Publications by authors named "Kye-Seong Kim"

71 Publications

The Role of Nkx3.1 in Cancers and Stemness.

Int J Stem Cells 2021 Feb 28. Epub 2021 Feb 28.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea.

The well-known androgen-regulated homeobox gene, , is located on the short arm of chromosome 8. It is the first known prostate epithelium-specific marker, and is a transcription factor involved in development of the testes and prostate. In addition to specifying the prostate epithelium and maintaining normal prostate secretory function, Nkx3.1 is an established marker for prostate cancer. Over the years, however, this gene has been implicated in various other cancers, and technological advances have allowed determination of its role in other cellular functions. Nkx3.1 has also been recently identified as a factor capable of replacing Oct4 in cellular reprogramming. This review highlights the role of this tumor suppressor and briefly describes its functions, ranging from prostate development to maintenance of stemness and cellular reprogramming.
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http://dx.doi.org/10.15283/ijsc20121DOI Listing
February 2021

Disease modeling and stem cell immunoengineering in regenerative medicine using CRISPR/Cas9 systems.

Comput Struct Biotechnol J 2020 19;18:3649-3665. Epub 2020 Nov 19.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea.

CRISPR/Cas systems are popular genome editing tools that belong to a class of programmable nucleases and have enabled tremendous progress in the field of regenerative medicine. We here outline the structural and molecular frameworks of the well-characterized type II CRISPR system and several computational tools intended to facilitate experimental designs. The use of CRISPR tools to generate disease models has advanced research into the molecular aspects of disease conditions, including unraveling the molecular basis of immune rejection. Advances in regenerative medicine have been hindered by major histocompatibility complex-human leukocyte antigen (HLA) genes, which pose a major barrier to cell- or tissue-based transplantation. Based on progress in CRISPR, including in recent clinical trials, we hypothesize that the generation of universal donor immune-engineered stem cells is now a realistic approach to tackling a multitude of disease conditions.
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http://dx.doi.org/10.1016/j.csbj.2020.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710510PMC
November 2020

E3 Ubiquitin Ligase APC/C Regulation of Phenylalanine Hydroxylase Stability and Function.

Int J Mol Sci 2020 Nov 28;21(23). Epub 2020 Nov 28.

Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C) is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.
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http://dx.doi.org/10.3390/ijms21239076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7729981PMC
November 2020

E3 Ubiquitin Ligase APC/C Negatively Regulates FAH Protein Stability by Promoting Its Polyubiquitination.

Int J Mol Sci 2020 Nov 18;21(22). Epub 2020 Nov 18.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.

Fumarylacetoacetate hydrolase (FAH) is the last enzyme in the degradation pathway of the amino acids tyrosine and phenylalanine in mammals that catalyzes the hydrolysis of 4-fumarylacetoacetate into acetoacetate and fumarate. Mutations of the gene are associated with hereditary tyrosinemia type I (HT1), resulting in reduced protein stability, misfolding, accelerated degradation and deficiency in functional proteins. Identifying E3 ligases, which are necessary for FAH protein stability and degradation, is essential. In this study, we demonstrated that the FAH protein level is elevated in liver cancer tissues compared to that in normal tissues. Further, we showed that the FAH protein undergoes 26S proteasomal degradation and its protein turnover is regulated by the anaphase-promoting complex/cyclosome-Cdh1 (APC/C) E3 ubiquitin ligase complex. APC/C acts as a negative stabilizer of FAH protein by promoting FAH polyubiquitination and decreases the half-life of FAH protein. Thus, we envision that Cdh1 might be a key factor in the maintenance of FAH protein level to regulate FAH-mediated physiological functions.
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http://dx.doi.org/10.3390/ijms21228719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7699203PMC
November 2020

Importance of Deubiquitination in Macrophage-Mediated Viral Response and Inflammation.

Int J Mol Sci 2020 Oct 29;21(21). Epub 2020 Oct 29.

Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 24341, Korea.

Ubiquitination and deubiquitination play a fundamental role in the signaling pathways associated with innate and adaptive immune responses. Macrophages are key sentinels for the host defense, triggering antiviral and inflammatory responses against various invading pathogens. Macrophages recognize the genetic material of these pathogens as pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) through the activation of its pattern recognition receptors (PRRs), initiating the cascade of immune signaling, which leads to the production of pro- and anti-inflammatory cytokines that initiates the appropriate immune response. Macrophage-mediated immune response is highly regulated and tightly controlled by the ubiquitin system since its abnormal activation or dysregulation may result in the severe pathogenesis of numerous inflammatory and autoimmune diseases. Deubiquitinating enzymes (DUBs) play a crucial role in reversing the ubiquitination and controlling the magnitude of the immune response. During infection, pathogens manipulate the host defense system by regulating DUBs to obtain nutrients and increase proliferation. Indeed, the regulation of DUBs by small molecule inhibitors has been proposed as an excellent way to control aberrant activation of immune signaling molecules. This review is focused on the complex role of DUBs in macrophage-mediated immune response, exploring the potential use of DUBs as therapeutic targets in autoimmune and inflammatory diseases by virtue of small molecule DUB inhibitors.
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http://dx.doi.org/10.3390/ijms21218090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662591PMC
October 2020

HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.

Biochim Biophys Acta Mol Cell Res 2020 12 27;1867(12):118835. Epub 2020 Aug 27.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea. Electronic address:

Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118835DOI Listing
December 2020

Protein Degradation and the Pathologic Basis of Phenylketonuria and Hereditary Tyrosinemia.

Int J Mol Sci 2020 Jul 15;21(14). Epub 2020 Jul 15.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.

A delicate intracellular balance among protein synthesis, folding, and degradation is essential to maintaining protein homeostasis or proteostasis, and it is challenged by genetic and environmental factors. Molecular chaperones and the ubiquitin proteasome system (UPS) play a vital role in proteostasis for normal cellular function. As part of protein quality control, molecular chaperones recognize misfolded proteins and assist in their refolding. Proteins that are beyond repair or refolding undergo degradation, which is largely mediated by the UPS. The importance of protein quality control is becoming ever clearer, but it can also be a disease-causing mechanism. Diseases such as phenylketonuria (PKU) and hereditary tyrosinemia-I (HT1) are caused due to mutations in and gene, resulting in reduced protein stability, misfolding, accelerated degradation, and deficiency in functional proteins. Misfolded or partially unfolded proteins do not necessarily lose their functional activity completely. Thus, partially functional proteins can be rescued from degradation by molecular chaperones and deubiquitinating enzymes (DUBs). Deubiquitination is an important mechanism of the UPS that can reverse the degradation of a substrate protein by covalently removing its attached ubiquitin molecule. In this review, we discuss the importance of molecular chaperones and DUBs in reducing the severity of PKU and HT1 by stabilizing and rescuing mutant proteins.
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http://dx.doi.org/10.3390/ijms21144996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404301PMC
July 2020

Advances in Deubiquitinating Enzyme Inhibition and Applications in Cancer Therapeutics.

Cancers (Basel) 2020 Jun 15;12(6). Epub 2020 Jun 15.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.

Since the discovery of the ubiquitin proteasome system (UPS), the roles of ubiquitinating and deubiquitinating enzymes (DUBs) have been widely elucidated. The ubiquitination of proteins regulates many aspects of cellular functions such as protein degradation and localization, and also modifies protein-protein interactions. DUBs cleave the attached ubiquitin moieties from substrates and thereby reverse the process of ubiquitination. The dysregulation of these two paramount pathways has been implicated in numerous diseases, including cancer. Attempts are being made to identify inhibitors of ubiquitin E3 ligases and DUBs that potentially have clinical implications in cancer, making them an important target in the pharmaceutical industry. Therefore, studies in medicine are currently focused on the pharmacological disruption of DUB activity as a rationale to specifically target cancer-causing protein aberrations. Here, we briefly discuss the pathophysiological and physiological roles of DUBs in key cancer-related pathways. We also discuss the clinical applications of promising DUB inhibitors that may contribute to the development of DUBs as key therapeutic targets in the future.
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http://dx.doi.org/10.3390/cancers12061579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352412PMC
June 2020

Genome-scale screening of deubiquitinase subfamily identifies USP3 as a stabilizer of Cdc25A regulating cell cycle in cancer.

Cell Death Differ 2020 Nov 15;27(11):3004-3020. Epub 2020 May 15.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea.

Conventional screening methods for deubiquitinating enzymes (DUBs) have important limitations. A loss-of-function study based on the knockout of DUB genes in mammalian cells can provide an excellent model for exploring DUB function. Here, we used CRISPR-Cas9 to perform genome-scale knockout of the entire set of genes encoding ubiquitin-specific proteases (USPs), a DUB subfamily, and then systematically screened for DUBs that stabilize the Cdc25A oncoprotein. USP3 was identified as a deubiquitinase of Cdc25A. USP3 depletion reduces the Cdc25A protein level, resulting in a significant delay in cell-cycle progression, and reduces the growth of cervical tumor xenografts in nude mice. Clinically, USP3 expression is positively correlated with Cdc25A protein expression and the poorest survival in breast cancer. We envision that our DUB knockout library kit will facilitate genome-scale screening of functional DUBs for target proteins of interest in a wide range of biomedical fields.
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http://dx.doi.org/10.1038/s41418-020-0557-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566649PMC
November 2020

The Role of Deubiquitinating Enzymes in Hematopoiesis and Hematological Malignancies.

Cancers (Basel) 2020 Apr 28;12(5). Epub 2020 Apr 28.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.

Hematopoietic stem cells (HSCs) are responsible for the production of blood cells throughout the human lifespan. Single HSCs can give rise to at least eight distinct blood-cell lineages. Together, hematopoiesis, erythropoiesis, and angiogenesis coordinate several biological processes, i.e., cellular interactions during development and proliferation, guided migration, lineage programming, and reprogramming by transcription factors. Any dysregulation of these processes can result in hematological disorders and/or malignancies. Several studies of the molecular mechanisms governing HSC maintenance have demonstrated that protein regulation by the ubiquitin proteasomal pathway is crucial for normal HSC function. Recent studies have shown that reversal of ubiquitination by deubiquitinating enzymes (DUBs) plays an equally important role in hematopoiesis; however, information regarding the biological function of DUBs is limited. In this review, we focus on recent discoveries about the physiological roles of DUBs in hematopoiesis, erythropoiesis, and angiogenesis and discuss the DUBs associated with common hematological disorders and malignancies, which are potential therapeutic drug targets.
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http://dx.doi.org/10.3390/cancers12051103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281754PMC
April 2020

The role of deubiquitinating enzymes in cancer drug resistance.

Cancer Chemother Pharmacol 2020 04 7;85(4):627-639. Epub 2020 Mar 7.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, 04763, South Korea.

Drug resistance is a well-known phenomenon leading to a reduction in the effectiveness of pharmaceutical treatments. Resistance to chemotherapeutic agents can involve various intrinsic cellular processes including drug efflux, increased resistance to apoptosis, increased DNA damage repair capabilities in response to platinum salts or other DNA-damaging drugs, drug inactivation, drug target alteration, epithelial-mesenchymal transition (EMT), inherent cell heterogeneity, epigenetic effects, or any combination of these mechanisms. Deubiquitinating enzymes (DUBs) reverse ubiquitination of target proteins, maintaining a balance between ubiquitination and deubiquitination of proteins to maintain cell homeostasis. Increasing evidence supports an association of altered DUB activity with development of several cancers. Thus, DUBs are promising candidates for targeted drug development. In this review, we outline the involvement of DUBs, particularly ubiquitin-specific proteases, and their roles in drug resistance in different types of cancer. We also review potential small molecule DUB inhibitors that can be used as drugs for cancer treatment.
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http://dx.doi.org/10.1007/s00280-020-04046-8DOI Listing
April 2020

Critical Roles of Deubiquitinating Enzymes in the Nervous System and Neurodegenerative Disorders.

Mol Cells 2020 Mar;43(3):203-214

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Korea.

Post-translational modifications play major roles in the stability, function, and localization of target proteins involved in the nervous system. The ubiquitin-proteasome pathway uses small ubiquitin molecules to degrade neuronal proteins. Deubiquitinating enzymes (DUBs) reverse this degradation and thereby control neuronal cell fate, synaptic plasticity,axonal growth, and proper function of the nervous system.Moreover, mutations or downregulation of certain DUBshave been found in several neurodegenerative diseases, as well as gliomas and neuroblastomas. Based on emerging findings, DUBs represent an important target for therapeutic intervention in various neurological disorders. Here, we summarize advances in our understanding of the roles of DUBs related to neurobiology.
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http://dx.doi.org/10.14348/molcells.2020.2289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7103888PMC
March 2020

Maintenance of hPSCs under Xeno-Free and Chemically Defined Culture Conditions.

Int J Stem Cells 2019 Nov;12(3):484-496

Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea.

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.
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http://dx.doi.org/10.15283/ijsc19090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6881038PMC
November 2019

O-cyclic phytosphingosine-1-phosphate stimulates HIF1α-dependent glycolytic reprogramming to enhance the therapeutic potential of mesenchymal stem cells.

Cell Death Dis 2019 08 5;10(8):590. Epub 2019 Aug 5.

Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science, and BK21 PLUS Program for Creative Veterinary Science Research, Seoul National University, Seoul, 08826, Republic of Korea.

O-cyclic phytosphingosine-1-phosphate (cP1P) is a novel chemically synthesized sphingosine metabolite derived from phytosphingosine-1-phosphate. Although structurally similar to sphingosine-1-phosphate (S1P), its biological properties in stem cells remain to be reported. We investigated the effect of cP1P on the therapeutic potential of mesenchymal stem cells (MSCs) and their regulatory mechanism. We found that, under hypoxia, cP1P suppressed MSC mitochondrial dysfunction and apoptosis. Metabolic data revealed that cP1P stimulated glycolysis via the upregulation of glycolysis-related genes. cP1P-induced hypoxia-inducible factor 1 alpha (HIF1α) plays a key role for MSC glycolytic reprogramming and transplantation efficacy. The intracellular calcium-dependent PKCα/mammalian target of the rapamycin (mTOR) signaling pathway triggered by cP1P regulated HIF1α translation via S6K1, which is critical for HIF1 activation. Furthermore, the cP1P-activated mTOR pathway induced bicaudal D homolog 1 expression, leading to HIF1α nuclear translocation. In conclusion, cP1P enhances the therapeutic potential of MSC through mTOR-dependent HIF1α translation and nuclear translocation.
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http://dx.doi.org/10.1038/s41419-019-1823-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683124PMC
August 2019

CRISPR-mediated upregulation of DR5 and downregulation of cFLIP synergistically sensitize HeLa cells to TRAIL-mediated apoptosis.

Biochem Biophys Res Commun 2019 04 9;512(1):60-65. Epub 2019 Mar 9.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea. Electronic address:

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as an anticancer therapy because it mediates apoptosis of several cancer cell types but not normal human cell types. In this study, we implemented genome editing techniques to upregulate DR5 and downregulate cFLIP in HeLa cells to stimulate TRAIL-induced apoptosis. We designed and validated sgRNAs to enrich the endogenous level of DR5 by dead Cas9 (dCas9). Similarly, we designed two sgRNAs to disrupt the cFLIP gene by CRISPR/Cas9. We analyzed the effect of TRAIL on tumor cells by co-transfecting HeLa cells with the best combinations of sgRNAs regulating DR5 and cFLIP genes. TRAIL-induced apoptosis in HeLa cells was evaluated by the γH2AX foci formation assay to check for double-strand break and propidium iodide and Annexin V staining to quantify apoptotic cells. Viable cells were identified by CCK-8 assay, and cleaved-PARP level was evaluated by Western blot. This is the first study to demonstrate that genome editing techniques can be used as an effective combinatorial treatment strategy to induce apoptosis of cancer cells. In particular, enhancement of DR5 expression and inhibition of cFLIP expression by genome editing had a synergistic effect of inhibiting proliferation and inducing apoptosis in TRAIL-resistant HeLa cells. These results suggest that combinatorial treatment strategies mediated by the CRISPR/Cas9 system may be effective for design of other human TRAIL-resistant cell types.
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http://dx.doi.org/10.1016/j.bbrc.2019.03.018DOI Listing
April 2019

Deubiquitinating enzymes as cancer biomarkers: new therapeutic opportunities?

BMB Rep 2019 Mar;52(3):181-189

Graduate School of Biomedical Science and Engineering, Department of Biomedical Science, Hanyang University, Seoul 04763; College of Medicine, Hanyang University, Seoul 04763, Korea.

Cancer remains a life-threatening disease and accounts for the major mortality rates worldwide. The practice of using biomarkers for early detection, staging, and customized therapy may increase cancer patients' survival. Deubiquitinating enzymes (DUBs) are a family of proteases that remove ubiquitin tags from proteins of interest undergoing proteasomal degradation. DUBs play several functional roles other than deubiquitination. One of the important roles of DUBs is regulation of tumor progression. Several reports have suggested that the DUB family members were highly-elevated in various cancer cells and tissues in different stages of cancer. These findings suggest that the DUBs could be used as drug targets in cancer therapeutics. In this review, we recapitulate the role of the DUB family members, including ubiquitinspecific protease, otubain protease, and important candidates from other family members. Our aim was to better understand the connection between DUB expression profiles and cancers to allow researchers to design inhibitors or gene therapies to improve diagnosis and prognosis of cancers. [BMB Reports 2019; 52(3): 181-189].
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6476481PMC
March 2019

The stability and oncogenic function of LIN28A are regulated by USP28.

Biochim Biophys Acta Mol Basis Dis 2019 03 10;1865(3):599-610. Epub 2018 Dec 10.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul 04763, South Korea. Electronic address:

RNA-binding protein LIN28A is often highly expressed in human malignant tumors and is involved in tumor metastasis and poor prognosis. Knowledge about post-translational regulatory mechanisms governing LIN28A protein stability and function is scarce. Here, we investigated the role of ubiquitination and deubiquitination on LIN28A protein stability and report that LIN28A protein undergoes ubiquitination. Ubiquitin-specific protease 28 (USP28), a deubiquitinating enzyme, interacts with and stabilizes LIN28A protein to extend its half-life. USP28, through its deubiquitinating activity, antagonizes LIN28A protein turnover by reversing its proteasomal degradation. Our study describes the consequential impacts of USP28-mediated stabilization of LIN28A protein on enhancing cancer cell viability, migration and ultimately augmenting LIN28A-mediated tumor progression. Overall, our data suggest that a synergistic, combinatorial approach of targeting LIN28A with USP28 would contribute to effective cancer therapeutics.
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http://dx.doi.org/10.1016/j.bbadis.2018.12.006DOI Listing
March 2019

Reprogramming mechanisms influence the maturation of hematopoietic progenitors from human pluripotent stem cells.

Cell Death Dis 2018 10 24;9(11):1090. Epub 2018 Oct 24.

Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon, Republic of Korea.

Somatic cell nuclear transfer (SCNT) or the forced expression of transcription factors can be used to generate autologous pluripotent stem cells (PSCs). Although transcriptomic and epigenomic comparisons of isogenic human NT-embryonic stem cells (NT-ESCs) and induced PSCs (iPSCs) in the undifferentiated state have been reported, their functional similarities and differentiation potentials have not been fully elucidated. Our study showed that NT-ESCs and iPSCs derived from the same donors generally displayed similar in vitro commitment capacity toward three germ layer lineages as well as proliferative activity and clonogenic capacity. However, the maturation capacity of NT-ESC-derived hematopoietic progenitors was significantly greater than the corresponding capacity of isogenic iPSC-derived progenitors. Additionally, donor-dependent variations in hematopoietic specification and commitment capacity were observed. Transcriptome and methylome analyses in undifferentiated NT-ESCs and iPSCs revealed a set of genes that may influence variations in hematopoietic commitment and maturation between PSC lines derived using different reprogramming methods. Here, we suggest that genetically identical iPSCs and NT-ESCs could be functionally unequal due to differential transcription and methylation levels acquired during reprogramming. Our proof-of-concept study indicates that reprogramming mechanisms and genetic background could contribute to diverse functionalities between PSCs.
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http://dx.doi.org/10.1038/s41419-018-1124-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200746PMC
October 2018

Different Methods of Delivering CRISPR/Cas9 Into Cells.

Prog Mol Biol Transl Sci 2018 12;159:157-176. Epub 2018 Jun 12.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea. Electronic address:

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) is comprised of repetitive bases followed by short fragments of DNA from a previously invading organism that provide immunity to the most prokaryotic organisms. An RNA-dependent spacer is required for CRISPR/Cas9 to recognize the target DNA. Delivery of the CRISPR/Cas9-guide RNA (gRNA) complex to any cell results in modification of the target sequence. The CRISPR/Cas9-mediated genome editing technique is currently in the spotlight and has several research interests, including molecular medicine and agriculture. There are several factors that hinder the delivery of this complex, such as the large size of the plasmid or high dosage of the chemical agent. There are several methods available to deliver CRISPR/Cas9 and its components to the target cells. It includes viral, non-viral and physical methods to deliver plasmid or ribonucleoprotein (RNP) of CRISPR components. But in vivo CRISPR/Cas9 delivery remains challenging to the researchers due to insertional mutagenesis, targeted delivery, immunogenicity, and off-targets. However, studies suggesting that the CRISPR/Cas9-RNP delivery can overcome these hurdles. Here, we review the various methods for delivery of CRISPR/Cas9 and gRNA to several cell lines, highlighting the limitations of each approach, and suggest possible alternative methods.
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http://dx.doi.org/10.1016/bs.pmbts.2018.05.001DOI Listing
July 2019

NFAT5 promotes in vivo development of murine melanoma metastasis.

Biochem Biophys Res Commun 2018 11 5;505(3):748-754. Epub 2018 Oct 5.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea. Electronic address:

Malignant melanoma is one of the most fatal and aggressive skin cancers, originating from pigment-containing melanocytes. Despite progress in clinical research, treatment options for malignant melanoma have been limited. The nuclear factor of activated T-cell 5 (NFAT5), originally identified as tonicity regulated transcription factor Ton/EBP, is now known as a carcinogenic gene in several types of cancer pathology. In this study, we knocked down NFAT5 to investigate its role in melanoma cancer. shRNA-mediated knockdown of NFAT5 led to a significant decrease in cell proliferation in vitro. Additionally, depletion of NFAT5 inhibited the cell migratory ability of B16BL6 melanoma cells and led to more accumulation at the G2/M phase of the cell cycle. Furthermore, NFAT5 was essential for the development of melanoma cancer pathophysiology in an in vivo mouse model. NFAT5 knockdown-induced tumor growth was slow and tumor volume was significantly reduced compared to mock controls. Moreover, NFAT5 knockdown was associated with a low number of metastatic nodules on the lung and liver. To our knowledge, our data demonstrate for the first time a role of NFAT5 in the development of melanoma. We provide evidence for NFAT5 as a marker of cell migration and metastasis, indicating that NFAT5 represents a novel therapeutic target in melanoma.
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http://dx.doi.org/10.1016/j.bbrc.2018.09.171DOI Listing
November 2018

Deubiquitinating enzymes in cancer stem cells: functions and targeted inhibition for cancer therapy.

Drug Discov Today 2018 12 1;23(12):1974-1982. Epub 2018 Jun 1.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, South Korea; College of Medicine, Hanyang University, Seoul, South Korea. Electronic address:

The ability of cancers to evade conventional treatments, such as chemotherapy and radiation therapy, has been attributed to a subpopulation of cancer stem cells (CSCs). CSCs are regulated by mechanisms similar to those that regulate normal stem cells (NSCs), including processes involving ubiquitination and deubiquitination enzymes (DUBs) that regulate the expression of various factors, such as Notch, Wnt, Sonic Hedgehog (Shh), and Hippo. In this review, we discuss the roles of various DUBs involved in the regulation of core stem cell transcription factors and CSC-related proteins that are implicated in the modulation of cellular processes and carcinogenesis. In addition, we discuss the various DUB inhibitors that have been designed to target processes relevant to cancer and CSC maintenance.
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http://dx.doi.org/10.1016/j.drudis.2018.05.035DOI Listing
December 2018

2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms.

Stem Cell Reports 2017 05 27;8(5):1312-1328. Epub 2017 Apr 27.

Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, Republic of Korea; Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul 04763, Republic of Korea. Electronic address:

Mouse embryonic stem cells (ESCs) are maintained in serum with leukemia inhibitory factor (LIF) to maintain self-renewal and pluripotency. Recently, a 2i culture method was reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintain ESCs in a naive ground state. How 2i maintains a ground state of ESCs remains elusive. Here we show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine (5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintain a naive ground state through JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation.
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http://dx.doi.org/10.1016/j.stemcr.2017.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425728PMC
May 2017

Transcriptional regulatory networks underlying the reprogramming of spermatogonial stem cells to multipotent stem cells.

Exp Mol Med 2017 04 14;49(4):e315. Epub 2017 Apr 14.

Hanyang University College of Medicine, Graduate School of Biomedical Science and Engineering, Seoul, Republic of Korea.

Spermatogonial stem cells (SSCs) are germline stem cells located along the basement membrane of seminiferous tubules in testes. Recently, SSCs were shown to be reprogrammed into multipotent SSCs (mSSCs). However, both the key factors and biological networks underlying this reprogramming remain elusive. Here, we present transcriptional regulatory networks (TRNs) that control cellular processes related to the SSC-to-mSSC reprogramming. Previously, we established intermediate SSCs (iSSCs) undergoing the transition to mSSCs and generated gene expression profiles of SSCs, iSSCs and mSSCs. By comparing these profiles, we identified 2643 genes that were up-regulated during the reprogramming process and 15 key transcription factors (TFs) that regulate these genes. Using the TF-target relationships, we developed TRNs describing how these TFs regulate three pluripotency-related processes (cell proliferation, stem cell maintenance and epigenetic regulation) during the reprogramming. The TRNs showed that 4 of the 15 TFs (Oct4/Pou5f1, Cux1, Zfp143 and E2f4) regulated cell proliferation during the early stages of reprogramming, whereas 11 TFs (Oct4/Pou5f1, Foxm1, Cux1, Zfp143, Trp53, E2f4, Esrrb, Nfyb, Nanog, Sox2 and Klf4) regulated the three pluripotency-related processes during the late stages of reprogramming. Our TRNs provide a model for the temporally coordinated transcriptional regulation of pluripotency-related processes during the SSC-to-mSSC reprogramming, which can be further tested in detailed functional studies.
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http://dx.doi.org/10.1038/emm.2017.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420799PMC
April 2017

Effects of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system-Based Deletion of miR-451 in Mouse Embryonic Stem Cells on Their Self-Renewal and Hematopoietic Differentiation.

Tissue Eng Regen Med 2017 Apr 8;14(2):179-185. Epub 2017 Mar 8.

1Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763 South Korea.

Pluripotent stem cells (PSCs) are a useful source of cells for exploring the role of genes related with early developmental processes and specific diseases due to their ability to differentiate into all somatic cell types. Recently, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system has proven to be a robust tool for targeted genetic modification. Here, we generated miR-451-deficient PSCs using the CRISPR/Cas9 system with PCR-based homologous recombination donor and investigated the impact of its deletion on self-renewal and hematopoietic development. CRISPR/Cas9-mediated miR-451 knockout did not alter the gene expressions of pluripotency, cellular morphology, and cell cycle, but led to impaired erythrocyte development. These findings propose that a combination of PSCs and CRISPR/Cas9 system could be useful to promote biomedical applications of PSCs by elucidating the function and manipulating of specific miRNAs during lineage specification and commitment.
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http://dx.doi.org/10.1007/s13770-017-0031-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171581PMC
April 2017

Concise Review: Fate Determination of Stem Cells by Deubiquitinating Enzymes.

Stem Cells 2017 01 6;35(1):9-16. Epub 2016 Jul 6.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea.

Post-translational modification by ubiquitin molecules is a key regulatory process for stem cell fate determination. Ubiquitination and deubiquitination are the major cellular processes used to balance the protein turnover of several transcription factors that regulate stem cell differentiation. Deubiquitinating enzymes (DUBs), which facilitate the processing of ubiquitin, significantly influence stem cell fate choices. Specifically, DUBs play a critical regulatory role during development by directing the production of new specialized cells. This review focuses on the regulatory role of DUBs in various cellular processes, including stem cell pluripotency and differentiation, adult stem cell signaling, cellular reprogramming, spermatogenesis, and oogenesis. Specifically, the identification of interactions of DUBs with core transcription factors has provided new insight into the role of DUBs in regulating stem cell fate determination. Thus, DUBs have emerged as key pharmacologic targets in the search to develop highly specific agents to treat various illnesses. Stem Cells 2017;35:9-16.
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http://dx.doi.org/10.1002/stem.2446DOI Listing
January 2017

Dissecting microRNA-mediated regulation of stemness, reprogramming, and pluripotency.

Cell Regen 2016 22;5. Epub 2016 Mar 22.

Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 04763 South Korea.

Increasing evidence indicates that microRNAs (miRNAs), endogenous short non-coding RNAs 19-24 nucleotides in length, play key regulatory roles in various biological events at the post-transcriptional level. Embryonic stem cells (ESCs) represent a valuable tool for disease modeling, drug discovery, developmental studies, and potential cell-based therapies in regenerative medicine due to their unlimited self-renewal and pluripotency. Therefore, remarkable progress has been made in recent decades toward understanding the expression and functions of specific miRNAs in the establishment and maintenance of pluripotency. Here, we summarize the recent knowledge regarding the regulatory roles of miRNAs in self-renewal of pluripotent ESCs and during cellular reprogramming, as well as the potential role of miRNAs in two distinct pluripotent states (naïve and primed).
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http://dx.doi.org/10.1186/s13619-016-0028-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802578PMC
March 2016

The Importance of Ubiquitination and Deubiquitination in Cellular Reprogramming.

Stem Cells Int 2016 6;2016:6705927. Epub 2016 Jan 6.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 133-791, Republic of Korea; College of Medicine, Hanyang University, Seoul 133-791, Republic of Korea.

Ubiquitination of core stem cell transcription factors can directly affect stem cell maintenance and differentiation. Ubiquitination and deubiquitination must occur in a timely and well-coordinated manner to regulate the protein turnover of several stemness related proteins, resulting in optimal embryonic stem cell maintenance and differentiation. There are two switches: an E3 ubiquitin ligase enzyme that tags ubiquitin molecules to the target proteins for proteolysis and a second enzyme, the deubiquitinating enzyme (DUBs), that performs the opposite action, thereby preventing proteolysis. In order to maintain stemness and to allow for efficient differentiation, both ubiquitination and deubiquitination molecular switches must operate properly in a balanced manner. In this review, we have summarized the importance of the ubiquitination of core stem cell transcription factors, such as Oct3/4, c-Myc, Sox2, Klf4, Nanog, and LIN28, during cellular reprogramming. Furthermore, we emphasize the role of DUBs in regulating core stem cell transcriptional factors and their function in stem cell maintenance and differentiation. We also discuss the possibility of using DUBs, along with core transcription factors, to efficiently generate induced pluripotent stem cells. Our review provides a relatively new understanding regarding the importance of ubiquitination/deubiquitination of stem cell transcription factors for efficient cellular reprogramming.
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http://dx.doi.org/10.1155/2016/6705927DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736574PMC
February 2016

Regulation of c-Myc Expression by Ahnak Promotes Induced Pluripotent Stem Cell Generation.

J Biol Chem 2016 Jan 23;291(2):752-61. Epub 2015 Nov 23.

From the Department of Life Sciences, Ewha Womans University, Seoul 120-750, Korea,

We have previously reported that Ahnak-mediated TGFβ signaling leads to down-regulation of c-Myc expression. Here, we show that inhibition of Ahnak can promote generation of induced pluripotent stem cells (iPSC) via up-regulation of endogenous c-Myc. Consistent with the c-Myc inhibitory role of Ahnak, mouse embryonic fibroblasts from Ahnak-deficient mouse (Ahnak(-/-) MEF) show an increased level of c-Myc expression compared with wild type MEF. Generation of iPSC with just three of the four Yamanaka factors, Oct4, Sox2, and Klf4 (hereafter 3F), was significantly enhanced in Ahnak(-/-) MEF. Similar results were obtained when Ahnak-specific shRNA was applied to wild type MEF. Of note, expressionof Ahnak was significantly induced during the formation of embryoid bodies from embryonic stem cells, suggesting that Ahnak-mediated c-Myc inhibition is involved in embryoid body formation and the initial differentiation of pluripotent stem cells. The iPSC from 3F-infected Ahnak(-/-) MEF cells (Ahnak(-/-)-iPSC-3F) showed expression of all stem cell markers examined and the capability to form three primary germ layers. Moreover, injection of Ahnak(-/-)-iPSC-3F into athymic nude mice led to development of teratoma containing tissues from all three primary germ layers, indicating that iPSC from Ahnak(-/-) MEF are bona fide pluripotent stem cells. Taken together, these data provide evidence for a new role for Ahnak in cell fate determination during development and suggest that manipulation of Ahnak and the associated signaling pathway may provide a means to regulate iPSC generation.
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http://dx.doi.org/10.1074/jbc.M115.659276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705395PMC
January 2016

PGC-Enriched miRNAs Control Germ Cell Development.

Mol Cells 2015 Oct 7;38(10):895-903. Epub 2015 Oct 7.

Graduate School of Biomedical Science and Engineering, Department of Biomedical Science, Hanyang University, Seoul 133-791, Korea.

Non-coding microRNAs (miRNAs) regulate the translation of target messenger RNAs (mRNAs) involved in the growth and development of a variety of cells, including primordial germ cells (PGCs) which play an essential role in germ cell development. However, the target mRNAs and the regulatory networks influenced by miRNAs in PGCs remain unclear. Here, we demonstrate a novel miRNAs control PGC development through targeting mRNAs involved in various cellular pathways. We reveal the PGC-enriched expression patterns of nine miRNAs, including miR-10b, -18a, -93, -106b, -126-3p, -127, -181a, -181b, and -301, using miRNA expression analysis along with mRNA microarray analysis in PGCs, embryonic gonads, and postnatal testes. These miRNAs are highly expressed in PGCs, as demonstrated by Northern blotting, miRNA in situ hybridization assay, and miRNA qPCR analysis. This integrative study utilizing mRNA microarray analysis and miRNA target prediction demonstrates the regulatory networks through which these miRNAs regulate their potential target genes during PGC development. The elucidated networks of miRNAs disclose a coordinated molecular mechanism by which these miRNAs regulate distinct cellular pathways in PGCs that determine germ cell development.
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http://dx.doi.org/10.14348/molcells.2015.0146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4625071PMC
October 2015

The role of deubiquitinating enzymes in spermatogenesis.

Cell Mol Life Sci 2015 Dec 8;72(24):4711-20. Epub 2015 Sep 8.

Graduate School of Biomedical Science and Engineering, Hanyang University, Seongdong-gu, Seoul, South Korea.

Spermatogenesis is a complex process through which spermatogonial stem cells undergo mitosis, meiosis, and cell differentiation to generate mature spermatozoa. During this process, male germ cells experience several translational modifications. One of the major post-translational modifications in eukaryotes is the ubiquitination of proteins, which targets proteins for degradation; this enables control of the expression of enzymes and structural proteins during spermatogenesis. It has become apparent that ubiquitination plays a key role in regulating every stage of spermatogenesis starting from gonocytes to differentiated spermatids. It is understood that, where there is ubiquitination, deubiquitination by deubiquitinating enzymes (DUBs) also exists to counterbalance the ubiquitination process in a reversible manner. Normal spermatogenesis is dependent on the balanced actions of ubiquitination and deubiquitination. This review highlights the current knowledge of the role of DUBs and their essential regulatory contribution to spermatogenesis, especially during progression into meiotic phase, acrosome biogenesis, quality sperm production, and apoptosis of germ cells.
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http://dx.doi.org/10.1007/s00018-015-2030-zDOI Listing
December 2015