model of the microbiome for HPV positive and HPV negative cancer cell lines.
Materials and Methods
Cervical cancer lines will be grown in-vitro using cell growth medium and factors. Cells will be transfected stably with plasmid vectors made to encode various PALB2 mutations. Cells will be transfected with factors Sox-2, Oct-4, and Nanog and supplemented with growth medium. Cells will then be treated with: 10ng/mL of Lactobacillus Acidophilus, then incubated for 24 hours in a 37-degree Celsius incubator. Cell lysates will be collected and analyzed using microarray (Affymetrix Gene Chip). Later, PALB2 mutations will be expressed in cell lines, and cells will be assayed for changes in gene expression. Finally, different cell lines will be co-cultured in a 2D model in the presence of an array of BSL-1 level microbes.
Expected Results
Changes in the microbiome will yield observable effects on cell expression profiles and function.
Summary
Our research will study the transcriptomic profiles of treated cervical cells. Environmental conditions such as media composition and temperature will be modified to assess the impact on gene expression.
References
1. Arbyn, Marc, et al. “Estimates of Incidence and Mortality of Cervical Cancer in 2018: a Worldwide Analysis.” The Lancet Global Health, vol. 8, no. 2, 2020, doi:10.1016/s2214- 109x(19)30482-6. 2. “Cervical Cancer - Symptoms and Signs.” Cancer.Net, 10 June, 2019, www.cancer.net/ cancer-types/cervical-cancer/symptoms-and-signs. 3. “Cervical Cancer.” Dana, www.dana-farber.org/cervical-cancer/diagnosis/. 4. Yang, Xi, et al. “Role of Lactobacillus in Cervical Cancer.” Cancer Management and Research, Dove Medical Press, 16 May, 2018, www.ncbi.nlm.nih.gov/pmc/articles/ PMC5962305/#b13-cmar-10-1219. 5. Human Papillomavirus (HPV) and Cervical Cancer.” World Health Organization, World Health Organization, www.who.int/news-room/fact-sheets/detail/human-papillomavirus- (hpv)-and-cervical-cancer.
Joycelyn Mejia is a junior, majoring in Biology. Dr. Victoria Mgbemena is an Assistant Professor in the Department of Biology with research interest in cancer.
