Evaluation of the cell cycle regulation in Tuberin knockout cell lines

Tuberin (TSC2) and its binding partner hamartin (TSC1) are tumour suppressor proteins that regulate cell growth and cell proliferation through the mTOR pathway and cell cycle, respectively. Mutations in these genes are related to many proliferative diseases such as Tuberous Sclerosis Complex (TSC) and cancers. Our lab has determined a novel role for TSC2 as a cell cycle regulator at the G2/M transition. TSC2 can bind to the G2/M cyclin, Cyclin B1 (CycB1), and retain this cyclin in the cytoplasm, delaying mitotic onset. My project focuses on further understanding the TSC2/CycB1 complex formation and the consequences of this interaction during the progression of the cell cycle. I hypothesized that cells lacking TSC2 will have an improper G2/M transition causing morphological and proliferative abnormalities. To test my hypothesis, I used TSC1 or/and TSC2 knockout HEK293 cell lines acquired from the TSC Biosample Repository to measure the rate of the G2/M transition, cell proliferation, and cell morphology. To further understand the TSC2 knockout consequences, the cells were synchronized using a thymidine block (S phase blocker) and the cell cycle profiles were analyzed following release. I found that KO cells exhibit a change in morphology and size when examined under a confocal microscope. When the cell cycle profiles were analyzed with flow cytometry, the double knockout cells are significantly decreased in the dead cell population under normal conditions while TSC1 KO cells are significantly decreased in the S phase population. Additionally, KO cells were prone to serum starvation-induced apoptosis and were never able to fully synchronize using a double thymidine block; however, a 2mM final concentration of thymidine provided a more uniform block than 0.2mM. Moreover, when analyzed with flow cytometry, the WT HEK293 were significantly larger than the KO cells and dilute BrdU faster than KO cells suggesting faster proliferation. The results of my project will shed light on the role of TSC2 as a cell cycle regulator and clarify its role as a tumour suppressor protein.

“Joining the Porter Lab has been one of the most rewarding experiences of my academic career. Before joining the Porter Lab, I wasn’t really sure in which direction I wanted to take my academic career. However, I knew that I liked science and I knew that this would be a great opportunity to explore my interests and work with highly skilled academics doing cutting-edge research. What I didn’t know, was that, on top of being mentored and pushed to achieve excellence, I would develop amazing connections with people who I’m very proud to call my colleagues. It has been a rollercoaster of emotions, to say the least. From the long frustrating days trying to troubleshoot experiments, to the many laughs and fun nights shared with lab mates, it has all been such an integral part of my university experience. I’m incredibly grateful for all of the support and guidance I have received over the years and especially during my thesis. The Porter Lab has been a place for my knowledge and interests to grow unrestricted and for me to be able to give back to the scientific community, even if only in a minute way. Even as I write this and look back on all of my successes and failures, I know it was accomplished by standing on the shoulders of giants with the unwavering support of everyone in the lab. I don’t know what the future holds for me, but I will forever look back on this experience with fond memories and will always consider myself a Porter lab rat.”

Spy1: A Closer Look at its Binding Mutants

Spy1 (SPEEDY; RINGO) has been previously identified as a novel cell-cycle regulator that is elevated in breast cancer, promoting progression through the cell cycle and increased cell proliferation. This atypical, cyclin-like protein is capable of binding directly to Cdk1 and Cdk2, activating their kinase activity to override cell-cycle checkpoints; however, the mechanism by which Spy1A activates Cdks has been resolved using only Cdk2 and Spy1A, a single-member within the Spy1 family. Spy1 can also promote the degradation of p27, a Cdk2 inhibitor, through its phosphorylation and binding between p27 and the Spy1-Cdk2 complex. Amino acid residues on Spy1 have been identified to be important for p27 and Cdk2 binding and Cdk2 activation. There is interest in determining whether these residues are conserved across Spy1 family members and further resolving the impact these mutations have on Spy1 function. We expressed these mutants in HEK-293 cells in the presence and absence of p27 to examine the effects of these mutants on cell growth and binding to its effectors. To investigate sequence homology between the different Spy1 family members, their amino acid sequences were aligned and compared using the Basic Local Alignment Search Tool (BLAST). Future work can be done to examine whether the function of these sites is conserved across the family members. The fulfillment of these objectives will provide a better understanding of Spy1 to target it in cancer treatments.

“I have nothing but great things to say about my two years in Dr. Porter’s lab. Whether it was interacting with fellow undergrads, the RA’s, or Dr. Porter herself, the entire team was welcoming from the beginning and always displayed a willingness to help. Despite the fact that Covid-19 disrupted my chances of returning to the lab, I managed to stay connected to the entire team throughout my thesis experience. Since joining Dr. Porter’s lab in my third year, I have acquired so many great skills that I will be taking with me on my future endeavors. While I am sad to be leaving the University of Windsor and such a great group of devoted researchers, I know my experiences will have a long-lasting impact on myself as a student, researcher, and future medical professional. Thank you everyone in the lab for your kindness, guidance, and encouragement.”

The Effects of Dexamethasone on Breast Cancer and Chemotherapeutic Treatment

Breast cancer is the second leading cause of cancer deaths in Canadian women. A particular aggressive subset of breast cancer, triple-negative breast cancer (TNBC), accounts for approximately 15% of all breast cancer diagnoses. Unlike other subtypes, it does not express well-defined molecular targets, such as hormone receptors, that allow for targeted treatment. TNBCs are therefore treated with cocktails of potent but general cytotoxic chemical therapies which have been known to cause harsh side effects such as hypersensitivity, nausea, and vomiting. These side effects are combatted by the addition of the synthetic glucocorticoid, Dexamethasone (Dex) to the treatment regime. It is of clinical significance to understand the impact of Dex on breast cancer behavior and how it influences chemotherapy treatment and patient response. Through the completion of a literature review, the role of Dex in promoting breast cancer progression through cell survival and metastasis was evaluated. Understanding the effects of glucocorticoids, such as Dex, on breast cancer cell biology and their interaction with chemotherapeutic agents can lead to alternative therapeutic strategies and improved patient outcomes.

“Joining the Porter Lab in my second year was a highlight of my university experience. Not only did I get to meet such talented and inspiring people, but I got to learn several skills that are applicable to many life situations. Even though my thesis project was unconventional due to the pandemic, I had such amazing mentors that ensured I learned as much as I would have if I had been able to physically be in the lab. I am grateful for the experience and the people that helped me succeed, and I am honored to have witnessed the many successes of my fellow lab members. I am looking forward to continuing to hear about the amazing work the Porter Lab Team is doing.”

Katie will be starting Optometry School at Waterloo University next fall.

The Tumour Suppressor Tuberin: Coordinating DNA Damage and Mitotic Onset

The cell cycle is a series of events that involve cell growth and division. It is regulated by cyclins and CDKs, as well as checkpoints that control its progression. Tuberin (TSC2 gene) is a tumour suppressor protein that controls cell growth and proliferation. Tuberin mutations are implicated in several cancers and diseases such as tuberous sclerosis complex (TSC), a multisystem genetic disorder that is characterized by the growth of hamartomas. Previous data from our lab has demonstrated tuberin’s role in controlling mitotic onset by binding to the G2/M cyclin, Cyclin B1 according to nutrient availability. My project explores the role of the tuberin in the G2/M transition during DNA damage repair. DNA damage can result from events such as radiation, X-ray, and reactive oxygen species which activates the G2/M checkpoint to restrict mitotic onset and ensure sufficient time to repair damaged DNA. In our studies, NIH3T3 p53 positive cells were transfected with TSC2-WT or clinical TSC2 mutants that present low affinity for cyclin B1 (C696Y) or lack a GAP domain. The transfected cells were treated with etoposide, a topoisomerase II drug that induces double-stranded DNA breaks during the G2/M transition. The changes in cell cycle phases were analyzed using flow cytometry. Our preliminary results have determined that following treatment with 2µM of etoposide at 8 hours, there is a significant decrease in the % of cells at the G2/M transition in C696Y transfected cells vs. those overexpressing tuberin. This project will help expand the role of tuberin during the mitotic onset and clarify mechanisms of proliferative diseases such as TSC and cancers. 

“My experience in the Porter Lab has been one that has been truly life-changing! By deciding to embark on this journey, I had the opportunity to really see science in action! Throughout our undergraduate career, we learn about these concepts and different experiments from listening to lectures and reading textbooks. Being able to go into the lab and perform my own experiments has shed light to truly how hard and rewarding science can be. The feeling of finally seeing the hard work pay off from repeating and troubleshooting an experiment countless times is one that is so fulfilling! While starting my 420 and performing my own research had its challenging moments, I was constantly supported and reassured by the rest of our lab every step of the way. My experience as a Porter Lab Rat has given me the opportunity to meet and work with so many amazing people. From Dr. Porter to the RAs, grad students, and other undergrads, everyone has been so passionate and dedicated in our pursuit of scientific advancement and it has been an honor being able to work with everyone. Joining the Porter Lab and doing a 420 has been such a gratifying learning experience for me and I highly encourage others to do the same!”

Kim is the recipient of the 1st place 10 Min Presentation for Cell and Genetics at the Ontario Biology Day. She will continue her research in our lab while coursing the 5th year undergrad studies at the University of Windsor.

The impact of HA-labeled nanoparticles on CD44+ Tumour Initiating Cells in Glioblastoma

Glioblastoma (GBM) is the most aggressive brain tumor with a median survival of only 15 months. Despite decades of research, GBM continues to pose a great therapy challenge due to its extreme genetic and phenotypic heterogeneity. The pools of stem-like tumour initiating cells (TICs) maintain and recapitulate the heterogeneity of GBM; their ability to self-renew fuels resistance to treatment and tumour recurrence. Furthermore, successful treatment of GBM is hindered by the poor penetration of the available therapeutics through a tightly regulated blood-brain barrier (BBB). Conjugated polymer nanoparticles (CPNs) are a class of nanotechnology of potential novel application for GBM treatment. CPNs can penetrate the BBB and can also be encapsulated with drugs for cargo delivery, in addition to functionalizing their surface with ligands complementary to protein receptors expressed on the glioma cell surface. In collaboration with the Rondeau- Gagné group, my project focuses on a diketopyrrolopyrrole (DPP)-based CPNs labeled with a fluorescently tagged hyaluronic acid (HA). HA is the primary ligand of the CD44 receptor present on the surface of TICs. CD44 overexpression is implicated in GBM progression and correlates with poor patient survival. Using the human U-251 MG GBM cells and patient-derived primary lines, I will investigate the effects of CPNs on CD44 signaling and stem cell properties of TICs in vitro as well as in vivo, using a Zebrafish model. Collectively, these results will not only further validate the CPN system as a potential future anti-GBM therapy but also contribute to a better understanding of CD44 biology in GBM.

“When I joined the Porter Lab at the start of my second year, I had no idea that it would end up being both the highlight of my undergraduate journey and the most rewarding experience I’ve had to date. Before joining the lab, I wasn’t familiar with the research at all, but by the end of my fourth year, it’s safe to say that research has become a genuine, lifelong passion of mine. My time in the lab has made it clear that I want to pursue a career involving research in the future. It’s been an opportunity for me to both learn and develop many valuable skills and traits like working as a team, problem-solving, and patience, as well as responding to failure and keeping determination. I’ve learned an incredible amount of information during my time in the lab and the desire to learn only continues to grow. Research can be very time-consuming, and it does require a lot of dedication and effort, but I can confidently say that I’d repeat my experience in the Porter Lab over and over again with no complaints. The time I spent here has felt like my family away from home. A big part of my research experience revolved around building relationships with my mentors, graduate students, and fellow lab mates, and it has been a surreal experience. These are relationships that I’ll be beyond grateful for forever; I’ll always remember being a Porter Lab Rat. I’ve caught myself thinking about how amazing it is to be able to contribute to cancer research and work towards elevating the health of our community. Even if my actions seem minuscule, I recognize now that these little steps are all vital in understanding the bigger picture. The Porter Lab is in good hands – I know every individual member is going to make some very important contributions to the field of research, and I hope I can be there to celebrate these accomplishments with them all!”

Sami is the recipient of 1st place undergraduate speaker at UWill Discover conference, NSERC USRA 2020 summer, and 2021 summer. He will continue his research in our lab during the summer.

The research in Porter Lab is divided into 4 main groups, one research associate fellow is responsible for the projects, grants, and students into each group. I’m Elizabeth Fidalgo, Ph.D., and I’m the leader of the Tuberin group. As the group name states our group studies a protein named Tuberin. This protein is a Tumour suppressor protein that controls cell growth and cell proliferation. Mutations in the Tuberin gene (TSC2) lead to several diseases, including Tuberous Sclerosis where patients present with large benign tumours (hamartomas) that affect primarily the skin, heart, brain and kidney and lead to compounding problems like autism and seizures. Mutations in Tuberin also result in Pulmonary Lymphangioleiomyomatosis (LAM), a cyst condition that affects the lungs and surrounding tissues in primarily young women. Mutations in TSC2 have been found in several cancers, including cancers of the skin, breast, kidney, and brain. As you can see, Tuberin is an essential protein for the control of cell growth and proliferation.

My group was the first one to report the role of Tuberin in the regulation of an important checkpoint in the cell cycle. Our results were published in the Cell Cycle 10: 3129-3139 with the title: The tumor suppressor tuberin regulates mitotic onset through the cellular localization of cyclin B1. Since then we have been investigating the mechanisms behind the essential control of cell division by Tuberin.  Understanding the basic biology is necessary for us to understand what this protein does in disease. This work is the foundation of our NSERC (Natural Science Engineering Research Council of Canada) work.

Tuberin is a large protein (200KDa) and is regulated by phosphorylation and it’s the main player of diverse pathways inside a cell.  It isn’t easy to work with it, so my group has developed tools to make a little bit easier to understand the functions of Tuberin towards the cell cycle. One of the first tools we developed using molecular biology techniques is a G2/M reporter, a vector that is transfected into the cell to monitor the Tuberin checkpoint. The cells turn blue when at the checkpoint, this way we can monitor the time that takes for the cell to move from one phase of the cell cycle to other. We’ve published the engineering steps for the construction of this reporter in Cytotechnology 2016 (1): 19-14 Title: Derivation of a novel G2 reporter system. This work was largely conducted by an undergraduate student in our lab, Sabrina Botsford.

Another tool under construction is the BiFC system (Bimolecular Fluorescence Complementation) where the cells turn yellow when Tuberin is actively participating of the checkpoint. These fluorescent tools are important because through them we can use time-lapse fluorescence microscopy and flow cytometry techniques to better understand the functional role of Tuberin in the cell cycle. This BiFC project was started by another undegraduate student, Marisa Market. We are also genetically modifying the genome of HEK293 (kidney) and HeLa (cervical tumour) cells using CRISPR-CAS system. We are introducing TSC2 clinical mutations in the genome of these cells lines to study the pathways affected by these mutations in hopes of revealing how these mutations regulate aberrant cell division. This basic information is essential for research to lead to new treatments for diseases ruled by Tuberin mutations.

This fundamental work supported by NSERC has provided the foundation for several other projects. We collaborate with Dr. James Gauld (Chemistry/Biochemistry UWindsor) on a Seeds4Hope grant sponsored by our local Windsor Cancer Centre Foundation (http://windsorcancerfoundation.org/seeds-4-hope/). This work is figuring out the important binding regions between Tuberin and its partners using computational (computer-based) approaches. In 2013 I was awarded a Seeds4Hope grant to study the role of Tuberin in the formation of Medulloblastoma, the primary brain cancer affecting children; an undergraduate student, Santo Spencer Briguglio, was supported to work on this project by the Brain Tumour Foundation of Canada. We are also currently working with Dr. Andrew Swan (Biology, UWindsor) to expand our cellular studies of Tuberin-Cyclin B1 into an in vivo Drosophila (fly) system. Our results have been presented at many local and international conferences.

One of the strengths of our group is the training of undergraduate and graduate students. We’ve trained several brilliant students, some of them have received awards/fellowships from our department, university, and the province and most have been successfully accepted to Medical and Pharmacology schools through Canada.

Being a tough protein to figure out we often joke that this project trains students to be strong! As an example, one of our past Tuberin undergrads Ryan Ard did his Ph.D. in the Allshire lab in the UK and is now a postdoc in the Marquardt lab in Denmark. Ryan received an international scholarship and has 7 publications including a Nature Communications paper. We are proud of our successful students! We currently have a great group: 2 undergrad thesis students (Jackie Fong and Gillian Denomme), an MSc student (Adam Pillon) and a Ph.D. student in collaboration with Dr. Andrew Swan (Mohammed Bourouh). We are looking for great things to come from this team.

Hope you enjoyed to know a little bit more about one of the research groups in the Porter Lab.

Please leave your comments and/or suggestions below, I’ll be happy to answer them.

Elizabeth

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