ISG15-USP18 Dysregulation by Oxidative Stress Promotes IFN-γ Secretion from CD8+ T Cells in Vitiligo.

Excessive oxidative stress is thought to play pathologic roles in cellular senescence and autoimmune disorders by inducing inflammation and breaking down immune tolerance. In this study, we sought to identify the factors linking oxidative stress to autoimmunity and cellular senescence in vitiligo, where elevated oxidative stress plays an important role. RNA sequencing analysis of hydrogen peroxide-treated melanocytes revealed upregulation of ISG15. The upregulation of ISG15 was observed in vitiligo skin tissues as well as in the blood of patients with vitiligo, whereas USP18 downregulation was observed in vitiligo melanocytes and vitiligo skin tissues. Oxidative stress induced hypermethylation of the USP18 promoter region in keratinocytes and melanocytes, and USP18 promoter hypermethylation was also confirmed in vitiligo skin tissues. Our results indicate that USP18 promoter hypermethylation caused by oxidative stress increases ISG15 expression in keratinocytes and melanocytes along with senescence changes, leading CD8+ T cells to produce IFN-γ, the main pathogenic cytokine in vitiligo. Therefore, the ISG15-USP18 network may be important in oxidative stress-induced autoimmunity and cellular senescence in vitiligo pathogenesis.

Stimulating macropinocytosis of peptide-drug conjugates through DNA-dependent protein kinase inhibition for treating KRAS-mutant cancer.

KRAS-mutant cancers, due to their protein targeting complexity, present significant therapeutic hurdles. The identification of the macropinocytic phenotype in these cancers has emerged as a promising alternative therapeutic target. Our study introduces MPD1, an macropinocytosis-targeting peptide-drug conjugates (PDC), which is developed to treat KRAS mutant cancers. This PDC is specifically designed to trigger a positive feedback loop through its caspase-3 cleavable characteristic. However, we observe that this loop is hindered by DNA-PK mediated DNA damage repair processes in cancer cells. To counter this impediment, we employ AZD7648, a DNA-PK inhibitor. Interestingly, the combined treatment of MPD1 and AZD7648 resulted in a 100% complete response rate in KRAS-mutant xenograft model. We focus on the synergic mechanism of it. We discover that AZD7648 specifically enhances macropinocytosis in KRAS-mutant cancer cells. Further analysis uncovers a significant correlation between the increase in macropinocytosis and PI3K signaling, driven by AMPK pathways. Also, AZD7648 reinforces the positive feedback loop, leading to escalated apoptosis and enhanced payload accumulation within tumors. AZD7648 possesses broad applications in augmenting nano-sized drug delivery and preventing DNA repair resistance. The promising efficacy and evident synergy underscore the potential of combining MPD1 with AZD7648 as a strategy for treating KRAS-mutant cancers.

Sustained potentiation of bystander killing via PTEN-loss driven macropinocytosis targeted peptide-drug conjugate therapy in metastatic triple-negative breast cancer.

While conventional approaches for PTEN-loss cancers mainly focus on turning off growth promoting process through modulation of PI3K/AKT pathways, no effective therapeutic treatments that target PTEN-loss cancer cells have yielded results. Moreover, conventional targeted therapies, which are potent against only a subset of cancer cells with limited specificity, bring on temporary response. Here, we report the development of albumin-binding caspase-3 cleavable peptide-drug conjugate (PDC), which utilizes the enhanced albumin metabolism pathway in PTEN-loss cancer cells to enhance the intracellular delivery of chemotherapeutic payload that could exert a bystander killing effect. Albumin metabolism-mediated apoptosis triggered expression of caspase-3 allows the continuous activation of the PDC, accumulation of payloads, sustained upregulation of tumoral caspase-3, and intensified in-situ apoptosis. Importantly, PDC strategy exerts potent therapeutic efficacy against PTEN-loss metastatic triple-negative breast cancer, the highly aggressive and heterogenous nature of which remains a challenge conventional targeted therapies need to overcome. This study thus presents a conceptually novel approach to treat PTEN-loss cancer and creates new translational perspectives of exploiting PTEN-loss for providing an avenue to advance current targeted therapy.

Interaction between CD36 and FABP4 modulates adipocyte-induced fatty acid import and metabolism in breast cancer.

Adipocytes influence breast cancer behaviour via fatty acid release into the tumour microenvironment. Co-culturing human adipocytes and breast cancer cells increased CD36 expression, with fatty acid import into breast cancer cells. Genetic ablation of CD36 attenuates adipocyte-induced epithelial-mesenchymal transition (EMT) and stemness. We show a feedforward loop between CD36 and STAT3; where CD36 activates STAT3 signalling and STAT3 binds to the CD36 promoter, regulating its expression. CD36 expression results in metabolic reprogramming, with a shift towards fatty acid oxidation. CD36 inhibition induces de novo lipogenesis in breast cancer cells. Increased CD36 expression occurs with increased FABP4 expression. We showed that CD36 directly interacts with FABP4 to regulate fatty acid import, transport, and metabolism. CD36 and FABP4 inhibition induces apoptosis in tumour cells. These results indicate that CD36 mediates fatty acid import from adipocytes into cancer cells and activates signalling pathways that drive tumour progression. Targeting CD36 may have a potential for therapy, which will target the tumour microenvironment.

A Variant of SLC1A5 Is a Mitochondrial Glutamine Transporter for Metabolic Reprogramming in Cancer Cells.

Glutamine is an essential nutrient that regulates energy production, redox homeostasis, and signaling in cancer cells. Despite the importance of glutamine in mitochondrial metabolism, the mitochondrial glutamine transporter has long been unknown. Here, we show that the SLC1A5 variant plays a critical role in cancer metabolic reprogramming by transporting glutamine into mitochondria. The SLC1A5 variant has an N-terminal targeting signal for mitochondrial localization. Hypoxia-induced gene expression of the SLC1A5 variant is mediated by HIF-2α. Overexpression of the SLC1A5 variant mediates glutamine-induced ATP production and glutathione synthesis and confers gemcitabine resistance to pancreatic cancer cells. SLC1A5 variant knockdown and overexpression alter cancer cell and tumor growth, supporting an oncogenic role. This work demonstrates that the SLC1A5 variant is a mitochondrial glutamine transporter for cancer metabolic reprogramming.

An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay.

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Inhibition of gap junctional intercellular communication by an anti-migraine agent, flunarizine.

Gap junctions (GJs), which consist of proteins called connexins, are intercellular channels that allow the passage of ions, second messengers, and small molecules. GJs and connexins are considered as emerging therapeutic targets for various diseases. Previously, we screened numerous compounds using our recently developed iodide yellow fluorescent protein gap junctional intercellular communication (I-YFP GJIC) assay and found that flunarizine (FNZ), used for migraine prophylaxis and as an add-on therapy for epilepsy, inhibits GJIC in LN215 human glioma cells. In this study, we confirmed that FNZ inhibits GJIC using the I-YFP GJIC assay. We demonstrated that FNZ inhibits GJ activities via a mechanism that is independent of calcium channels and dopaminergic D2, histaminergic H1, or 5-HT receptors. In addition, we showed that FNZ significantly increases connexin 43 (Cx43) phosphorylation on the cell surface, but does not alter the total amount of Cx43. The beneficial effects of FNZ on migraines and epilepsy might be related to GJ inhibition.

Generation of trichogenic adipose-derived stem cells by expression of three factors.

BACKGROUND: Previous studies demonstrated that adipose-derived stem cells (ASCs) can promote hair growth, but unmet needs exist for enhancing ASC hair inductivity. OBJECTIVE: Therefore, we introduced three trichogenic factors platelet-derived growth factor-A, SOX2, and ß-catenin to ASCs (tfASCs) and evaluated whether tfASCs have similar characteristics as dermal papilla (DP) cells. METHOD: Global gene expression was examined using NGS analysis. Telogen-to-anagen induction, vibrissae hair follicle organ culture and patch assay were used. RESULTS: tfASC cell size is smaller than that of ASCs, and they exhibit short doubling time. tfASCs also resist aging and can be expanded until passage 12. Cell proportion in S and G2/M increases in tfASCs, and tfASCs express high mRNA levels of cell cycle related genes. The mRNA expression of DP markers was notably higher in tfASCs. Moreover, NGS analysis revealed that the global gene expression of tfASCs is similar to that of DP cells. The injection of tfASCs accelerated the telogen-to-anagen transition and conditioned medium of tfASCs increased the anagen phase of vibrissal hair follicles. Finally, we found that the injection of 3D-cultured tfASCs at p 9 generated new hair follicles in nude mice. CONCLUSION: Collectively, these results indicate that 1) tfASCs have similar characteristics as DP cells, 2) tfASCs have enhanced hair-regenerative potential compared with ASCs, and 3) tfASCs even at late passage can make new hair follicles in a hair reconstitution assay. Because DP cells are difficult to isolate/expand and ASCs have low hair inductivity, tfASCs and tfASC-CM are clinically good candidates for hair regeneration.