8 minute read
Studying the Role of The Kynurenine Metabolic Pathway in Cervical Cancer Cell Lines
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.
STUDYING THE ROLE OF THE KYNURENINE METABOLIC PATHWAY IN CERVICAL CANCER CELL LINES
Larecia Thomas and Victoria Mgbemena | Department of Biology, Brailsford College of Arts and Sciences, Prairie View A&M University
Introduction
The Kynurenine pathway is one of the routes that tryptophan degradation occurs in. It is catalyzed by IDO (Indoleamine 2, 3-dioxygenase) or TDO (tryptophan-2,3-dioxygenase). IDO can suppress immune systems creating a suitable environment for tumor cells to spread into. This is due to IDO expression being correlated to glioma cell motility. This is caused by IDO releasing Kynurenine, which then activates AhR (aryl hydrocarbon receptor). Increased Expression of IDO can be used as an indicator of cervical cancer due to it being found in the tumors (1). The Kynurenine pathway produces many different metabolites that can harm the body. Some of the results of a kynurenine pathway that has had a gain of function are inflammation, immune system suppression, and neurodegenerative conditions such as Alzheimer’s.
Hypothesis: If the regulation of the Kynurenine Metabolic Pathway and its byproducts is directly related to the survival or demise of cervical cancer cell lines, then we expect to see differences in the amounts of IDO or TDO in the cells in the presence of germline, somatic or epigenetic mutations.
Specific Aim 1: Determine the effect of different germline PALB2 mutations on IDO and TDO expression in cervical cell lines. Specific Aim 2: Determine the effect of IDO overexpression in the presence of different germline and somatic PALB2 mutations in cervical cell lines and cervical cancer cell lines. Specific Aim 3: Assess the effect of IDO and TDO overexpression on cancer cell invasion in cervical cancer cell lines.
Experimental design: We will culture and study normal cervical cell lines and compare to cervical cancer cell lines and a cervical cell line that has overexpressed tryptophan degradation (2). We will then assess the presence of IDO and TDO and take quantitative data of the expression of IDO and TDO in each cell line.
Experimental Materials and Methods
Cells will be seeded on a coated culture plate (made of DMEM HG (Gibco). They were supplemented with FBS (10%; Biowest), penicillin/ streptomycin, and gentamycin). Cells will be kept at physiological temperature standards. Cultures will be observed under microscopes. Cell lines will be transfected with plasmids encoding for mutations in DNA repair genes, such as PALB2 and FANCD2, and a Western Immunoblot will be conducted to determine the presence of IDO and TDO.
TDO will be overexpressed in the tryptophan degradation pathway in the presence and absence of DNA repair mutations. An immunofluorescence assay will be conducted to determine the location of proteins directly associated with the Kynurenine Pathway. The amount of tryptophan degradation (5) and secretion immunoregulatory compounds will be assayed using the enzyme-linked immunosorbent assay (ELISA).
Materials
Cell lines include SiHa, C33-A, normal human cervical, DoTc, HeLa. Six-well culture plates will be used, and cells will be cultured in a 5% CO2, 37-degree Celsius incubator.
Expected Results and Summary
After conducting the necessary experiments, it is expected that an increased amount of IDO and TDO in the cervical cancer cell lines would be observed in comparison to the standard cervical cell lines. It is also expected to see an increased number of immunoregulatory compounds that are a product of the Kynurenine Pathway.
Summary
IDO has been studied in both neuronal conditions and cancer models (1,2, 3,4). It has also been connected to an increase in pro-inflammatory cytokine production. It is currently unclear if non-commensal microorganisms in the cervical niche can contribute to elevated states of inflammation (6). Whether this inflammation also increases the risk of conditions like cervicitis and cervical cancer. Our group aims to study the potential impact of quinolinic acid and IDO production via the kynurenine metabolic pathway on cervical cell gene expression. Our research will study the transcriptomic profiles of infected cervical cells.
References
1. Hornyák, Lilla et al. “The Role of Indoleamine-2,3- Dioxygenase in Cancer Development, Diagnostics, and Therapy.” Frontiers in immunology vol. 9 151. 31 Jan. 2018, doi:10.3389/ fimmu.2018.00151 2. Du, Lisha et al. “Both IDO1 and TDO contribute to the malignancy of gliomas via the Kyn-AhR-AQP4 signaling pathway.” Signal transduction and targeted therapy vol. 5 10. 21 Feb. 2020, doi:10.1038/s41392-019-0103-4 3. Janik, Karolina et al. “Efficient and simple approach to in vitro culture of primary epithelial cancer cells.” Bioscience reports vol. 36,6 e00423. 9 Dec. 2016, doi:10.1042/BSR20160208 4. Mei, Xionge et al. “Radiosensitization by Hyperthermia: The Effects of Temperature, Sequence, and Time Interval in Cervical Cell Lines.” Cancers vol. 12,3 582. 3 Mar. 2020, doi:10.3390/ cancers12030582 5. Sadok, Ilona, et al. “Application of the Optimized and Validated LC-MS Method for Simultaneous Quantification of Tryptophan Metabolites in Culture Medium from Cancer Cells.” Journal of Pharmaceutical and Biomedical Analysis, Elsevier, 6 Aug. 2019, www.sciencedirect.com/science/article/abs/pii/ S0731708519306594?via=ihub. 6. Stadler, G., et al. “Development of Standardized Cell Culture Conditions for Tumor Cells with Potential Clinical Application.” Cytotherapy, Elsevier, 20 Dec. 2012, www.sciencedirect.com/ science/article/abs/pii/S1465324907701111.
Larecia Thomas is a senior, majoring in Biology. Dr. Voctoria Mgbemena is an Assistant Professor with research interest in cancer.
