Elucidating Mechanisms of Cancer Cell Growth And Invasion Using 2d And 3d Cell Culture Models

The Role of Germline Mutations in DNA repair genes during Cancer Stem Cell Development and Renewal Larecia Thomas, Aubrianna Jordan and Victoria Mgbemena

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. .

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.

Cells

Supernatant

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.

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.

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.

The Partner and localizer of BRCA2 (PALB2) encode for a protein that may function as a tumor suppressor (1). It binds to BRCA2 and DNA repair protein Rad 51 homolog 1 (Rad51) at nuclear damage foci and is assumed to facilitate their accumulation during homologous recombination repair of DNA breaks (2). As such, mutations in PALB2 have also been tied to breast, ovarian, and pancreatic cancers. 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 (3, 4, 5, 6) reoccurring germline mutations PALB2 in cervical cancer and others remain unknown.

The Partner and localizer of the BRCA2 (PALB2) function as a tumor suppressor gene. When there is DNA damage, along with the BRCA2 gene, Rad51 is a protein that plays a role in classical Non-Homologous End Joining (C-NHEJ) and Homologous Recombination (HR) to repair double-strand breaks in the DNA. The expression of the DNA repair complex proteins increases in response to DNA damage.

One focus of this research is to study the effects of mutations on the function of cells in a different stratum of cervical tissue. These tissues of the cervix are made of different cellular compositions (squamous and columnar epithelium). Relatively recently, there has been a move to examine intercellular and intracellular signaling mechanisms, cell migration and adhesion events using two-dimensional (2D) cultures, three-dimensional (3D) cultures, and eventually three-dimensional (4D) cultures in order to investigate the contribution of the dynamic microenvironment on cancer.

Two-dimensional cell cultures have long-since been a standard application for investigation of cell dynamics in physiological studies. Still, it is generally known that the culture conditions do not completely recapitulate actual living conditions and can induce changes in cells at the genetic and phenotypic levels, which may not be seen at the organismal level. There are, however, modifications that can be made to the system to examine cell receptor signaling cascades. Recent advancements have made it possible to culture organoids, which can mimic complex in vivo conditions using cell and fiber-based scaffolds (7,8,9).

The 3D cultures provide a more relevant model for viewing the interactions that occur in the human tissues. We propose 3D cultures to highlight the transcriptomic and proteomic expressions of the BRCA gene. We expect that should be a change in the way that the cells interact with each other in the matrigel in the presence of germline, somatic, and epigenetic alterations. The level of sensitivity within the tumors will impact the amount and rate of change during the observation period. We seek to observe these potential changes by looking at variables such as oxygen, metabolites, nutrients, and other molecules needed for the cell to grow and mature.

Specific Aim 1: Determine the effect of different germline PALB2 mutations on cervical stem-like cell survival. Specific Aim 2: Assess the metastatic potential of mutant cell lines in a mirrored tumor environment using 3-D systems. Specific Aim 3: Assess cervical cancer stem-like cell formation and maintenance after treatment.

Matrigel is a non-expensive gel that contains growth factors and regulatory molecules that help the process of protein extraction (7, 8, 9). It is also in a liquid form at certain temperatures (below 22°C) and should be kept inside of a controlled area under a hood, waiting to pipet. Once the matrigel is removed from the freezer, pipet (However many ml) into a silo so that it can assume its shape. In other studies, it was shown to improve conductivity between the ice and the silo if you place the silo on an aluminum sheet/ block that also stops the silo from clotting up, Once you have added all of your materials to the silo place into a centrifuge tube and centrifuge for about five-ten minutes. Place the contents of the centrifuge tube inside of a petri dish and allow gas exchange to occur and incubation. Once the matrigel is semi-solidified in the incubator, the cervical cells should be placed into the silo and cleaned with a phenol-free media at least four times a week.

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Using the 2D method, we expect to see a monolayer of cells growing inside of the culture, as well as these, being the lowest maintenance of all cell cultures; there should be an alteration in the morphology. We will begin with a co-culture experiment, so there may be two different growth patterns occurring at once. The culture is intended to mock an in vivo condition to provide an alternative to modeling the animal model cells. In using a 3D and a 2D cell culture model, there will be an observed difference in the number of cells, cell growth, and proliferation, as well as the ultrastructure of the cells.

The 3D cell cultures allow for cells to proliferate at a faster rate than a 2D culture. The 3D culture may provide a more accurate look at cell-to-cell interactions, tumor characteristics, and yield insights towards the development of different therapeutic drugs.

This project will primarily address germline mutations on cancers of the epithelium of the reproductive tracts. The molecular requirements for uterine cancer stem cell differentiation and renewal in the presence of a synonymous, missense mutation requires exploration. One objective of this research program would be to assess the roles of different PALB2 mutations during stem cell transformation and assay for DNA- damage and cell death by measuring expression of factors such as p53, RAD51, PALB2, FANCD2 (10), and labeling double-strand breaks.

1. Bing Xia, Qing Sheng, Koji Nakanishi, Akihiro Ohashi, Jianmin Wu, Nicole Christ, Xinggang Liu, Maria Jasin, Fergus J. Couch, David M. Livingston. (2006). Control of BRCA2 Cellular and Clinical Functions by a Nuclear Partner, PALB2, Molecular Cell, Volume 22, Issue 6, Pages 719-729, 1097-2765. 2. Mersch, Jacqueline et al. “Cancers associated with BRCA1 and BRCA2 mutations other than breast and ovarian.” Cancer vol. 121,2 (2015): 269-75. doi:10.1002/cncr.29041 3. Sellors JW, Mahony JB, Kaczorowski J, et al. Prevalence and predictors of human papillomavirus infection in women in Ontario, Canada. Survey of HPV in Ontario Women (SHOW) Group. CMAJ. 2000;163:503-508. 4. Howlader N, Noone AM, Krapcho M, Miller D, Bishop K, Kosary CL, Yu M, Ruhl J, Tatalovich Z, Mariotto A, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2014, National Cancer Institute. Bethesda, MD, https://seer. cancer.gov/csr/1975_2014/, based on November 2016 SEER data submission, was posted to the SEER website in April 2017. 5. International Collaboration of Epidemiological Studies of Cervical Cancer. Cervical cancer and reproductive factors: Collaborative reanalysis of individual data on 16,563 women with cervical carcinoma and 33,542 women without cervical carcinoma from 25 epidemiological studies. Int J Cancer. 2006; 119:1108-1124. 6. Kataja V, Syrjänen S, Yliskoski M, et al. Risk factors associated with cervical human papillomavirus infections: a case-control study. Am J Epidemiol. 1993;138:735-745. 7. Karolina Zuk, Anna et al. “Modeling and validating three-dimensional human normal cervix and cervical cancer tissues in vitro.” Journal of biomedical research vol. 31,3 (2017): 240-247. doi:10.7555/JBR.31.20160150 8. Miao, S., Cui, H., Esworthy, T., Mahadik, B., Lee, S. ‐j., Zhou, X., Hann, S. Y., Fisher, J. P., Zhang, L. G., 4D Self‐Morphing Culture Substrate for Modulating Cell Differentiation. Adv. Sci. 2020, 7, 1902403. https://doi.org/10.1002/advs.201902403 9. de Souza, N. Organoids. Nat Methods 15, 23 (2018). https://doi.org/10.1038/ nmeth.4576 10. Park, J. Y., Singh, T. R., Nassar, N., Zhang, F., Freund, M., Hanenberg, H., … Andreassen, P. R. (2014). Breast cancer-associated missense mutants of the PALB2 WD40 domain directly bind RAD51C, RAD51, and BRCA2, and disrupt DNA repair. Oncogene, 33(40), 4803–4812. doi:10.1038/onc.2013.421

Aubrianna Jordan is a senior, majoring in Biology. Dr. Victoria Mgbemena is an Assistant Professor in the Department of Biology with research interest in cancer.