Cancer
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/32539
Browse
Browsing Cancer by Author "Bunnell, Bruce"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Characterization of Estrogen Receptor Mutant Breast Cancer Three-Dimensional Cell Cultures(2024-03-21) Wright, Jordan; Mayer, Olivia; Rinderle, Caroline; Bunnell, BruceBackground: Breast cancer is one of the leading types of cancer among women globally. Many primary breast cancers are estrogen receptor-positive (ER+) and responsive to anti-estrogenic therapies; however, after long-term treatment, these tumors can mutate the estrogen receptor to survive. These mutations make the tumors more triple-negative-like and, therefore, more dangerous. Triple-negative breast cancer (TNBC) has been particularly challenging to treat due to its lack of estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) receptors. Due to the lack of treatment options, TNBC has a poor prognosis and contributes to a significant percentage of breast cancer mortalities. These ER mutants act more like TNBC, resulting in worse clinical outcomes. Current research on these ER mutants has been conducted using two-dimensional (2D) monolayer cell culture, which does not translate effectively in animal models and humans. Three-dimensional (3D) cell culture, which allows for the formation of spheroids, mimics actual tumors and provides results more consistent with actual tumor treatment in vitro. Due to the lack of research on these ER mutants in 3D culture, they first must be characterized to determine baseline gene expression and behavior. After characterization, identifying changes resulting from drug treatment will be possible. Methods: Three different MCF-7 ER+ mutant cells (D538G, E380Q, and Y537S) were seeded at a density of 3000 cells per well in a low-attachment, round-bottomed 96-well plate. After seven days of culture, spheroids were imaged. Spheroids were measured, and size differences were quantified compared to the control, wild-type, ER+ MCF-7 cell line. RNA was extracted from spheroids, quantified, and reverse transcribed to make cDNA. cDNA was used to perform qRT-PCR to determine baseline gene expression differences between ER+ control MCF-7s and ER+ mutant MCF-7 cell lines. Results: The wild-type ER+ spheres have smaller diameters, spherocity values closer to one, and are more compact. They express different levels of EMT markers from the controls, indicating alterations to signaling pathways in the mutant lines. These 3D cultures also vary in expression from the 2D cultures of the same cell lines. Conclusions: MCF-7 ER+ breast cancer cells aggregate more readily than ER+ mutants. The ER must be involved in signaling that promotes aggregation, as reduced ER signaling decreases the ability for spheroid formation. This phenomenon and the differences in gene expression may explain why these mutants tend to behave in a more triple-negative manner in humans. Cells cultured in 3D express some genes to different extents, confirming the importance of 3D culture to identify future therapies – cells behave differently in different culturing contexts, 3D being more consistent with actual tumor behavior. Therefore, characterizing ER+ mutants before beginning drug treatment studies is crucial to understanding how compounds affect cancer cells and identifying differences in different ER+ mutants to know better how to treat them in the future.Item GW525701 Treatment Alters Cellular Morphology and Decreases Triple-Negative Breast Cancer Viability(2024-03-21) Rinderle, Caroline; Bunnell, BrucePurpose: Breast cancer is the second leading cause of death in women in the United States. Triple-negative breast cancer (TNBC) is named for its lack of estrogen (ER) and progesterone (PR) receptors, as well as HER2. The lack of receptors makes these tumors increasingly more difficult to treat, resulting in worser clinical outcomes compared to other receptor-positive subtypes. Kinase inhibitor compounds have been of great interest for treating TNBC patients. Specific kinase inhibitors have been successful against other cancer subtypes, namely chronic myeloid leukemia (CML), which has substantially bettered the outcomes associated with this disease. However, in total, only 72 kinase inhibitors have been approved for clinical use, many of which are not used as cancer therapeutics at all, let alone for breast cancer. Much more research remains to determine the role of other, understudied kinases in cancer proliferation and survival. One kinase inhibitor compound, GW525701, targets STK10, TNIK, SLK, MAP4K4, MINK, and DDR1, none of which are targeted by FDA-approved therapeutics. If treating cancer cells with this kinase inhibitor compound reduces cancer survival and proliferation, then it may be a potential future therapeutic, and will give better insight into the mechanisms controlling TNBC survival. Methods: Breast cancer cells (4IC, BT-549, MCF-7) were treated with GW525701 for 72 hours at a range of concentrations (1nM, 10nM, 100nM, 1µM, 10µM), then stained with phalloidin to visualize cytoskeletal changes. Cellular proliferation and viability were tested using a Cell Titer Glo assay. Migration changes were visualized and quantified using a Boyden migration chamber and ImageJ analysis. All treatment groups were compared to vehicle-treated controls. Results:GW525701 treatment at all concentrations caused morphologic changes consistent with senescence and apoptotic cell death. Cellular proliferation and viability were inversely correlated with GW525701 treatment concentration. Conclusions: Treatment with GW525701 may give insight into the roles of STK10, TNIK, SLK, MAP4K4, MINK, and DDR1 in TNBC survival. This information will be crucial to determining an effective means of targeting TNBC in a therapeutic manner in the future. The specific kinase(s) responsible for the phenotypic outcomes in this experiment will be elucidated in the future as well.