Nam Yong Lee

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• Choi, A. S., Jenkins-Lane, L. M., Barton, W., Kumari, A., Lancaster, C., Raulerson, C., Ji, H., Altomare, D., Starr, M. D., Whitaker, R., Phaeton, R., Arend, R., Shtutman, M., Nixon, A. B., Hempel, N., Lee, N. Y., & Mythreye, K. (2024). Glycosaminoglycan modifications of betaglycan regulate ectodomain shedding to fine-tune TGF-β signaling responses in ovarian cancer. Cell communication and signaling : CCS, 22(1), 128.
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  In pathologies including cancer, aberrant Transforming Growth Factor-β (TGF-β) signaling exerts profound tumor intrinsic and extrinsic consequences. Intense clinical endeavors are underway to target this pathway. Central to the success of these interventions is pinpointing factors that decisively modulate the TGF-β responses. Betaglycan/type III TGF-β receptor (TβRIII), is an established co-receptor for the TGF-β superfamily known to bind directly to TGF-βs 1-3 and inhibin A/B. Betaglycan can be membrane-bound and also undergo ectodomain cleavage to produce soluble-betaglycan that can sequester its ligands. Its extracellular domain undergoes heparan sulfate and chondroitin sulfate glycosaminoglycan modifications, transforming betaglycan into a proteoglycan. We report the unexpected discovery that the heparan sulfate glycosaminoglycan chains on betaglycan are critical for the ectodomain shedding. In the absence of such glycosaminoglycan chains betaglycan is not shed, a feature indispensable for the ability of betaglycan to suppress TGF-β signaling and the cells' responses to exogenous TGF-β ligands. Using unbiased transcriptomics, we identified TIMP3 as a key inhibitor of betaglycan shedding thereby influencing TGF-β signaling. Our results bear significant clinical relevance as modified betaglycan is present in the ascites of patients with ovarian cancer and can serve as a marker for predicting patient outcomes and TGF-β signaling responses. These studies are the first to demonstrate a unique reliance on the glycosaminoglycan chains of betaglycan for shedding and influence on TGF-β signaling responses. Dysregulated shedding of TGF-β receptors plays a vital role in determining the response and availability of TGF-βs', which is crucial for prognostic predictions and understanding of TGF-β signaling dynamics.
• Flores, P. C., Ahmed, T., Podgorski, J., Ortiz, H. R., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2024). Phosphoproteomic profiling identifies DNMT1 as a key substrate of beta IV spectrin-dependent ERK/MAPK signaling in suppressing angiogenesis. Biochemical and biophysical research communications, 711, 149916.
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  βIV-spectrin is a membrane-associated cytoskeletal protein that maintains the structural stability of cell membranes and integral proteins such as ion channels and transporters. Its biological functions are best characterized in the brain and heart, although recently we discovered a fundamental new role in the vascular system. Using cellular and genetic mouse models, we reported that βIV-spectrin acts as a critical regulator of developmental and tumor-associated angiogenesis. βIV-spectrin was shown to selectively express in proliferating endothelial cells (EC) and suppress VEGF/VEGFR2 signaling by enhancing receptor internalization and degradation. Here we examined how these events impact the downstream kinase signaling cascades and target substrates. Based on quantitative phosphoproteomics, we found that βIV-spectrin significantly affects the phosphorylation of epigenetic regulatory enzymes in the nucleus, among which DNA methyltransferase 1 (DNMT1) was determined as a top substrate. Biochemical and immunofluorescence results showed that βIV-spectrin inhibits DNMT1 function by activating ERK/MAPK, which in turn phosphorylates DNMT1 at S717 to impede its nuclear localization. Given that DNMT1 controls the DNA methylation patterns genome-wide, and is crucial for vascular development, our findings suggest that epigenetic regulation is a key mechanism by which βIV-spectrin suppresses angiogenesis.
• Javed, Z., Shin, D. H., Pan, W., White, S. R., Elhaw, A. T., Kim, Y. S., Kamlapurkar, S., Cheng, Y. Y., Benson, J. C., Abdelnaby, A. E., Phaëton, R., Wang, H. G., Yang, S., Sullivan, M. L., St Croix, C. M., Watkins, S. C., Mullett, S. J., Gelhaus, S. L., Lee, N., , Coffman, L. G., et al. (2024). Drp1 splice variants regulate ovarian cancer mitochondrial dynamics and tumor progression. EMBO reports, 25(10), 4281-4310.
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  Aberrant mitochondrial fission/fusion dynamics are frequently associated with pathologies, including cancer. We show that alternative splice variants of the fission protein Drp1 (DNM1L) contribute to the complexity of mitochondrial fission/fusion regulation in tumor cells. High tumor expression of the Drp1 alternative splice variant lacking exon 16 relative to other transcripts is associated with poor outcome in ovarian cancer patients. Lack of exon 16 results in Drp1 localization to microtubules and decreased association with mitochondrial fission sites, culminating in fused mitochondrial networks, enhanced respiration, changes in metabolism, and enhanced pro-tumorigenic phenotypes in vitro and in vivo. These effects are inhibited by siRNAs designed to specifically target the endogenously expressed transcript lacking exon 16. Moreover, lack of exon 16 abrogates mitochondrial fission in response to pro-apoptotic stimuli and leads to decreased sensitivity to chemotherapeutics. These data emphasize the pathophysiological importance of Drp1 alternative splicing, highlight the divergent functions and consequences of changing the relative expression of Drp1 splice variants in tumor cells, and strongly warrant consideration of alternative splicing in future studies focused on Drp1.
• Kwak, E. A., Ahmed, T., Flores, P. C., Ortiz, H. R., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2023). Beta IV spectrin inhibits the metastatic growth of melanoma by suppressing VEGFR2-driven tumor angiogenesis. Cancer medicine, 12(18), 18981-18987.
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  Tumor-associated angiogenesis mediates the growth and metastasis of most solid cancers. Targeted therapies of the VEGF pathways can effectively block these processes but often fail to provide lasting benefits due to acquired resistance and complications.
• Ortiz, H. R., Cruz Flores, P., Podgorski, J., Ramonett, A., Ahmed, T., Hempel, N., Charest, P. G., Ellis, N. A., Langlais, P. R., Montfort, W. R., Mythreye, K., Kumar, S., & Lee, N. Y. (2024). Extracellular signals induce dynamic ER remodeling through αTAT1-dependent microtubule acetylation. Neoplasia (New York, N.Y.), 53, 101003.
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  Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to various growth factors and cytokines including TGF-β and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT1-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT1 pathway may be a key target in ER stress and dysfunction.
• Ahmed, T., Ramonett, A., Kwak, E. A., Kumar, S., Flores, P. C., Ortiz, H. R., Langlais, P. R., Hund, T. J., Mythreye, K., & Lee, N. Y. (2023). Endothelial tip/stalk cell selection requires BMP9-induced β-spectrin expression during sprouting angiogenesis. Molecular biology of the cell, 34(7), ar72.
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  β-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, β-spectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing β-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of β-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce β-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that β-spectrin acts upstream of these pathways as loss of β-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate β-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.
• Ahmed, T., Cruz-Flores, P., Pan, C. C., Ortiz, H. R., Lee, Y. S., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). EPDR1 is a noncanonical effector of insulin-mediated angiogenesis regulated by an endothelial-specific TGF-β receptor complex.. Journal of Biological Chemistry.
• Kwak, E. A., Pan, C. C., Ramonett, A., Kumar, S., Cruz-Flores, P., Ahmed, T., Ortiz, H. R., Lochhead, J. L., Ellis, N., Mouneimne, G., Lee, Y. S., Vanderah, T. W., Milnes, T. M., Mohler, P. J., Hund, T. J., Langlais, P. R., Mythreye, K., & Lee, N. Y. (2022). BIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature Communications.
• Lee, N. Y., Mythreye, K., Langlais, P. R., Hund, T. J., Mohler, P. J., Milnes, T. M., Vanderah, T. W., Lee, Y. S., Mouneimne, G., Ellis, N., Lochhead, J. L., Ortiz, H. R., Ahmed, T., Cruz-Flores, P., Kumar, S., Ramonett, A., Pan, C. C., & Kwak, E. A. (2021). BIV-spectrin as a stalk cell-intrinsic regulator of VEGF signaling. Nature Communications.
• Lee, N. Y., Mythreye, K., Langlais, P. R., Kashatus, D. F., Milnes, T. M., Vanderah, T. W., Lee, Y. S., Ortiz, H. R., Flores, P. C., Ahmed, T., Kwak, E. A., & Ramonett, A. (2022). Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport. Molecular biology of the cell, 33(1), ar4.
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  Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.
• Long, V. P., Bonilla, I. M., Baine, S., Glynn, P., Kumar, S., Schober, K., Mowrey, K., Weiss, R., Lee, N. Y., Mohler, P. J., Györke, S., Hund, T. J., Fedorov, V. V., & Carnes, C. A. (2020). Chronic heart failure increases negative chronotropic effects of adenosine in canine sinoatrial cells via A1R stimulation and GIRK-mediated I. Life sciences, 240, 117068.
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  Bradycardia contributes to tachy-brady arrhythmias or sinus arrest during heart failure (HF). Sinoatrial node (SAN) adenosine A1 receptors (ADO A1Rs) are upregulated in HF, and adenosine is known to exert negative chronotropic effects on the SAN. Here, we investigated the role of A1R signaling at physiologically relevant ADO concentrations on HF SAN pacemaker cells.
• Kim, Y. S., Gupta Vallur, P., Jones, V. M., Worley, B. L., Shimko, S., Shin, D. H., Crawford, L. C., Chen, C. W., Aird, K. M., Abraham, T., Shepherd, T. G., Warrick, J. I., Lee, N. Y., Phaeton, R., Mythreye, K., & Hempel, N. (2019). Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells. Oncogene.
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  Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.
• Mukhopadhyay, H., & Lee, N. Y. (2019). Multifaceted roles of TAK1 signaling in cancer. Oncogene.
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  Context-specific signaling is a prevalent theme in cancer biology wherein individual molecules and pathways can have multiple or even opposite effects depending on the tumor type. TAK1 represents a particularly notable example of such signaling diversity in cancer progression. Originally discovered as a TGF-β-activated kinase, over the years it has been shown to respond to numerous other stimuli to phosphorylate a wide range of downstream targets and elicit distinct cellular responses across cell and tissue types. Here we present a comprehensive review of TAK1 signaling and provide important therapeutic perspectives related to its function in different cancers.
• Parker, S. S., Krantz, J., Kwak, E. A., Barker, N. K., Deer, C. G., Lee, N. Y., Mouneimne, G., & Langlais, P. R. (2019). Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes. Molecular & cellular proteomics : MCP, 18(7), 1363-1381.
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  Insulin-stimulated glucose uptake is known to involve microtubules, although the function of microtubules and the microtubule-regulating proteins involved in insulin action are poorly understood. CLASP2, a plus-end tracking microtubule-associated protein (+TIP) that controls microtubule dynamics, was recently implicated as the first +TIP associated with insulin-regulated glucose uptake. Here, using protein-specific targeted quantitative phosphoproteomics within 3T3-L1 adipocytes, we discovered that insulin regulates phosphorylation of the CLASP2 network members G2L1, MARK2, CLIP2, AGAP3, and CKAP5 as well as EB1, revealing the existence of a previously unknown microtubule-associated protein system that responds to insulin. To further investigate, G2L1 interactome studies within 3T3-L1 adipocytes revealed that G2L1 coimmunoprecipitates CLASP2 and CLIP2 as well as the master integrators of +TIP assembly, the end binding (EB) proteins. Live-cell total internal reflection fluorescence microscopy in adipocytes revealed G2L1 and CLASP2 colocalize on microtubule plus-ends. We found that although insulin increases the number of CLASP2-containing plus-ends, insulin treatment simultaneously decreases CLASP2-containing plus-end velocity. In addition, we discovered that insulin stimulates redistribution of CLASP2 and G2L1 from exclusive plus-end tracking to "trailing" behind the growing tip of the microtubule. Insulin treatment increases α-tubulin Lysine 40 acetylation, a mechanism that was observed to be regulated by a counterbalance between GSK3 and mTOR, and led to microtubule stabilization. Our studies introduce insulin-stimulated microtubule stabilization and plus-end trailing of +TIPs as new modes of insulin action and reveal the likelihood that a network of microtubule-associated proteins synergize to coordinate insulin-regulated microtubule dynamics.
• Kwak, E. A., & Lee, N. Y. (2018). Synergetic roles of TGF-β signaling in tissue engineering. Cytokine, 115, 60-63.
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  Recent advances in tissue engineering highlight biomaterial designs with context-specific growth factors, cytokines and various small molecules to better mimic the natural extracellular matrix (ECM) microenvironments. These efforts have led to direct improvements in cell-cell and cell-ECM interactions while mitigating undesirable cellular and immunogenic responses. In this short review, we focus on the crucial roles and regulation of transforming growth factor β (TGF-β) signaling in biomaterial applications during tissue repair and regeneration.
• Lee, N. Y. (2018). GPx3 supports ovarian cancer progression by manipulating the extracellular redox environment. Redox Biology.
• Lee, N. Y. (2018). Mediator of kinase CDK8/CDK19 drives YAP1 dependent BMP4 induced EMT in cancer. Oncogene.
• Lee, N. Y. (2018). Regulation of Microtubule Acetylation and Intracellular Transport by TGF-beta Activated Kinase 1 (TAK1). Nature Communications.
• Lee, N. Y. (2018). Tumor-derived Inhibin is a new paracrine factor of angiogenesis. Cancer Research.
• Pan, C. C., Shah, N., Kumar, S., Wheeler, S. E., Cinti, J., Hoyt, D. G., Beattie, C. E., An, M., Mythreye, K., Rakotondraibe, L. H., & Lee, N. Y. (2017). Angiostatic actions of capsicodendrin through selective inhibition of VEGFR2-mediated AKT signaling and disregulated autophagy. Oncotarget, 8(8), 12675-12685.
• Varadaraj, A., Jenkins, L. M., Singh, P., Chanda, A., Snider, J., Lee, N. Y., Amsalem-Zafran, A. R., Ehrlich, M., Henis, Y. I., & Mythreye, K. (2017). TGF-β triggers rapid fibrillogenesis via a novel TβRII-dependent fibronectin-trafficking mechanism. Molecular biology of the cell, 28(9), 1195-1207.
• Wheeler, S. E., & Lee, N. Y. (2017). Emerging Roles of Transforming Growth Factor β Signaling in Diabetic Retinopathy. Journal of cellular physiology, 232(3), 486-489.