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Browsing by Author "Roy, Gitanjali"
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Item IB-DNQ and Rucaparib dual treatment alters cell cycle regulation and DNA repair in triple negative breast cancer cells(bioRxiv, 2024-05-18) Runnebohm, Avery M.; Wijeratne, H. R. Sagara; Peck Justice, Sarah A.; Wijeratne, Aruna B.; Roy, Gitanjali; Singh, Naveen; Hergenrother, Paul; Boothman, David A.; Motea, Edward A.; Mosley, Amber L.; Biochemistry and Molecular Biology, School of MedicineBackground: Triple negative breast cancer (TNBC), characterized by the lack of three canonical receptors, is unresponsive to commonly used hormonal therapies. One potential TNBC-specific therapeutic target is NQO1, as it is highly expressed in many TNBC patients and lowly expressed in non-cancer tissues. DNA damage induced by NQO1 bioactivatable drugs in combination with Rucaparib-mediated inhibition of PARP1-dependent DNA repair synergistically induces cell death. Methods: To gain a better understanding of the mechanisms behind this synergistic effect, we used global proteomics, phosphoproteomics, and thermal proteome profiling to analyze changes in protein abundance, phosphorylation and protein thermal stability. Results: Very few protein abundance changes resulted from single or dual agent treatment; however, protein phosphorylation and thermal stability were impacted. Histone H2AX was among several proteins identified to have increased phosphorylation when cells were treated with the combination of IB-DNQ and Rucaparib, validating that the drugs induced persistent DNA damage. Thermal proteome profiling revealed destabilization of H2AX following combination treatment, potentially a result of the increase in phosphorylation. Kinase substrate enrichment analysis predicted altered activity for kinases involved in DNA repair and cell cycle following dual agent treatment. Further biophysical analysis of these two processes revealed alterations in SWI/SNF complex association and tubulin / p53 interactions. Conclusions: Our findings that the drugs target DNA repair and cell cycle regulation, canonical cancer treatment targets, in a way that is dependent on increased expression of a protein selectively found to be upregulated in cancers without impacting protein abundance illustrate that multi-omics methodologies are important to gain a deeper understanding of the mechanisms behind treatment induced cancer cell death.Item In-depth bioinformatics analysis of the phosphoproteome of triple negative breast cancer treated with a tumor selective NQO1 bioactivatable drug(2021-01) Roy, Gitanjali; Mosley, Amber L.; Georgiadis, Millie M.; Goebl, MarkItem SAT074 Induction Of Insulin Hypersecretion Uncovers Distinctions Between Adaptive And Maladaptive Endoplasmic Reticulum Stress Response In Beta Cells(The Endocrine Society, 2023-10-05) Roy, Gitanjali; Rodrigues dos Santos, Karina; Kwakye, Michael B.; Tan, Zhiyong; Johnson, Travis S.; Kalwat, Michael A.; Biostatistics and Health Data Science, School of MedicinePancreatic islet β-cells release insulin to maintain glucose homeostasis. β-cells must translate, package, and secrete large amounts of insulin. During this process the unfolded protein response of the endoplasmic reticulum (UPRER) is induced to maintain these functions. However, stimuli that force β-cell to secrete insulin at enhanced rates and for prolonged durations risk inducing the terminal UPRER and eventual apoptosis. In a chemical screen for insulin secretion modulators, we discovered SW016789 which caused hypersecretion of insulin and led to a transient induction of the UPRER, but not apoptosis. In contrast, SERCA2 ER Ca2+ pump inhibitor thapsigargin induces the terminal UPRER. We hypothesized that SW016789 can be used as a tool compound to discover genes involved in β-cell adaptation to hypersecretion-induced stress. We performed time course transcriptomics in MIN6 β-cells exposed to SW016789 (5 µM) or thapsigargin (100 nM) from 0-24 h. Unbiased analyses using a Dirichlet process Gaussian process (DPGP) method revealed clusters of genes temporally co-regulated and the genes within these clusters were distinct between SW016789 and thapsigargin treatments. In particular, after 6 h of SW016789-induced hypersecretion we found a highly induced cluster of genes (SW cluster 3) enriched in adaptive UPRER factors (e.g. Manf). Conversely, most of the thapsigargin-induced genes clustered at 24 h and were enriched for terminal UPRER factors (e.g. Txnip). Pathway analysis of SW cluster 3 indicated that genes involved in in regulation of mRNA methylation and ER-associated degradation were also induced by SW016789 sooner and with greater amplitude than by thapsigargin, suggesting distinct differences in the handling of protein translation and degradation. From the SW cluster 3 genes we selected proteins known to be ER-associated or secreted and generated stable transgenic or CRISPR knockout MIN6 β-cell lines for each. Our data suggest altered expression of these factors may impair glucose-stimulated insulin secretion responses and alter cell viability in presence or absence of ER stressors including cytokines, thapsigargin, and tunicamycin. In conclusion, we have successfully shown that pharmacological induction of insulin hypersecretion can induce a distinct transcriptional outcome from that of canonically-induced UPRER and that we can take advantage of this property to discover new β-cell regulatory pathways and targets. We envision this dataset as a resource for the secretory biology and islet biology communities.Item Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function(Oxford University Press, 2022) Rodrigues-dos-Santos, Karina; Roy, Gitanjali; Binns, Derk D.; Grzemska, Magdalena G.; Barella, Luiz F.; Armoo, Fiona; McCoy, Melissa K.; Huynh, Andy V.; Yang, Jonathan Z.; Posner, Bruce A.; Cobb, Melanie H.; Kalwat, Michael A.; Medicine, School of MedicinePancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.