Browsing by Author "Corson, Timothy"

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    Dissecting the Role of Novel O-GlcNAcylation of NF-κB in Pancreatic Cancer
    (2024-06) Motolani, Aishat Abiola; Lu, Tao; Safa, Ahmad; Dong, Charlie; Pollok, Karen; Corson, Timothy
    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, with a mere 5-year survival of ~10%. This highlights the urgent need for innovative treatment options for PDAC patients. The nuclear factor κB (NF-κB) is a crucial transcription factor that is constitutively activated in PDAC. It mediates the transcription of oncogenic and inflammatory genes that facilitate multiple PDAC phenotypes. Thus, a better understanding of the mechanistic underpinnings of NF-κB activation holds great promise for PDAC diagnosis and effective therapeutics. Here, we report a novel finding that the p65 subunit of NF-κB is O-GlcNAcylated at serine 550 and 551 upon NF-κB activation. Importantly, the overexpression of either serine-to-alanine (S-A) single mutant (S550A or S551A) or double mutant (S550A/S551A) of p65 in PDAC cells impaired NF-κB nuclear translocation, p65 phosphorylation, and transcriptional activity, independent of IκBα degradation. Moreover, the p65 mutants downregulate a category of NF-κB-target genes, which play a role in perpetuating major cancer hallmarks. We further show that overexpression of the p65 mutants inhibited PDAC cellular proliferation, migration, and anchorage-independent growth compared to WT-p65. We also show that inhibition of NF-κB O-GlcNAcylation may mitigate gemcitabine resistance and enhance its efficacy in PDAC cells. Collectively, our study uncovers a novel aspect of NF-κB regulation, which could aid future therapeutic development by targeting O-GlcNAc transferase (OGT) in pancreatic cancer.
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    Human Stem Cell Differentiated Retinal Ganglion Cells for Developing Glaucoma Neuroprotection and Cell Replacement Strategies
    (2024-07) Anbarasu, Kavitha; Das, Arupratan; Corson, Timothy; Meyer, Jason; Graham, Brett; Janga, Sarath
    Progressive loss of retinal ganglion cells (RGCs) leads to glaucoma. Early diagnosis offers an opportunity to protect existing RGCs. In advanced glaucoma, most RGCs are lost causing blindness and cell replacement therapy the only option. We used a human stem cell-based RGC differentiation model to develop neuroprotection by restoring mitochondrial homeostasis and enhancing RGC differentiation efficiency to increase the success of cell replacement therapy. Unmyelinated axons in RGCs require high levels of ATP, making disrupted mitochondria a risk factor in glaucoma. Our goal was to restore mitochondrial homeostasis through mitophagy (mitochondrial autophagy) and mitobiogenesis (mitochondrial biogenesis). Mutations in the mitophagy protein Optineurin (OPTNE50K) are found in patients with normal tension glaucoma and hence, we also used RGCs with the E50K mutation. We discovered that hRGCE50Ks suffer from mitobiogenesis issues, Parkin/Pink mediated mitophagy defects, and have OPTNE50K-Tank binding kinase-1 (TBK1) aggregates. hRGCE50Ks have lower mitochondrial mass and a higher mitochondrial load. We inhibited TBK1 to induce mitochondrial biogenesis and dissolve OPTNE50K-TBK1 aggregates. Our results show TBK1 inhibition triggered mitobiogenesis, dissolved aggregates, decreased mitochondrial ATP production load, and increased spare respiratory capacity, leading to neuroprotection. With complete RGC loss, enhancing differentiation to progenitor cells with lower cell division capacity can improve the success of cell replacement therapy and reduce teratoma formation and poor tissue integration. We observed that stem cells use proteasomes for mitochondrial degradation, while hRGCs use the lysosomal mitophagy pathway. Our results indicate that proteasomal activity declines during differentiation to hRGCs. Inhibition of proteasomal activity during early differentiation resulted in higher and faster RGC differentiation, with similar effects seen in motor neuron differentiation. We did not observe metabolic reprogramming in differentiating cells upon proteasomal activity inhibition but saw changes in cell cycle distribution, specifically an increase in the number of cells in the G1 phase. Proteomics analysis post-inhibitory treatment showed elevated neuronal differentiation proteins. Our results can be translated to minimize injection cell numbers and other risks of cell replacement therapy. In summary, my research identifies novel mechanisms for restoring mitochondrial homeostasis for neuroprotection in glaucomatous RGCs and develops an enhanced differentiation strategy to aid the success of cell replacement therapy.
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    Identification and Characterization of TONSL as an Immortalizing Oncogene
    (2024-11) Khatpe, Aditi Sanjay; Nakshatri, Harikrishna; Nephew, Kenneth; Motea, Edward; Cornett, Evan; Corson, Timothy
    The global issue of exponentially increasing breast cancer cases necessitates investigating the early genomic aberrations leading to tumorigenesis. To address this, we employed our unique biobank of healthy breast tissue to generate an isogeneic cell line model system, comprising primary breast epithelial cells and their immortalized counterparts. By comparing the genetic alterations between these cell types, we discovered that TONSL upregulation is one of the early events during breast tumorigenesis. TONSL is a Tonsoku-like DNA repair protein located on chromosome 8q24.3. Our findings reveal that TONSL is an immortalizing oncogene, capable of transforming primary breast epithelial cells in conjunction with defined oncogenes, resulting in Estrogen Receptor-positive breast adenocarcinomas. Furthermore, we observed that TONSL-amplified breast cancer cells are dependent on TONSL for tumor growth, and these TONSLHigh cells and tumors exhibit an upregulated homologous recombination DNA repair pathway, which may contribute to chemotherapy resistance. It is noteworthy that higher levels of TONSL protein in primary breast cancers, particularly in ER+ breast cancers, are associated with poor outcomes. Approximately 20% of breast cancers display recurring genomic amplification involving chromosome 8q24.3, with TONSL amplification potentially being the initial hit leading to tumorigenesis. In an attempt to target these TONSL/chr. 8q24.3 amplified breast tumors, we observed that breast cancer cells with this amplification are sensitive to CBL0137 – a TONSL-FACT complex inhibitor, both in vitro and in vivo. TONSL interacts with multiple proteins and functions in multiple cellular processes. To study the TONSL specific interactome, we performed immunoprecipitation with a TONSL antibody using protein lysates from TONSL-immortalized primary breast epithelial cells, followed by mass spectrometry analysis of the immunoprecipitates. Our results identified several proteins selectively enriched with the TONSL antibody, with the most significant being ETS variant transcription factor 6 (ETV6). ETV6 is known to play a role as a transcriptional repressor during embryonic development and hematopoiesis. Further studies on TONSL/chromosome 8q24.3 amplification will contribute to our understanding of breast tumor initiation, progression, and metastasis processes, as well as facilitate the development of novel therapeutic agents targeting the TONSL interactome.