Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth.

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression

Upregulation of GALNT7 in prostate cancer modifies O-glycosylation and promotes tumour growth

Prostate cancer is the most common cancer in men and it is estimated that over 350,000 men worldwide die of prostate cancer every year. There remains an unmet clinical need to improve how clinically significant prostate cancer is diagnosed and develop new treatments for advanced disease. Aberrant glycosylation is a hallmark of cancer implicated in tumour growth, metastasis, and immune evasion. One of the key drivers of aberrant glycosylation is the dysregulated expression of glycosylation enzymes within the cancer cell. Here, we demonstrate using multiple independent clinical cohorts that the glycosyltransferase enzyme GALNT7 is upregulated in prostate cancer tissue. We show GALNT7 can identify men with prostate cancer, using urine and blood samples, with improved diagnostic accuracy than serum PSA alone. We also show that GALNT7 levels remain high in progression to castrate-resistant disease, and using in vitro and in vivo models, reveal that GALNT7 promotes prostate tumour growth. Mechanistically, GALNT7 can modify O-glycosylation in prostate cancer cells and correlates with cell cycle and immune signalling pathways. Our study provides a new biomarker to aid the diagnosis of clinically significant disease and cements GALNT7-mediated O-glycosylation as an important driver of prostate cancer progression

Investigating the Cellular Effects of Mitochondrial DNA Damage Using Targeted Chemical Probes

Mitochondria are compartments within eukaryotic cells that produce energy to power cellular metabolism. Uniquely amongst mammalian organelles, mitochondria contain a small amount of their own genetic material (mtDNA), the proper maintenance and expression of which is essential for mitochondrial function. Mitochondrial DNA damage has been linked to several pathophysiological processes, but the cellular effects of mtDNA damage remain poorly understood. In order to better understand mtDNA damage, our group has pioneered the development of selective mitochondria-targeted DNA damaging agents using positively charged, hydrophobic delivery vectors known as mitochondria-penetrating peptides (MPPs). In my first study, an analogue of the commonly used anticancer drug cisplatin was conjugated to an MPP, yielding a mitochondria-targeted platinum DNA-damaging agent (mtPt) that induced specific damage to mtDNA. Using mtPt, I showed that selective mtDNA platinum damage induced apoptotic cell death associated with reactive oxygen species production, demonstrating that mtDNA damage is sufficient to mediate the cytotoxic activity of a platinum drug. In another work, I aimed to use mitochondria-targeted DNA damaging agents in a screen to characterize novel factors in mtDNA replication and repair. By knocking down expression of known DNA repair genes, and monitoring synergistic effects of gene knockdown on the activity of an mtDNA-oxidizing agent, I identified a series of genes with novel roles in mtDNA repair and replication. Included amongst these hits was DNA Polymerase θ (Polθ), a novel mtDNA polymerase. In a further study, I found that Polθ plays an essential role in facilitating mtDNA replication under conditions of oxidative stress. Finally, I conducted a genome-wide CRISPR screen to comprehensively identify genes whose knockout sensitizes cells to mitochondria-localized DNA damage. The hits of this screen were heavily enriched for genes involved in mitochondrial translation, implying the importance of mitochondrial protein synthesis in mediating an adaptive response to mtDNA damage. These studies demonstrate the application of mitochondria-targeted DNA damaging agents to the study of a wide range of significant areas in mitochondrial biology. Going forward, my thesis work shows mtDNA-damaging chemical probes to be essential tools for achieving better understanding of mtDNA damage in health and disease.Ph.D.2018-12-19 00:00:0

Lysosome Targeting Chimeras (LYTACs) for the Degradation of Secreted and Membrane Proteins

Targeted protein degradation is a powerful strategy to address the canonically undruggable proteome. However, current technologies are limited to targets with cytosolically-accessible and ligandable domains. Here, we designed and synthesized conjugates capable of binding both a cell surface lysosome targeting receptor and the extracellular domain of a target protein. These lysosome targeting chimeras (LYTACs) consist of an antibody fused to agonist glycopeptide ligands for the cation-independent mannose-6-phosphate receptor (CI-M6PR). LYTACs enabled a CRISPRi knockdown screen revealing the biochemical pathway for CI-M6PR-mediated cargo internalization. We demonstrated that LYTACs mediate efficient degradation of Apolipoprotein-E4, epidermal growth factor receptor (EGFR), CD71, and programmed death-ligand 1 (PD-L1). LYTACs represent a modular strategy for directing secreted and membrane proteins for degradation in the context of both basic research and therapy. </p

Targeting mitochondrial DNA with a platinum-based anticancer agent.

An analog of the anticancer drug cisplatin (mtPt) was&nbsp;delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt&nbsp;induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to&nbsp;mediate the activity of a platinum-based chemotherapeutic. This study demonstrates the specific delivery of a platinum drug to mitochondria and investigates the effects of directing this agent outside the nucleus

DNA Polymerase θ Increases Mutational Rates in Mitochondrial DNA

Replication and maintenance of mitochondrial DNA (mtDNA) is essential for cellular function, yet few DNA polymerases are known to function in mitochondria. Here, we conclusively demonstrate that DNA polymerase θ (Polθ) localizes to mitochondria and explore whether this protein is overexpressed in patient-derived cells and tumors. Polθ appears to play an important role in facilitating mtDNA replication under conditions of oxidative stress, and this error-prone polymerase was found to introduce mutations into mtDNA. In patient-derived cells bearing a pathogenic mtDNA mutation, Polθ expression levels were increased, indicating that the oxidative conditions in these cells promote higher expression levels for Polθ. Heightened Polθ expression levels were also associated with elevated mtDNA mutation rates in a selected panel of human tumor tissues, suggesting that this protein can influence mutational frequencies in tumors. The results reported indicate that the mitochondrial function of Polθ may have relevance to human disease

Targeting Mitochondrial DNA with a Platinum-Based Anticancer Agent

An analog of the anticancer drug cisplatin (mtPt) was delivered to mitochondria of human cells using a peptide specifically targeting this organelle. mtPt induces apoptosis without damaging nuclear DNA, indicating that mtDNA damage is sufficient to mediate the activity of a platinum-based chemotherapeutic. This study demonstrates the specific delivery of a platinum drug to mitochondria and investigates the effects of directing this agent outside the nucleus.National Cancer Institute (U.S.) (Grant CA034992)David H. Koch Graduate Fellowshi