IU Indianapolis ScholarWorks :: Browsing by Subject "NF-kB"

Browsing by Subject "NF-kB"

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Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells
(MDPI, 2022-12-15) Champion, Jesse D.; Dodd, Kayleigh M.; Lam, Hilaire C.; Alzahrani, Mohammad A. M.; Seifan, Sara; Rad, Ellie; Scourfield, David Oliver; Fishel, Melissa L.; Calver, Brian L.; Ager, Ann; Henske, Elizabeth P.; Davies, David Mark; Kelley, Mark R.; Tee, Andrew R.; Pediatrics, School of Medicine
Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.
FASN Negatively Regulates NF-kB/P65 Expression in Breast Cancer Cells by Disrupting Its Stability
(2020-02) Barlow, Lincoln James; Lu, Tao; Zhang, Jian-Ting; Fehrenbacher, Jill; Herbert, Brittney-Shea; Safa, Ahmad
The overexpression of the multi-domain enzyme fatty acid synthase (FASN) has long been associated with poor clinical prognosis and treatment outcome in various cancers. Previous research in the Zhang lab has determined a role for FASN in mediating increases in non-homologous end-joining (NHEJ) DNA double-strand break repair activity allowing for increased cancer cell survival, and this mechanism was found to involve inhibition of NF-kB/p65. The mechanism responsible for the regulation of NF-kB/p65 by FASN in cancer cells, however, remains unknown. To this end, I was able to determine that FASN negatively regulates both the expression and activity of NF-kB/p65 in breast cancer cells, and that this effect was likely mediated by the 16-carbon saturated fatty acid palmitate, the end product of FASN catalytic activity. Specifically, FASN was found to negatively regulate p65 expression by disrupting its protein stability as a result of an increase in poly-ubiquitination of p65 protein and subsequent proteasomal degradation. Further, I found that the phosphorylation site Thr254 of p65 is involved in the regulation of p65 protein stability by FASN, in that mutation of this residue resulted in a disruption in p65 stability. Finally, I was able to determine that FASN likely inhibits the ability of the peptidyl-prolyl cis/trans isomerase Pin1 to assist in maintaining p65 stability, in that both siRNA knockdown and pharmacological inhibition of Pin1 resulted in a reduction of p65 expression in FASN shRNA knockdown cells. The determination of this signaling mechanism serves to expand our understanding of the role of FASN in breast cancer cells and has the potential to assist in uncovering more effective ways to target the oncogenic FASN pathway to kill breast tumor cells and to overcome resistance to drug treatment.
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, Karen
The 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.
In vitro and In vivo evidence demonstrating chronic absence of Ref-1 Cysteine 65 impacts Ref-1 folding configuration, redox signaling, proliferation and metastasis in pancreatic cancer
(Elsevier, 2024) Mijit, M.; Kpenu, E.; Chowdhury, N. N.; Gampala, S.; Wireman, R.; Liu, S.; Babb, O.; Georgiadis, M. M.; Wan, J.; Fishel, M. L.; Kelley, M. R.; Pediatrics, School of Medicine
Ref-1/APE1 (Redox Effector/Apurinic Endonuclease 1) is a multifunctional enzyme that serves as a redox factor for several transcription factors (TFs), e.g., NF-kB, HIF-1α, which in an oxidized state fail to bind DNA. Conversion of these TFs to a reduced state serves to regulate various biological responses such as cell growth, inflammation, and cellular metabolism. The redox activity involves a thiol exchange reaction for which Cys65 (C65) serves as the nucleophile. Using CRISPR editing in human pancreatic ductal adenocarcinoma (PDAC) cells, we changed C65 to Ala (C65A) in Ref-1 to evaluate alteration of Ref-1 redox dynamics as well as chronic loss of Ref-1 redox activity on cell signaling pathways, specifically those regulated by NF-kB and HIF-1α. The redox activity of Ref-1 requires partial unfolding to expose C65, which is buried in the folded structure. Labeling of Ref-1 with polyethylene glycol-maleimide (PEGm) provides a readout of reduced Cys residues in Ref-1 and thereby an assessment of partial unfolding in Ref-1. In comparing Ref-1WT vs Ref-1C65A cell lines, we found an altered distribution of oxidized versus reduced states of Ref-1. Accordingly, activation of NF-kB and HIF-1α in Ref-1C65A lines was significantly lower compared to Ref-1WT lines. The bioinformatic data revealed significant downregulation of metabolic pathways including OXPHOS in Ref-1C65A expressing clones compared to Ref-1WT line. Ref-1C65A also demonstrated reduced cell proliferation and use of tricarboxylic acid (TCA) substrates compared to Ref-1WT lines. A subcutaneous as well as PDAC orthotopic in vivo model demonstrated a significant reduction in tumor size, weight, and growth in the Ref-1C65A lines compared to the Ref-1WT lines. Moreover, mice implanted with Ref-1C65A redox deficient cells demonstrate significantly reduced metastatic burden to liver and lung compared to mice implanted with Ref-1 redox proficient cells. These results from the current study provide direct evidence that the chronic absence of Cys65 in Ref-1 results in redox inactivity of the protein in human PDAC cells, and subsequent biological results confirm a critical involvement of Ref-1 redox signaling and tumorigenic phenotype.
Inhibition of PRMT5 by market drugs as a novel cancer therapeutic avenue
(Elsevier, 2023-01) Prabhu, Lakshmi; Martin, Matthew; Chen, Lan; Demir, Özlem; Jin, Jiamin; Huang, Xiumei; Motolani, Aishat; Sun, Mengyao; Jiang, Guanglong; Nakshatri, Harikrishna; Fishel, Melissa L.; Sun, Steven; Safa, Ahmad; Amaro, Rommie E.; Kelley, Mark R.; Liu, Yunlong; Zhang, Zhong-Yin; Lu, Tao; Radiation Oncology, School of Medicine
Market drugs, such as Food and Drug Administration (FDA) or European Medicines Agency (EMA)-approved drugs for specific indications provide opportunities for repurposing for newer therapeutics. This potentially saves resources invested in clinical trials that verify drug safety and tolerance in humans prior to alternative indication approval. Protein arginine methyltransferase 5 (PRMT5) overexpression has been linked to promoting the tumor phenotype in several cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal cancer (CRC), and breast cancer (BC), making PRMT5 an important target for cancer therapy. Previously, we showed that PRMT5-mediated methylation of the nuclear factor (NF)-κB, partially contributes to its constitutive activation observed in cancers. In this study, we utilized an AlphaLISA-based high-throughput screening method adapted in our lab, and identified one FDA-approved drug, Candesartan cilexetil (Can, used in hypertension treatment) and one EMA-approved drug, Cloperastine hydrochloride (Clo, used in cough treatment) that had significant PRMT5-inhibitory activity, and their anti-tumor properties were validated using cancer phenotypic assays . Furthermore, PRMT5 selective inhibition of methyltransferase activity was confirmed by reduction of both NF-κB methylation and its subsequent activation upon drug treatment. Using prediction, we identified critical residues on PRMT5 targeted by these drugs that may interfere with its enzymatic activity. Finally, Clo and Can treatment have exhibited marked reduction in tumor growth . Overall, we provide basis for pursuing repurposing Clo and Can as anti-PRMT5 cancer therapies. Our study offers potential safe and fast repurposing of previously unknown PRMT5 inhibitors into clinical practice.
Purification of SIMPL Antibody and Immunofluorescence of SIMPL Sub-Cellular Localization in Response to TNFα- and IL-1
(2011-03-10) Cogill, Steven B.; Harrington, Maureen A.; Goebl, Mark, 1958-; Sanghani, Sonal P.
SIMPL is a transcriptional co-activator that alters the activity of transcription factor, NF-κB. In response to pathogens, cytokines such as Interleukin-1 (IL-1) and Tumor Necrosis Factor (TNF) signal through the IL-1 and TNF-α receptors, respectively, which are found on various cell types. Activation of these receptors can result in the nuclear localization of NF-κB where it enables the transcription of several different genes key in the innate immune response. Endogenous co-localization of the SIMPL protein with NF-κB in response to these same cytokine signals has yet to be demonstrated. Polyclonal antibody generated against a truncated version of the SIMPL protein was purified from the sera obtained from immunized rabbits using affinity chromatography. The antibody was found to have a high specificity for both the native and denatured form of the protein as demonstrated by the lack of nonspecific bands observed in immunoprecipitations and Western blotting. The antibody was utilized in immunofluorescence experiments on mouse endothelial cells that were either unstimulated or were stimulated (IL-1 or TNF-α). In the absence of cytokine, SIMPL was localized in both the cytoplasm and the nucleus as opposed to NF-κB which was almost exclusively localized in the cytoplasm. In the presence of IL-1, the concentration of SIMPL in the nucleus was increased, and in the presence of TNF-α, the concentration of SIMPL in the nucleus was even greater. Results of this study identified future routes for SIMPL antibody isolation as well as to demonstrate that endogenous SIMPL protein nuclear localization may not be solely dependent upon TNF-α signaling.
Targeting Protein Arginine Methyltransferase 5 as a Novel Therapeutic Approach in Pancreatic & Colorectal Cancer
(2018-12) Prabhu, Lakshmi Milind; Lu, Tao; Safa, Ahmad; Pollok, Karen; Skaar, Todd; Zhang, Jian-Ting
Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both PDAC and CRC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of cellular immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. In a previous study, we identified a new mechanism by which NF-kB activity is regulated by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 not only activated NF-kB, but also significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both PDAC and CRC cells. Moreover, in order to examine the therapeutic potential of PRMT5 in these cancers, we adapted the state-of-the-art AlphaLISA technique into a high throughput screen (HTS) platform to screen for PRMT5 inhibitors. As a result, we successfully identified the small molecule PR5-LL-CM01 as our lead hit. Further validation experiments confirmed that PR5-LL-CM01 is a potent and specific PRMT5 inhibitor that exhibits significant anti-tumor efficacy in both in vitro and in vivo models of PDAC and CRC. Additionally, in a second screen, we discovered two natural compounds, P1608K04 and P1618J22, that can also function as the PRMT5 inhibitors. These findings further highlight the robustness of the PRMT5- specific AlphaLISA HTS technique. To conclude, we describe here for the first time a novel role of PRMT5 as a tumor-promoting factor in PDAC and CRC through NF-kB activation. By successfully developing and applying an innovative AlphaLISA HTS technique, we discovered PR5-LL-CM01, P1608K04, and P1618J22 as novel PRMT5 inhibitors, with PR5-LL-CM01 showing the strongest potency in both PDAC and CRC models. Therefore, we demonstrated that PRMT5 is a promising therapeutic target in PDAC and CRC, and the novel PRMT5 inhibitor PR5-LL-CM01 could serve as a promising basis for new drug development in PDAC and CRC.
Validation-based insertional mutagenesis (VBIM) technology identifies adenomatous polypossis coli (APC) like protein (ALP) as a novel negative regulator of NF-κB
(2015-01) Mundade, Rasika S.; Lu, Tao; Sullivan, William J., Jr.; Safa, Ahmad R.
Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. The nuclear factor κB (NF-κB) is an important family of transcription factors whose aberrant activation has been found in many types of cancer, including CRC. Therefore, understanding the regulation of NF-κB is of ultimate importance for cancer therapy. Using a novel validation-based insertional mutagenesis (VBIM) strategy, our lab has identified the novel adenomatous polyposis coli (APC) like protein (ALP) gene as a negative regulator of NF-κB. Preliminary studies from our lab demonstrated that overexpression of ALP led to decreased NF-κB activity by κB reporter assay and electrophoresis mobility gel shift assay (EMSA). The current project aims to further evaluate the role of ALP in the regulation of NF-κB signaling in CRC cells. We found that overexpression of ALP in human CRC HT29 cells greatly reduced both the number and the size of colonies that were formed in a soft agar assay. ALP overexpression also decreased the cell growth rate and cell migration ability, while shRNA mediated knockdown of ALP showed opposite effects, confirming that ALP is a tumor suppressor in CRC HT29 cells. Overexpression of ALP led to decreased NF-κB activity by κB reporter assay and condition media assay in CRC HT29 cells. Furthermore, immunohistochemical analysis with human colon vii tissues revealed that there is a gradual loss of ALP protein with tumor progression. We also found that ALP predominantly localizes in the cytoplasm, and binds to the p65 subunit of NF-κB, and might be functioning downstream of IκB kinase (IKK). In summary, in this study, we provide evidence regarding the tumor suppressor role of ALP in CRC by functioning as novel negative regulator of NF-κB. This discovery could lead to the establishment of ALP as a potential biomarker and therapeutic target in CRC.

Browsing by Subject "NF-kB"

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