LMP student seminars: 1 April

Each week during term time, MSc and PhD candidates in the Department of Laboratory Medicine and Pathobiology present their research.

Anyone is welcome. No need to register.

Location: Medical Sciences Building, rooms 4171 or 4279, see below.

As part of the core research curriculum, students taking LMP1001/2/3: Graduate Seminars in Laboratory Medicine and Pathobiology will present their projects. Please see abstracts below.

3. Cardiovascular, Physiology and Metabolism/ Molecular and Cell Biology and Regenerative Medicine

Location: MSB 4279

Viktor Prifti

• Title: Novel Mechanism of Thrombosis and Hemostasis: Investigating the Role of Platelet α-Dystroglycan
• Supervisor: Dr. Heyu Ni

2. Cancer, Development, and Aging

Location: MSB 4171

Nan Jiang

• Title: Investigating the Functional Role of Renal Stromal Factor CXCL12 in Murine Kidney Development
• Supervisor: Dr. Norman Rosenblum

Wai Lam Mok

• Title: Eliminate Colorectal Cancer Cells in the Drug-Tolerant Persister State: Combined Inhibition of Autophagy and Induction of Apoptosis
• Supervisor: Dr. Catherine O'Brien

Abstracts

Viktor Prifti: Novel Mechanism of Thrombosis and Hemostasis: Investigating the Role of Platelet α-Dystroglycan

Patients with abnormal alpha-dystroglycan (αDG) such as in muscular dystrophy experience increased bleeding diathesis during major surgeries. αDG is a glycosylated cell surface protein that binds extracellular matrix proteins and is also expressed on platelets. Platelets are essential for controlling bleeding through adhesion, activation, and aggregation at sites of vascular injury. αDG has been reported to be shed from activated platelets but the role of αDG in hemostasis and thrombosis has not been previously investigated. We aimed to explore the mechanism of platelet αDG in hemostasis and thrombosis by blocking αDG with monoclonal antibodies during platelet activation, agglutination, and aggregation assays. We then studied candidate binding proteins of αDG which were hypothesized to be major platelet activation/adhesion receptors (αIIbβ3, GPIbα). We found anti-αDG mAb significantly decreased platelet aggregation and agglutination induced by various agonists. We also detected anti-αDG mAb significantly decreased platelet activation via degranulation upon agonist induction. We then utilized bio-layer interferometry (BLI) to detect a mild binding affinity between recombinant human αDG and platelet αIIbβ3. Interestingly, a high affinity interaction was detected between αDG and GPIbα with BLI, which was subsequently confirmed with isothermal titration calorimetry. We then observed αDG binding to live human platelets which was diminished upon enzymatically removing GPIbα. Our data suggests αDG plays an important role in platelet activation and aggregation. Through interaction with αDG, GPIbα may deliver outside-in signalling for platelet activation. Our findings may improve care for muscular dystrophy patients and promote novel anti-thrombotic therapies.

Nan Jiang: Investigating the Functional Role of Renal Stromal Factor CXCL12 in Murine Kidney Development

Background: The kidney performs vital functions by excreting metabolic waste and regulating homeostatic functions via nephrons connected to a collecting system through which urine is transmitted to the bladder. Kidney formation occurs in utero via reciprocal interactions between stromal, nephrogenic and ureteric cell lineages and is complete before birth. Malformations of the kidney and urinary tract constitute a spectrum of diverse disorders termed CAKUT which are the major cause of childhood chronic kidney disease. The functional impact of CAKUT is determined, in large part, by the number and integrity of nephrons formed during embryogenesis. Despite the critical contribution of nephron formation to health and disease, mechanisms that control nephron number are largely undefined.

Hedgehog (Hh) signaling, actuated by secreted Hedgehog family proteins which bind to the cell surface and signal to control GLI family transcription factors, is required during formation of the kidney and nonrenal tissues. Our lab has demonstrated that constitutive activation of the Hedgehog signaling pathway in the embryonic renal stroma causes renal hypoplasia, defined as decreased nephron number with normal tissue patterning. Single cell RNA sequencing of mutant kidneys demonstrated upregulation of stromal CXCL12 expression, a homeostatic chemokine responsible of lymphopoiesis and tumorigenesis. Previous studies suggested that CXCL12 expression in the developing renal endothelium is crucial to vasculature development. However, the developmental role of CXCL12 in the stroma remains poorly understood.

Method: To generate stromal-specific Cxcl12 knockout mice, I will use the Foxd1Cre-GFP; Cxcl12^loxP/loxPmouse strain, where FOXD1 is a marker of renal stromal cells. Kidneys are characterized by immunostaining at various embryonic stages. Gene expression alterations are assessed via bulk RNA sequencing at E15.5.

Result: My data shows that mice with stromal Cxcl12-deficiency exhibited renal hypoplasia, characterized by a 25% reduction in kidney to body weight ratio (n=15 kidneys/genotype) and a 28% reduction in mature nephron count (n=4) as demonstrated by PAS staining for P4 kidney sections. Further immunostaining for NCAM+/LIM1+ nephron intermediate structures at E18.5 showed a 26% and 24% reduction in comma and S-shaped bodies, suggesting impaired nephron precursor differentiation. In addition, ureteric bud tip count is reduced by 34% at E14.5 in mutant kidneys, which could account for the reduction in nephron number and overall kidney size seen later in development. On the other hand, Immunostaining for stromal markers PBX1 and RALDH2 showed that kidney organization and morphology was normal in stromal Cxcl12-deficient kidneys, suggesting that stromal CXCL12 signaling does not play a role in renal dysplasia pathogenesis.

Conclusion: Overall, my project shows that stromal CXCL12 signaling is important for nephron formation and ureteric branching. To date, the role of the renal stroma in regulating kidney development remains poorly understood. This study will help unravel the molecular mechanism through which the stroma regulates renal organogenesis. This project will generate a disease model of CAKUT and shed light on its molecular pathogenesis. In addition, this project will provide a potential gene target for genetic screening and gene therapy in the prevention and treatment of CAKUT patients.

Wai Lam Mok: Eliminate Colorectal Cancer Cells in the Drug-Tolerant Persister State: Combined Inhibition of Autophagy and Induction of Apoptosis

Colorectal cancer (CRC) is the second most fatal cancer worldwide. Emerging evidence has demonstrated that cancer cells can enter a reversible drug-tolerant persister (DTP) state to escape death from chemotherapy, and this cannot be explained on the basis of genetic mutations. After chemotherapy ceases, DTPs regrow though they remain sensitive to chemotherapy. Thus, targeting DTPs prior to the development of irreversible drug resistance elicited by long-term chemotherapy represents a potential therapeutic opportunity. We previously showed that key macroautophagy/autophagy genes were upregulated in DTPs. However, preliminary data suggested autophagy inhibition alone was not efficient in preventing cancer regrowth as xenograft tumors started to regrow within a month after chemotherapy stopped. Additionally, pharmacogenomic profiling identified Navitoclax (ABT-263), an inhibitor of the anti-apoptotic proteins from the Bcl-2 family, as one of the top potential drug targets for DTPs in CRC. Here, we hypothesized that the survival of CRC cells in the DTP state is dependent on the combined activation of autophagy mediated by Unc-51-like kinases 1 (ULK1), which is the autophagy initiator, and suppression of apoptosis through the enhanced activity of anti-apoptotic Bcl-2 proteins. Using CRC patient-derived xenograft models, we generated a genetic knockout (KO) of ULK1 and pharmacologically inhibited the anti-apoptotic Bcl-2 proteins using ABT-263. Using Annexin V/Sytox staining, a flow cytometry-based apoptosis assay showed that there were significantly more dead cells in ULK1 KO treated with the chemotherapy irinotecan (CPT-11) in combination with ABT-263. We also found that the combined inhibition of autophagy and anti-apoptotic pathways in DTPs resulted in no cancer regrowth in vitro, and 7 out of 10 tumors did not grow back in vivo after chemotherapy was stopped for months. However, regrowth was observed when one of the two pathways was inhibited alone. Interestingly, suppression of anti-apoptotic activity did not elicit the upregulation of autophagy, suggesting autophagy does not compensate for the loss of anti-apoptotic activity. Taken together, our study reveals a novel therapeutic strategy for targeting DTPs and provides insight into preventing cancer recurrence.

Contact

No need to register.

Contact lmp.grad@utoronto.ca with any questions