SIRT1 ISGylation accelerates tumor progression by unleashing SIRT1 from the inactive state to promote its deacetylase activity.

ISG15 is an interferon-stimulated ubiquitin-like protein (UBL) with multifaceted roles as a posttranslational modifier in ISG15 conjugation (ISGylation). However, the mechanistic consequences of ISGylation in cancer have not been fully elucidated, largely due to a lack of knowledge on the ISG15 target repertoire. Here, we identified SIRT1, a nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase, as a new target for ISGylation. SIRT1 ISGylation impairs the association of SIRT1 with its negative regulator, deleted in breast cancer 1 (DBC1), which unleashes SIRT1 from its inactive state and leads to an increase in its deacetylase activity. Importantly, SIRT1 ISGylation promoted lung cancer progression and limited lung cancer cell sensitivity to DNA damage-based therapeutics in vivo and in vitro models. The levels of ISG15 mRNA and protein were significantly higher in lung cancer tissues than in adjacent normal tissues. Accordingly, elevated expression of SIRT1 and ISG15 was associated with poor prognosis in lung cancer patients, a finding that could be translated for lung cancer patient stratification and disease outcome evaluation. Taken together, our findings provide a mechanistic understanding of the regulatory effect of SIRT1 ISGylation on tumor progression and therapeutic efficacy in lung cancer.

WT1 Pulsed Human CD141+ Dendritic Cell Vaccine Has High Potential in Solid Tumor-Targeted Immunotherapy.

Dendritic cells (DC) are powerful cells that play critical roles in anti-tumor immunity, and their use in cancer immunotherapy unlocks hidden capabilities as an effective therapeutic. In order to maximize the full potential of DC, we developed a DC vaccine named CellgramDC-WT1 (CDW). CDW was pulsed with WT1, an antigen commonly expressed in solid tumors, and induced with zoledronate to aid DC maturation. Although our previous study focused on using Rg3 as an inducer of DC maturation, problems with quality control and access led us to choose zoledronate as a better alternative. Furthermore, CDW secreted IL-12 and IFN-γ, which induced the differentiation of naïve T cells to active CD8+ T cells and elicited cytotoxic T lymphocyte (CTL) response against cancer cells with WT1 antigens. By confirming the identity and function of CDW, we believe CDW is an improved DC vaccine and holds promising potential in the field of cancer immunotherapy.

Neuroprotective Effects of Licochalcone D in Oxidative-Stress-Induced Primitive Neural Stem Cells from Parkinson's Disease Patient-Derived iPSCs.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases caused by the loss of dopaminergic neurons in the substantia nigra pars compacta. Although the etiology of PD is still unclear, the death of dopaminergic neurons during PD progression was revealed to be associated with abnormal aggregation of α-synuclein, elevation of oxidative stress, dysfunction of mitochondrial functions, and increased neuroinflammation. In this study, the effects of Licochalcone D (LCD) on MG132-induced neurotoxicity in primitive neural stem cells (pNSCs) derived from reprogrammed iPSCs were investigated. A cell viability assay showed that LCD had anti-apoptotic properties in MG132-induced oxidative-stressed pNSCs. It was confirmed that apoptosis was reduced in pNSCs treated with LCD through 7-AAD/Annexin Ⅴ staining and cleaved caspase3. These effects of LCD were mediated through an interaction with JunD and through the EGFR/AKT and JNK signaling pathways. These findings suggest that LCD could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.

The diverse repertoire of ISG15: more intricate than initially thought.

ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein (UBL), which plays multifaceted roles not only as a free intracellular or extracellular molecule but also as a post-translational modifier in the process of ISG15 conjugation (ISGylation). ISG15 has only been identified in vertebrates, indicating that the functions of ISG15 and its conjugation are restricted to higher eukaryotes and have evolved with IFN signaling. Despite the highlighted complexity of ISG15 and ISGylation, it has been suggested that ISG15 and ISGylation profoundly impact a variety of cellular processes, including protein translation, autophagy, exosome secretion, cytokine secretion, cytoskeleton dynamics, DNA damage response, telomere shortening, and immune modulation, which emphasizes the necessity of reassessing ISG15 and ISGylation. However, the underlying mechanisms and molecular consequences of ISG15 and ISGylation remain poorly defined, largely due to a lack of knowledge on the ISG15 target repertoire. In this review, we provide a comprehensive overview of the mechanistic understanding and molecular consequences of ISG15 and ISGylation. We also highlight new insights into the roles of ISG15 and ISGylation not only in physiology but also in the pathogenesis of various human diseases, especially in cancer, which could contribute to therapeutic intervention in human diseases.

Acceleration of Mesenchymal-to-Epithelial Transition (MET) during Direct Reprogramming Using Natural Compounds.

Induced pluripotent stem cells (iPSCs) can be generated from somatic cells using Oct4, Sox2, Klf4, and c-Myc (OSKM). Small molecules can enhance reprogramming. Licochalcone D (LCD), a flavonoid compound present mainly in the roots of Glycyrrhiza inflata, acts on known signaling pathways involved in transcriptional activity and signal transduction, including the PGC1-α and MAPK families. In this study, we demonstrated that LCD improved reprogramming efficiency. LCD-treated iPSCs (LCD-iPSCs) expressed pluripotency-related genes Oct4, Sox2, Nanog, and Prdm14. Moreover, LCD-iPSCs differentiated into all three germ layers in vitro and formed chimeras. The mesenchymal-to-epithelial transition (MET) is critical for somatic cell reprogramming. We found that the expression levels of mesenchymal genes (Snail2 and Twist) decreased and those of epithelial genes (DSP, Cldn3, Crb3, and Ocln) dramatically increased in OR-MEF (OG2+/+/ROSA26+/+) cells treated with LCD for 3 days, indicating that MET effectively occurred in LCD-treated OR-MEF cells. Thus, LCD enhanced the generation of iPSCs from somatic cells by promoting MET at the early stages of reprogramming.

Deoxypodophyllotoxin Induces ROS-Mediated Apoptosis by Modulating the PI3K/AKT and p38 MAPK-Dependent Signaling in Oral Squamous Cell Carcinoma.

Deoxypodophyllotoxin (DPT), a naturally occurring flavonolignan, possesses several pharmacological properties, including anticancer property. However, the mechanisms underlying DPT mode of action in oral squamous cell carcinoma (OSCC) remain unknown. This study aimed to investigate the anticancer effects of DPT on OSCC and the underlying mechanisms. Results of the MTT assay revealed that DPT significantly reduced the cell viability in a time- and dose-dependent manner. Flow cytometry analysis revealed that DPT induces apoptosis in OSCC cells in a dose-dependent manner. Moreover, DPT enhanced the production of mitochondrial reactive oxygen species (ROS) in OSCC cells. Mechanistically, DPT induced apoptosis in OSCC cells by suppressing the PI3K/AKT signaling pathway while activating the p38 MAPK signaling to regulate the expression of apoptotic proteins. Treatment with SC79, an AKT activator, reversed the effects of DPT on AKT signaling in OSCC cells. Taken together, these results provide the basis for the use of DPT in combination with conventional chemotherapy for the treatment of oral cancer.

Licochalcone H Induces Cell Cycle Arrest and Apoptosis in Human Skin Cancer Cells by Modulating JAK2/STAT3 Signaling.

Licochalcone H (LCH) is a phenolic compound synthetically derived from licochalcone C (LCC) that exerts anticancer activity. In this study, we investigated the anticancer activity of LCH in human skin cancer A375 and A431 cells. The 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell viability assay was used to evaluate the antiproliferative activity of LCH. Cell cycle distribution and the induction of apoptosis were analyzed by flow cytometry. Western blotting assays were performed to detect the levels of proteins involved in cell cycle progression, apoptosis, and the JAK2/STAT3 signaling pathway. LCH inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay revealed that LCH induced apoptosis, and the LCH-induced apoptosis was accompanied by cell cycle arrest in the G1 phase. Western blot analysis showed that the phosphorylation of JAK2 and STAT3 was decreased by treatment with LCH. The inhibition of the JAK2/STAT3 signaling pathway by pharmacological inhibitors against JAK2/STAT3 (cryptotanshinone (CTS) and S3I-201) simulated the antiproliferative effect of LCH suggesting that LCH induced apoptosis by modulating JAK2/STAT3 signaling.

Global Proteomic Analysis of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells via Connective Tissue Growth Factor Treatment under Chemically Defined Feeder-Free Culture Conditions.

Stem cells can be applied usefully in basic research and clinical field due to their differentiation and self-renewal capacity. The aim of this study was to establish an effective novel therapeutic cellular source and create its molecular expression profile map to elucidate the possible therapeutic mechanism and signaling pathway. We successfully obtained a mesenchymal stem cell population from human embryonic stem cells (hESCs) cultured on chemically defined feeder-free conditions and treated with connective tissue growth factor (CTGF) and performed the expressive proteomic approach to elucidate the molecular basis. We further selected 12 differentially expressed proteins in CTGF-induced hESC-derived mesenchymal stem cells (C-hESC-MSCs), which were found to be involved in the metabolic process, immune response, cell signaling, and cell proliferation, as compared to bone marrow derived-MSCs(BM-MSCs). Moreover, these up-regulated proteins were potentially related to the Wnt/ß-catenin pathway. These results suggest that C-hESC-MSCs are a highly proliferative cell population, which can interact with the Wnt/ß-catenin signaling pathway; thus, due to the upregulated cell survival ability or downregulated apoptosis effects of C-hESC-MSCs, these can be used as an unlimited cellular source in the cell therapy field for a higher therapeutic potential. Overall, the study provided valuable insights into the molecular functioning of hESC derivatives as a valuable cellular source.

3'-UTR Polymorphisms in Thymidylate Synthase with Colorectal Cancer Prevalence and Prognosis.

Colorectal cancer (CRC) is the third most common type of cancer and the second leading cause of cancer-related mortality in Western countries. Polymorphisms in one-carbon metabolism and angiogenesis-related genes have been shown to play important roles in tumor development, progression, and metastasis for many cancers, including CRC. Moreover, recent studies have reported that polymorphisms in specific microRNA (miRNA)-binding regions, which are located in the 3'-untranslated region (UTR) of miRNA-regulated genes, are present in a variety of cancers. Here, we investigated the association between two thymidylate synthase (TYMS or TS) 3'-UTR polymorphisms, 1100T>C [rs699517] and 1170A>G [rs2790], and CRC susceptibility and progression in Korean patients. A total of 450 CRC patients and 400 healthy controls were enrolled in this study, and genotyping at the TS locus was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or TaqMan allelic discrimination assays. We found that TS 1170A>G genotypes, as well as the TS 1100T-1170G and 1100C-1170A haplotypes, are strongly associated with CRC. The TS 1100TC+CC type was associated with a poor survival (OS and RFS) rate. In addition, levels of the TS 1100C and TS 1170G allele were found to be significantly increased in CRC tissue. Our study provides the first evidence for 3'-UTR variants in TS genes as potential biomarkers of CRC prognosis and prevention.

How Is the Fidelity of Proteins Ensured in Terms of Both Quality and Quantity at the Endoplasmic Reticulum? Mechanistic Insights into E3 Ubiquitin Ligases.

The endoplasmic reticulum (ER) is an interconnected organelle that plays fundamental roles in the biosynthesis, folding, stabilization, maturation, and trafficking of secretory and transmembrane proteins. It is the largest organelle and critically modulates nearly all aspects of life. Therefore, in the endoplasmic reticulum, an enormous investment of resources, including chaperones and protein folding facilitators, is dedicated to adequate protein maturation and delivery to final destinations. Unfortunately, the folding and assembly of proteins can be quite error-prone, which leads to the generation of misfolded proteins. Notably, protein homeostasis, referred to as proteostasis, is constantly exposed to danger by flows of misfolded proteins and subsequent protein aggregates. To maintain proteostasis, the ER triages and eliminates terminally misfolded proteins by delivering substrates to the ubiquitin-proteasome system (UPS) or to the lysosome, which is termed ER-associated degradation (ERAD) or ER-phagy, respectively. ERAD not only eliminates misfolded or unassembled proteins via protein quality control but also fine-tunes correctly folded proteins via protein quantity control. Intriguingly, the diversity and distinctive nature of E3 ubiquitin ligases determine efficiency, complexity, and specificity of ubiquitination during ERAD. ER-phagy utilizes the core autophagy machinery and eliminates ERAD-resistant misfolded proteins. Here, we conceptually outline not only ubiquitination machinery but also catalytic mechanisms of E3 ubiquitin ligases. Further, we discuss the mechanistic insights into E3 ubiquitin ligases involved in the two guardian pathways in the ER, ERAD and ER-phagy. Finally, we provide the molecular mechanisms by which ERAD and ER-phagy conduct not only protein quality control but also protein quantity control to ensure proteostasis and subsequent organismal homeostasis.

Quantitative Proteomic Analysis of Primitive Neural Stem Cells from LRRK2 G2019S-Associated Parkinson's Disease Patient-Derived iPSCs.

Parkinson's disease (PD) is a common neurodegenerative disease, causing movement defects. The incidence of PD is constantly increasing and this disease is still incurable. Thus, understanding PD pathophysiology would be pivotal for the development of PD therapy, and various PD models have thus been already developed. Through recent advances in reprogramming techniques, a primitive neural stem cell (pNSC) derived from PD patient induced pluripotent stem cells (iPSCs) could be potentially used as a reproducible and reliable experimental system to analyze the effect of the leucine-rich repeat kinase 2 G2019S mutation (LK2GS) in neural cells. Here, we investigated the advantages of such a model system through quantitative proteomic analysis of pNSCs from normal control iPSCs and familial PD patient iPSCs harboring LK2GS. We confirmed that the expression of molecules known to be involved in PD pathogenesis, such as oxidative stress-, cell adhesion-, and cytoskeleton-related proteins, were altered in the LK2GS pNSC. In addition, we showed that down-regulation of Ku80, which was found in the proteomic analysis with LK2GS pNSCs, resulted in apoptosis induced by DNA damage response. Taken together, we suggest that pNSCs from PD iPSCs could provide a reliable and useful model system to study PD. Moreover, the highly expandable pNSC is suitable for multi-omics approaches to understand PD pathologies and discover therapeutic targets for PD.

Emerging Roles of USP18: From Biology to Pathophysiology.

Eukaryotic proteomes are enormously sophisticated through versatile post-translational modifications (PTMs) of proteins. A large variety of code generated via PTMs of proteins by ubiquitin (ubiquitination) and ubiquitin-like proteins (Ubls), such as interferon (IFN)-stimulated gene 15 (ISG15), small ubiquitin-related modifier (SUMO) and neural precursor cell expressed, developmentally downregulated 8 (NEDD8), not only provides distinct signals but also orchestrates a plethora of biological processes, thereby underscoring the necessity for sophisticated and fine-tuned mechanisms of code regulation. Deubiquitinases (DUBs) play a pivotal role in the disassembly of the complex code and removal of the signal. Ubiquitin-specific protease 18 (USP18), originally referred to as UBP43, is a major DUB that reverses the PTM of target proteins by ISG15 (ISGylation). Intriguingly, USP18 is a multifaceted protein that not only removes ISG15 or ubiquitin from conjugated proteins in a deconjugating activity-dependent manner but also acts as a negative modulator of type I IFN signaling, irrespective of its catalytic activity. The function of USP18 has become gradually clear, but not yet been completely addressed. In this review, we summarize recent advances in our understanding of the multifaceted roles of USP18. We also highlight new insights into how USP18 is implicated not only in physiology but also in pathogenesis of various human diseases, involving infectious diseases, neurological disorders, and cancers. Eventually, we integrate a discussion of the potential of therapeutic interventions for targeting USP18 for disease treatment.

Neuroprotective Effects of Cryptotanshinone in a Direct Reprogramming Model of Parkinson's Disease.

Parkinson's disease (PD) is a well-known age-related neurodegenerative disease. Considering the vital importance of disease modeling based on reprogramming technology, we adopted direct reprogramming to human-induced neuronal progenitor cells (hiNPCs) for in vitro assessment of potential therapeutics. In this study, we investigated the neuroprotective effects of cryptotanshinone (CTN), which has been reported to have antioxidant properties, through PD patient-derived hiNPCs (PD-iNPCs) model with induced oxidative stress and cell death by the proteasome inhibitor MG132. A cytotoxicity assay showed that CTN possesses anti-apoptotic properties in PD-hiNPCs. CTN treatment significantly reduced cellular apoptosis through mitochondrial restoration, such as the reduction in mitochondrial reactive oxygen species and increments of mitochondrial membrane potential. These effects of CTN are mediated via the nuclear factor erythroid 2-related factor 2 (NRF2) pathway in PD-hiNPCs. Consequently, CTN could be a potential antioxidant reagent for preventing disease-related pathological phenotypes of PD.

Iroquois Homeobox Protein 2 Identified as a Potential Biomarker for Parkinson's Disease.

The diagnosis of Parkinson's disease (PD) is initiated after the occurrence of motor symptoms, such as resting tremors, rigidity, and bradykinesia. According to previous reports, non-motor symptoms, notably gastrointestinal dysfunction, could potentially be early biomarkers in PD patients as such symptoms occur earlier than motor symptoms. However, connecting PD to the intestine is methodologically challenging. Thus, we generated in vitro human intestinal organoids from PD patients and ex vivo mouse small intestinal organoids from aged transgenic mice. Both intestinal organoids (IOs) contained the human LRRK2 G2019S mutation, which is the most frequent genetic cause of familial and sporadic PD. By conducting comprehensive genomic comparisons with these two types of IOs, we determined that a particular gene, namely, Iroquois homeobox protein 2 (IRX2), showed PD-related expression patterns not only in human pluripotent stem cell (PSC)-derived neuroectodermal spheres but also in human PSC-derived neuronal cells containing dopaminergic neurons. We expected that our approach of using various cell types presented a novel technical method for studying the effects of multi-organs in PD pathophysiology as well as for the development of diagnostic markers for PD.

Licochalcone C induces cell cycle G1 arrest and apoptosis in human esophageal squamous carcinoma cells by activation of the ROS/MAPK signaling pathway.

Along with changes in dietary habits and lifestyle, the incidence of esophageal cancer is increasing around the world. Since chemotherapy for esophageal cancer has significant side effects, phytochemicals have attracted attention as an alternative medicine. Licochalcone C (LCC) is a flavonoid compound extracted from Licorice, with a variety of clinical uses including anti-cancer, anti-inflammatory and anti-oxidant effects. Treatment with LCC for 48 h significantly decreased cell viability of esophageal squamous cell carcinoma (ESCC) cells in a dose- and time-dependent manner with IC50 values of 28 µM (KYSE 30), 36 µM (KYSE 70), 19 µM (KYSE 410), 28 µM (KYSE 450) and 26 µM (KYSE 510). LCC induced G1 arrest accompanied by decreased cyclin D1 expression and an increase in the levels of p21 and p27. LCC increased the levels of intracellular ROS, cytochrome C release, and multi-caspase activity, and decreased mitochondrial membrane potential. LCC induced the protein expression of ER stress markers (GRP78 and CHOP) and phosphorylation JNK, c-Jun and p38. We investigated the expression of pro-apoptotic and anti-apoptotic proteins to elucidate the mechanism of apoptosis. Our findings contribute to the understanding of apoptosis mechanism underlying LCC in ESCC cells and provide new insights into the potential clinical opportunities of LCC for ESCC treatment.

Proteostasis In The Endoplasmic Reticulum: Road to Cure.

The endoplasmic reticulum (ER) is an interconnected organelle that is responsible for the biosynthesis, folding, maturation, stabilization, and trafficking of transmembrane and secretory proteins. Therefore, cells evolve protein quality-control equipment of the ER to ensure protein homeostasis, also termed proteostasis. However, disruption in the folding capacity of the ER caused by a large variety of pathophysiological insults leads to the accumulation of unfolded or misfolded proteins in this organelle, known as ER stress. Upon ER stress, unfolded protein response (UPR) of the ER is activated, integrates ER stress signals, and transduces the integrated signals to relive ER stress, thereby leading to the re-establishment of proteostasis. Intriguingly, severe and persistent ER stress and the subsequently sustained unfolded protein response (UPR) are closely associated with tumor development, angiogenesis, aggressiveness, immunosuppression, and therapeutic response of cancer. Additionally, the UPR interconnects various processes in and around the tumor microenvironment. Therefore, it has begun to be delineated that pharmacologically and genetically manipulating strategies directed to target the UPR of the ER might exhibit positive clinical outcome in cancer. In the present review, we summarize recent advances in our understanding of the UPR of the ER and the UPR of the ER-mitochondria interconnection. We also highlight new insights into how the UPR of the ER in response to pathophysiological perturbations is implicated in the pathogenesis of cancer. We provide the concept to target the UPR of the ER, eventually discussing the potential of therapeutic interventions for targeting the UPR of the ER for cancer treatment.

Direct Reprogramming to Human Induced Neuronal Progenitors from Fibroblasts of Familial and Sporadic Parkinson's Disease Patients.

In Parkinson's disease (PD) research, human neuroblastoma and immortalized neural cell lines have been widely used as in vitro models. The advancement in the field of reprogramming technology has provided tools for generating patient-specific induced pluripotent stem cells (hiPSCs) as well as human induced neuronal progenitor cells (hiNPCs). These cells have revolutionized the field of disease modeling, especially in neural diseases. Although the direct reprogramming to hiNPCs has several advantages over differentiation after hiPSC reprogramming, such as the time required and the simple procedure, relatively few studies have utilized hiNPCs. Here, we optimized the protocol for hiNPC reprogramming using pluripotency factors and Sendai virus. In addition, we generated hiNPCs of two healthy donors, a sporadic PD patient, and a familial patient with the LRRK2 G2019S mutation (L2GS). The four hiNPC cell lines are highly proliferative, expressed NPC markers, maintained the normal karyotype, and have the differentiation potential of dopaminergic neurons. Importantly, the patient hiNPCs show different apoptotic marker expression. Thus, these hiNPCs, in addition to hiPSCs, are a favorable option to study PD pathology.

Protein Quality Control in the Endoplasmic Reticulum and Cancer.

The endoplasmic reticulum (ER) is an essential compartment of the biosynthesis, folding, assembly, and trafficking of secretory and transmembrane proteins, and consequently, eukaryotic cells possess specialized machineries to ensure that the ER enables the proteins to acquire adequate folding and maturation for maintaining protein homeostasis, a process which is termed proteostasis. However, a large variety of physiological and pathological perturbations lead to the accumulation of misfolded proteins in the ER, which is referred to as ER stress. To resolve ER stress and restore proteostasis, cells have evolutionary conserved protein quality-control machineries of the ER, consisting of the unfolded protein response (UPR) of the ER, ER-associated degradation (ERAD), and autophagy. Furthermore, protein quality-control machineries of the ER play pivotal roles in the control of differentiation, progression of cell cycle, inflammation, immunity, and aging. Therefore, severe and non-resolvable ER stress is closely associated with tumor development, aggressiveness, and response to therapies for cancer. In this review, we highlight current knowledge in the molecular understanding and physiological relevance of protein quality control of the ER and discuss new insights into how protein quality control of the ER is implicated in the pathogenesis of cancer, which could contribute to therapeutic intervention in cancer.