Determining the Dependency of Spy1 based on Rb Status in Triple Negative Breast Cancer
Breast cancer is the second most common cancer worldwide and the most common cancer among women. Triple Negative Breast Cancer (TNBC) is a particularly aggressive form of breast cancer with many subtypes based on gene expression profiles. There are currently no targeted treatments for TNBC due to its molecular characteristics, urging the discovery of new therapeutic targets. Potential therapeutic avenues are the cell cycle and its mediators which play an important role in cancer formation and progression. Spy1, a cyclin-like protein, promotes cell proliferation through the G1/S and G2/M checkpoints. Spy1 promotes proliferation even in the presence of DNA damage, overriding checkpoints and increasing cancer susceptibility. While Spy1 has been found to be elevated in breast cancer, its unique binding structure makes for an ideal candidate for cell cycle inhibition therapy. The retinoblastoma tumor suppressor protein (Rb) is known to regulate the DNA damage response system and is key in regulating the cell cycle. However, studies have shown that Rb is often mutated in TNBC inducing deregulated cell cycle progression potentially leading to tumor development. For some breast cancer subtypes the presence or absence of Rb (Rb status) can dictate response to treatment by cell cycle inhibitor drugs. Using in vitro TNBC models (MDA-MB-231 & Bt549 cell lines), this study aims to determine if Spy1 can override checkpoints independently of Rb status, and if elevated levels of Spy1 alter this response. These results could provide further guidance in developing cell cycle inhibition targeted therapies and potentially better TNBC patient outcomes.
“Before transferring to the University of Windsor, I wasn’t sure of how to get involved in research. Being in second year at the time, I was intimidated by research and unsure about my capabilities as a scientist. When I enrolled in CURES (cancer undergraduate research experience students), a course with Dr. Lisa Porter, I learned about the diversity of local cancer research taking place in Windsor-Essex. This course fuelled my motivation to learn, and genuine passion to contribute in this field. I found my confidence as a researcher when I joined Porter Lab in my fourth year, where I was thrilled to be given the opportunity to complete my undergraduate thesis. Up until my last year of University, I never knew myself to be as confident and disciplined as I am now. Being in a research lab has accelerated my learning, competency, and knowledge translation in such a short time. The atmosphere of Porter Lab is extremely motivating, and the people are supportive and patient. Porter Lab offers each member numerous opportunities to move forward science within the community and has helped me adapt to becoming a leader in science- a field I never felt I fit in before. During my time in lab, I have learned a multitude about the nature of science and what is involved in basic science research, including all the time invested in planning experiments and writing grants. Completing my undergraduate thesis was extremely rewarding and I am excited to continue my research in a master’s degree with Porter Lab, working alongside all the Porter Lab rats!”
The Impact of Cyclin B1 on Tuberin Stabilization
Tuberous Sclerosis Complex (TSC) is an autosomal dominant disorder caused by mutations in either TSC1 or TSC2, genes that encode the proteins Hamartin and Tuberin respectively. Hamartomas (benign tumours), skin lesions, neurological symptoms, renal dysfunctions, and retinal malformations are often present in TSC patients with varying severity. Tuberin and Hamartin regulate protein synthesis through mTORC1 inhibition. Tuberin can also delay mitotic onset at the G2/M cell cycle transition by binding to Cyclin B1. Hamartin has been shown to stabilize Tuberin by inhibiting its ubiquitination by HERC1 and subsequent degradation. Preliminary data from our lab suggests that Cyclin B1 may also contribute to the stabilization of Tuberin levels during the G2/M transition. Phosphorylation status of the cytoplasmic retention sequence (CRS) of Cyclin B1 plays an important role in the formation of the Tuberin/Cyclin B1 complex. The unphosphorylated CRS form of Cyclin B1 (Cyclin B1 5xA) binds stronger to Tuberin compared to the phosphorylated form (Cyclin B1 5xE). My thesis investigates the role of Cyclin B1 in Tuberin stabilization. HEK293-TSC1 null cell lines (IC2) were transfected with varying concentrations of Cyclin B1 5xA DNA and Tuberin protein levels were quantified by Western blot techniques. After blocking HEK293 cells in G2/M using thymidine and collecting them at different timepoints, endogenous Tuberin levels at different stages of the cell cycle were also quantified with Western blot techniques. Understanding the role of Cyclin B1 in Tuberin stabilization will shed light on cell proliferation and growth mechanisms that underlie tumorigenic disorders.
Characterization of Multiple Myeloma Cells Treated with CDK Inhibitors
Multiple myeloma (MM) is an aggressive hematopoietic cancer that is caused by the abnormal growth of plasma cells in the bone marrow. MM has a poor prognosis and most patients will eventually relapse with a more aggressive and untreatable form of the disease.
Cyclin Dependent Kinase Inhibitors (CKis) are an emerging targeted therapeutic option for Relapsed MM and have been investigated in many clinical trials. Flavopiridol is a first-generation non-specific CKI that inhibits CDK1, CDK2, CDK4, and CDK7. Flavopiridol has had high pre-therapeutic efficacy, but limited clinical success as a monotherapy. Dinaciclib is a second-generation CKI that is more selective and inhibits CDK1, CDK2, CDK5, and CDK9. Dinaciclib has also had low efficacy in clinical trials. In this study, we compared MM cells (U266B1) treated with Dinaciclib to Flavopiridol in-vitro by examining cell proliferation, apoptosis, and cell cycle profiles. Our data shows that there is no significant difference in cell proliferation or apoptosis of U266B1 cells treated with Dinaciclib and Flavopiridol after 24 hours, 48 hours, and 72 hours. Cells treated with Flavopiridol were more frequently arrested at the G2M checkpoint compared to Dinaciclib after 72 hours. Better characterization of MM cells treated with different types of CKIs will help to better understand their lack of clinical success as a monotherapy.