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Browsing by Author "Jerde, Travis"
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Item APE1/REF-1 redox signaling regulates HIF1A-mediated CA9 expression in hypoxic pancreatic cancer cells : combination treatment in patient-derived pancreatic tumor model(2017-12-14) Logsdon, Derek Paul; Kelly, Mark; Fishel, Melissa; Jerde, Travis; Vasko, Michael; Fehrenbacher, JillPancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock-down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex-vivo 3-dimensional tumor co-culture model. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.Item eIF3a Regulates De Novo Fatty Acid Synthesis as an Alternative Mechanism in Cisplatin Response in Non-Small Cell Lung Cancer Cells(2024-08) Gu, Boqing; Jerde, Travis; Lu, Tao; Safa, Ahmad R.; Zhang, Jian-Ting; Wek, Ronald C.eIF3a is known to modulate DNA damage repair and cancer chemotherapy resistance partially via translational regulation of Raptor and its downstream mTOR pathway activity. Fatty acid synthase (FASN) has recently been reported to exert negative feedback on the mTOR signaling pathway, and FASN overexpression is associated with reduced chemotherapy efficiency in multiple cancer types. Here, we show that eIF3a exerts additional regulation on mTOR signaling pathway and chemotherapy resistance in non-small cell lung cancer by inhibiting FASN-mediated de novo lipid synthesis. Through genetic and chemical manipulations, we demonstrate that eIF3a physically interacts with the 5’-UTR of FASN mRNA to prevent FASN protein synthesis. Furthermore, FASN downregulation by eIF3a results in accumulation of malonyl-CoA, a substrate for fatty acid synthesis, which in turn directly inhibits mTOR activity of mTORC1 complex, decreasing NER protein level and cellular sensitivity to cisplatin in an eIF3a-dependent manner in addition to eIF3a-regulated expression of Raptor subunit in mTORC1. Taken together, our findings reveal a direct translational control of FASN-mediated fatty acid metabolism, suggesting a multi-level eIF3a regulatory paradigm on NER protein synthesis and activity during cancer cell response to cisplatin treatment.Item Function of a Unique Dually Localized EF-Hand Domain Containing Protein, TgEFP1, During the Lytic Cycle of the Human Parasite Toxoplasma Gondii(2022-08) Dave, Noopur Kirti; Arrizabalaga, Gustavo; Absalon, Sabrina; Fehrenbacher, Jill; Gilk, Stacey; Jerde, Travis; Mastracci, TeresaThe pathogenesis associated with toxoplasmosis is attributed to repeated rounds of the parasite lytic cycle, which has been shown to be regulated by calcium fluxes. However, little is known about the calcium homeostatic mechanisms utilized by T. gondii. Recently, our lab has identified a novel protein-TgEFP1 (TGGT1_255660), which is predicted to bind Ca2+ through its two EF-hand domains. Interestingly, TgEFP1 showed a unique dual localization at the PLV/ELC and the PV of the parasite. Previous work showed that the PLV/ELC harbors other ion binding and conducting proteins that are important for parasite survival and propagation. However, the function of this compartment in the parasite is unknown. Therefore, I hypothesize that the PLV/ELC, through the function of TgEFP1, plays a key role in calcium homeostasis of T. gondii. To test this hypothesis, we sought to characterize the function of TgEFP1 during the parasite lytic cycle and determine TgEFP1 interacting proteins that also localize to the PLV/ELC. Partial permeabilization and ultrastructure expansion microscopy techniques confirmed the dual localization of TgEFP1 at the PLV/ELC and the PV. TgEFP1 knockout parasites exhibited several phenotypic defects including a faster lytic rate, shorter intracellular cycle, and were more sensitive to calcium ionophore treatment. Signal peptide deletion led to a mislocalization of TgEFP1 as cytosolic puncta, while mutations at key calcium coordinating residues lead to exclusive localization of TgEFP1 at the PV. Lastly, immunoprecipitation assays followed by LC-MS/MS identified a novel lectin-like protein- TgLectin (TGGT1_258950) as a direct interactor of TgEFP1-HA. Collectively, these findings support that through the function of TgEFP1, the PLV/ELC, plays a key role in calcium-dependent processes during the lytic cycle of the parasite.Item Genetic Approach to Discover ARMC4 as a Novel NF-κB Negative Regulator and Tumor Suppressor in Colorectal Cancer(2020-04) Martin, Matthew Peter; Lu, Tao; Safa, Ahmad; Corson, Tim; Jerde, Travis; Pollok, KarenThe nuclear factor κB (NF-κB) plays pivotal roles in inflammatory and immune responses and in cancer. Therefore, understanding its regulation holds great promise for disease therapy. Using validation-based insertional mutagenesis (VBIM), a powerful technique established by us, we discovered armadillo repeat containing protein 4 (ARMC4) as a novel negative regulator of NF-κB in colorectal cancer (CRC). ARMC4 is a rarely studied protein only known to date for its role in primary ciliary dyskinesia (PCD) and mouse spermatogenesis. Thus, my work reveals a completely new facet of ARMC4 function that has never been reported before. We showed that ARMC4 overexpression downregulated the expression of NF-κB-dependent genes, many of which are related to cancer. Additionally, compared to the vector control group, overexpression of ARMC4 in HEK293 cells or CRC HT29, DLD1, and HCT116 cells dramatically reduced NF-κB activity, cellular proliferation, anchorage-independent growth, and migratory ability in vitro, and unsurprisingly, significantly decreased xenograft tumor growth in vivo. In contrast, shARMC4 knockdown cells showed quite opposite effect. Furthermore, co-immunoprecipitation (Co-IP) experiment confirmed that ARMC4 may form a complex with the p65 subunit of NF-κB. Importantly, immunohistochemistry (IHC) data exhibited much lower ARMC4 expression level in CRC patient tumor tissues compared to normal tissues, indicating that ARMC4 may function as a tumor suppressor in CRC. To conclude, my important findings for the first time uncovered the negative regulatory function of ARMC4 in NF-κB signaling, and present ARMC4 as an innovative therapeutic target in CRC treatment.Item Inflammation by Toxoplasma gondii Infection Induces Prostatic Hyperplasia and Accompanying Urinary Dysfunction(2024-08) Stanczak, Emily F.; Arrizabalaga, Gustavo; Bauer, Margaret; Jerde, Travis; Nakshatri, HarikrishnaBenign prostatic hyperplasia is the non-cancerous enlargement of the prostate. This syndrome develops as men age and affects 50% of men by the age of 50 and 80% of men by the age of 80. BPH is associated with a pattern of symptoms and pathology in patients that can be painful, problematic, and lower the quality of life. BPH is a multifactorial disease which may develop due to lifestyle choices, genetics, metabolic disorder, and potentially infection of the organ. Interestingly, previous work from our research group showed that the common parasite T. gondii can infect and establish a chronic infection in the prostate of mice resulting in histological hyperplasia and glandular nodule formation, reminiscent of that observed in men with BPH. This leads to the hypothesis that T. gondii contributes to BPH in humans. In this work, we investigated whether there is a correlation between T. gondii infection and BPH using a cohort of age matched BPH diagnosed and non-BPH diagnosed control donors. My data show that men diagnosed with BPH are significantly more likely to have evidence of T. gondii infection than undiagnosed controls. Additionally, men with antibodies against T. gondii and BPH had significantly more severe pathology in several categories including and most notably in epithelial and glandular nodules seen only in a steroid hormone model of the disease. Moreover, I have determined that mice infected with parasites demonstrated abnormal urination pattern behavior indicating lower urinary tract dysfunction. Based on these results, we conclude that T. gondii can contribute to the development and severity of BPH and BPH pathology in humans. In addition, T. gondii can potentially be used as a model for BPH and BPH-like symptoms and pathology in mice and other model organisms.Item Interplay between collapsin response mediator protein 2 (CRMP2) phosphorylation and sumoylation modulates NaV1.7 trafficking(2015-07-06) Dustrude, Erik Thomas; Brustovetsky, Nickolay; Khanna, Rajesh; Cummins, Theodore R.; Jerde, Travis; Obukhov, AlexanderThe voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.Item Investigating the Mechanisms of Resistance to Dual PI3K/MTOR Inhibitor in PIK3CA Mutant Basal Like Bladder Cancer(2019-03) Elbanna, May F.M.; Pili, Roberto; Jerde, Travis; Zhang, Jian-Ting; Lu, Tao; Fishel, MelissaMuscle invasive bladder cancer (MIBC) carries a poor prognosis where the overall 5 year survival ranges from 48% to 66%. To date, targeted therapies, except for immune checkpoint inhibitors have not been shown to be effective in the management of this disease, where conventional chemotherapy (i.e. cisplatin) continues to be the standard of care. Therefore, the challenge lies in identifying key molecular events that can predict response to targeted therapies and thereby provide patients with maximal clinical benefit. Using two MIBC patient derived xenograft models (PDX) that carry alterations in PI3K signaling, one of the most dysregulated signaling pathways in bladder cancer, we studied determinants of response to PI3K targeted inhibition and mechanisms of resistance. We found that PIK3CA mutation status as well as tumor subtype (luminal-like or basal-like) play cooperative role in driving treatment response, where PIK3CA E542K mutation in basal-like tumors is associated with resistance to PI3K inhibition. Resistance is driven by feedback activation of alternative feedback signaling such as RAS-MAPK pathway. Based on the mechanistic changes induced upon resistance, we tested different drug combinations that can overcome resistance to PI3K targeted inhibition. Interestingly, we observed bromodomain inhibition by JQ1 to be the most effective strategy to re-sensitize resistant cells to PI3K targeted therapy. Overall, this project provides a predictive paradigm of response to PI3K targeted inhibition in PIK3CA mutant MIBC and sets the stage for future rational clinical trial design.Item Investigations into the function of Elp3 in Toxoplasma gondii(2017-05-04) Padgett, Leah Rausch; Arrizabalaga, Gustavo; Jerde, Travis; Mosley, Amber; Nass, Richard M.; Sullivan, William J., Jr.The parasite Toxoplasma gondii causes life-threatening infection in immunocompromised individuals. Our lab has determined that Toxoplasma Elongator protein-3 (TgElp3) is required for parasite viability. While catalytic domains are conserved, TgElp3 is the only component of the six-subunit Elongator complex present in Toxoplasma; moreover, TgElp3 localizes to the outer mitochondria membrane (OMM). These unusual features suggest that TgElp3 may have unique roles in parasite biology that could be useful in drug targeting. The goals of this thesis were to determine the function of TgElp3 and how the protein traffics to the OMM. In other species, Elp3 mediates lysine acetylation of histones and alphatubulin, and its radical S-adenosyl methionine (rSAM) domain is important for the formation of tRNA modifications, which enhance translation efficiency and fidelity. Given its location, histones would not be an expected substrate, and we further determined that tubulin acetylation in Toxoplasma is mediated by a different enzyme, TgATAT. We found that overexpression of TgElp3 at the parasite’s mitochondrion results in a significant replication defect, but overexpression of TgElp3 lacking the transmembrane domain (TMD) or with a mutant rSAM domain is tolerated. We identified one such modification, 5-methoxycarbonylmethyl-2thiouridine (mcm5S2U) that is likely mediated by TgElp3. These findings signify the importance of TgElp3’s rSAM domain for protein function, and confirms TgElp3 activity at the OMM is essential for Toxoplasma viability as previously reported. To determine how TgElp3 traffics to the OMM, we performed a bioinformatics survey that discovered over 50 additional “tail-anchored” proteins present in Toxoplasma. Mutational analyses found that targeting of these TA proteins to specific parasite organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.Item Lansoprazole and its Metabolites in the Treatment of TNBC and the Contribution of ABCG2 to CC-115 Resistance(2019-08) Beebe, Jennifer Diane; Zhang, Jian-Ting; Jerde, Travis; Safa, Ahmad; Xie, Jingwu; Fishel, MelissaTriple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer with a dismal prognosis. Targeted therapies for breast cancer with expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) are currently available; however, due to the lack of ER, PR, and HER2 in TNBC, targeted therapies are limited. While surgery and traditional chemotherapy remain the standard of care, development of a new treatment strategy for TNBC is needed to improve clinical outcomes. Fatty acid synthase (FASN) has been implicated as a metabolic oncogene and has given cancer cells a survival advantage by increasing NHEJ repair. Recently, it has been shown that FDA-approved proton pump inhibitors, used for the treatment of acid related digestive diseases, have antitumor effects. Here, I show that a metabolite of lansoprazole, 5-hydroxy lansoprazole sulfide, has increased potency over parent compound lansoprazole. 5-hydroxy lansoprazole sulfide inhibits FASN, leading to a decrease in PARP and NHEJ DNA repair activity in TNBC. Ultimately, this leads to an increase in DNA damage and cell death via apoptosis. These findings suggest that 5-hydroxy lansoprazole sulfide, as a metabolite of lansoprazole, may have better activity in suppressing TNBC cells and that 5-hydroxy lansoprazole sulfide may be developed as a therapeutic for TNBC treatment. Furthermore, due to the role of FASN in increasing NHEJ repair, we hypothesized that FASN played a role in resistance to CC-115, a dual mTOR/DNA-PK inhibitor currently in clinical trials, by increasing DNA-PK activity. However, it was found that ABCG2, an ATP-binding cassette transporter, and not FASN, has a role in CC-115 resistance. ABCG2 effluxes CC-115 from cancer cells, increasing resistance to treatment. Inhibition of ABCG2 by FTC or PZ39C8 led to accumulation of CC-115 within cells and sensitization to treatment. Therefore, ABCG2 status should be assessed to stratify patients into treatment groups, increasing the efficacy of CC-115 treatment.Item Optimization of Survivin Dimerization Inhibitors for the Treatment of Docetaxel-Resistant Prostate Cancer(2020-01) Peery, Robert Craig; Jerde, Travis; Zhang, Jian-Ting; Pili, Roberto; Safa, Ahmad; Sullivan, William J., Jr.Despite therapeutic advancements, prostate cancer remains the second most common cause of cancer-related mortality in men. Docetaxel is the first cytotoxic agent to show modest improvements in overall survival rate in patients with metastatic prostate cancer. Unfortunately, over half of these patients do not respond to treatment and ultimately all develop resistance. The mechanism mediating docetaxel resistance remains unknown. Survivin has a classical biological role in cancer, in fact survivin has been shown to be overexpressed in almost every solid tumor and is associated with drug resistance and clinically aggressive disease. In these studies I demonstrate that docetaxel resistant cells have overexpression of survivin compared to sensitive parental cells, knockdown of survivin decreases docetaxel resistance, and stable overexpression of survivin increases resistance to docetaxel. The data in these studies suggest that survivin is likely implicated in docetaxel resistance and treatment with a direct survivin inhibitor may sensitize resistant cells to docetaxel. To this end the evaluation and optimization of two different backbones of survivin inhibitors was performed. One such inhibitor identified is LQZ-7-3 which decreases survivin level via proteasome degradation, leads to apoptosis of cells, and showed efficacy in a prostate cancer xenograft model in vivo when given in an oral formulation. LQZ- 7-3 showed strong specificity to survivin versus other IAP family members at the protein level. Another inhibitor, LQZ-7F-1, demonstrated nanomolar inhibition of cancer cell growth and similar effects on survivin. Both compounds synergized with docetaxel in vitro warranting future in vivo efficacy studies as a combinatorial therapy. Overall, our findings indicate survivin is a significant contributor to docetaxel resistance in metastatic prostate cancer at the molecular level and survivin inhibitors may prove efficacious as a new therapy to sensitize cancer cells to chemotherapies.