PKCα-LSD1-NF-κB-Signaling Cascade Is Crucial for Epigenetic Control of the Inflammatory Response.

The inflammatory response mediated by nuclear factor κB (NF-κB) signaling is essential for host defense against pathogens. Although the regulatory mechanism of NF-κB signaling has been well studied, the molecular basis for epigenetic regulation of the inflammatory response is poorly understood. Here we identify a new signaling axis of PKCα-LSD1-NF-κB, which is critical for activation and amplification of the inflammatory response. In response to excessive inflammatory stimuli, PKCα translocates to the nucleus and phosphorylates LSD1. LSD1 phosphorylation is required for p65 binding and facilitates p65 demethylation, leading to enhanced stability. In vivo genetic analysis using Lsd1SA/SA mice with ablation of LSD1 phosphorylation and chemical approaches in wild-type mice with inhibition of PKCα or LSD1 activity show attenuated sepsis-induced inflammatory lung injury and mortality. Together, we demonstrate that the PKCα-LSD1-NF-κB signaling cascade is crucial for epigenetic control of the inflammatory response, and targeting this signaling could be a powerful therapeutic strategy for systemic inflammatory diseases, including sepsis.

DNA damage-induced RORα is crucial for p53 stabilization and increased apoptosis.

A critical component of the DNA damage response is the p53 tumor suppressor, and aberrant p53 function leads to uncontrolled cell proliferation and malignancy. Several molecules have been shown to regulate p53 stability; however, genome-wide systemic approaches for determining the affected, specific downstream target genes have not been extensively studied. Here, we first identified an orphan nuclear receptor, RORα, as a direct target gene of p53, which contains functional p53 response elements. The functional consequences of DNA damage-induced RORα are to stabilize p53 and activate p53 transcription in a HAUSP/Usp7-dependent manner. Interestingly, microarray analysis revealed that RORα-mediated p53 stabilization leads to the activation of a subset of p53 target genes that are specifically involved in apoptosis. We further confirmed that RORα enhances p53-dependent, in vivo apoptotic function in the Drosophila model system. Together, we determined that RORα is a p53 regulator that exerts its role in increased apoptosis via p53.

Loss-of-function screens of druggable targetome against cancer stem-like cells.

Cancer stem-like cells (CSLCs) contribute to the initiation and recurrence of tumors and to their resistance to conventional therapies. In this study, small interfering RNA (siRNA)-based screening of ∼4800 druggable genes in 3-dimensional CSLC cultures in comparison to 2-dimensional bulk cultures of U87 glioma cells revealed 3 groups of genes essential for the following: survival of the CSLC population only, bulk-cultured population only, or both populations. While diverse biologic processes were associated with siRNAs reducing the bulk-cultured population, CSLC-eliminating siRNAs were enriched in a few functional categories, such as lipid metabolism, protein metabolism, and gene expression. Interestingly, siRNAs that selectively reduced CSLC only were found to target genes for cholesterol and unsaturated fatty acid synthesis. The lipidomic profile of CSLCs revealed increased levels of monounsaturated lipids. Pharmacologic blockage of these target pathways reduced CSLCs, and this effect was eliminated by addition of downstream metabolite products. The present CSLC-sensitive target categories provide a useful resource that can be exploited for the selective elimination of CSLCs.-Song, M., Lee, H., Nam, M.-H., Jeong, E., Kim, S., Hong, Y., Kim, N., Yim, H. Y., Yoo, Y.-J., Kim, J. S., Kim, J.-S., Cho, Y.-Y., Mills, G. B., Kim, W.-Y., Yoon, S. Loss-of-function screens of druggable targetome against cancer stem-like cells.

Elevated Response to Type I IFN Enhances RANKL-Mediated Osteoclastogenesis in Usp18-Knockout Mice.

A balance between bone formation and bone resorption is critical for the maintenance of bone mass. In many pathological conditions, including chronic inflammation, uncontrolled activation of osteoclast differentiation often causes excessive bone resorption that results in osteoporosis. In this study, we identified the osteopenia phenotype of mice lacking Usp18 (also called Ubp43), which is a deISGylating enzyme and is known as a negative regulator of type I IFN signaling. The expression of Usp18 was induced in preosteoclasts upon receptor activator of NF-κB ligand (RANKL) treatment. In an in vitro osteoclast-differentiation assay, bone marrow macrophages from Usp18-deficient mice exhibited an enhanced differentiation to multinucleated cells, elevated activation of NFATc1, and an increased expression of osteoclast marker genes upon RANKL treatment. Furthermore, in vitro quantification of bone resorption revealed a great increase in osteoclastic activities in Usp18-deficient cells. Interestingly, proinflammatory cytokine genes, such as IP-10 (CXCL10), were highly expressed in Usp18-deficient bone marrow macrophages upon RANKL treatment compared with wild-type cells. In addition, serum cytokine levels, especially IP-10, were significantly high in Usp18-knockout mice. In sum, we suggest that, although type I IFN is known to restrict osteoclast differentiation, the exaggerated activation of the type I IFN response in Usp18-knockout mice causes an osteopenia phenotype in mice.

RORalpha attenuates Wnt/beta-catenin signaling by PKCalpha-dependent phosphorylation in colon cancer.

Wnt family members play diverse roles in development and disease. Noncanonical Wnt ligands can inhibit canonical Wnt signaling depending on the cellular context; however, the underlying mechanism of this antagonism remains poorly understood. Here we identify a specific mechanism of orphan nuclear receptor RORalpha-mediated inhibition of canonical Wnt signaling in colon cancer. Wnt5a/PKCalpha-dependent phosphorylation on serine residue 35 of RORalpha is crucial to link RORalpha to Wnt/beta-catenin signaling, which exerts inhibitory function of the expression of Wnt/beta-catenin target genes. Intriguingly, there is a significant correlation of reduction of RORalpha phosphorylation in colorectal tumor cases compared to their normal counterpart, providing the clinical relevance of the findings. Our data provide evidence for a role of RORalpha, functioning at the crossroads between the canonical and the noncanonical Wnt signaling pathways, in mediating transrepression of the Wnt/beta-catenin target genes, thereby providing new approaches for the development of therapeutic agents for human cancers.

The mitochondrial pathway and reactive oxygen species are critical contributors to interferon-α/ß-mediated apoptosis in Ubp43-deficient hematopoietic cells.

UBP43 (also known as USP18) plays a role in the negative regulation of interferon-α/ß signaling, and bone marrow cells in Ubp43-deficient mice exhibited hypersensitivity to interferon-α/ß-mediated apoptosis. Here, we show that the mitochondrial apoptotic pathway and reactive oxygen species are major contributors to the elevated interferon-α/ß-mediated apoptosis in Ubp43-deficient mouse bone marrow cells and in UBP43-knockdown THP-1 cells. Furthermore, TRAIL and FASL, which were proposed as apoptosis inducers upon interferon-α/ß treatment in UBP43-knockdown adherent cancer cells, did not cause apoptosis in these hematopoietic cells. Therefore, although UBP43 depletion can cause hypersensitivity to interferon-α/ß-mediated apoptosis in a broad range of cell types, the downstream pathway may vary depending on the cell type.

Biodistribution of Exosomes and Engineering Strategies for Targeted Delivery of Therapeutic Exosomes.

Exosomes are cell-secreted nano-sized vesicles which deliver diverse biological molecules for intercellular communication. Due to their therapeutic potential, exosomes have been engineered in numerous ways for efficient delivery of active pharmaceutical ingredients to various target organs, tissues, and cells. In vivo administered exosomes are normally delivered to the liver, spleen, kidney, lung, and gastrointestinal tract and show rapid clearance from the blood circulation after systemic injection. The biodistribution and pharmacokinetics (PK) of exosomes can be modulated by engineering various factors such as cellular origin and membrane protein composition of exosomes. Recent advances accentuate the potential of targeted delivery of engineered exosomes even to the most challenging organs including the central nervous system. Major breakthroughs have been made related to various imaging techniques for monitoring in vivo biodistribution and PK of exosomes, as well as exosomal surface engineering technologies for inducing targetability. For inducing targeted delivery, therapeutic exosomes can be engineered to express various targeting moieties via direct modification methods such as chemically modifying exosomal surfaces with covalent/non-covalent bonds, or via indirect modification methods by genetically engineering exosome-producing cells. In this review, we describe the current knowledge of biodistribution and PK of exosomes, factors determining the targetability and organotropism of exosomes, and imaging technologies to monitor in vivo administered exosomes. In addition, we highlight recent advances in strategies for inducing targeted delivery of exosomes to specific organs and cells.

Cardiac glycosides display selective efficacy for STK11 mutant lung cancer.

Although STK11 (LKB1) mutation is a major mediator of lung cancer progression, targeted therapy has not been implemented due to STK11 mutations being loss-of-function. Here, we report that targeting the Na(+)/K(+)-ATPase (ATP1A1) is synthetic lethal with STK11 mutations in lung cancer. The cardiac glycosides (CGs) digoxin, digitoxin and ouabain, which directly inhibit ATP1A1 function, exhibited selective anticancer effects on STK11 mutant lung cancer cell lines. Restoring STK11 function reduced the efficacy of CGs. Clinically relevant doses of digoxin decreased the growth of STK11 mutant xenografts compared to wild type STK11 xenografts. Increased cellular stress was associated with the STK11-specific efficacy of CGs. Inhibiting ROS production attenuated the efficacy of CGs, and STK11-AMPK signaling was important in overcoming the stress induced by CGs. Taken together, these results show that STK11 mutation is a novel biomarker for responsiveness to CGs. Inhibition of ATP1A1 using CGs warrants exploration as a targeted therapy for STK11 mutant lung cancer.

Integrated analysis of transcriptomes of cancer cell lines and patient samples reveals STK11/LKB1-driven regulation of cAMP phosphodiesterase-4D.

The recent proliferation of data on large collections of well-characterized cancer cell lines linked to therapeutic drug responses has made it possible to identify lineage- and mutation-specific transcriptional markers that can help optimize implementation of anticancer agents. Here, we leverage these resources to systematically investigate the presence of mutation-specific transcription markers in a wide variety of cancer lineages and genotypes. Sensitivity and specificity of potential transcriptional biomarkers were simultaneously analyzed in 19 cell lineages grouped into 228 categories based on the mutational genotypes of 12 cancer-related genes. Among a total of 1,455 category-specific expression patterns, the expression of cAMP phosphodiesterase-4D (PDE4D) with 11 isoforms, one of the PDE4(A-D) subfamilies, was predicted to be regulated by a mutant form of serine/threonine kinase 11 (STK11)/liver kinase B1 (LKB1) present in lung cancer. STK11/LKB1 is the primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK). Subsequently, we found that the knockdown of PDE4D gene expression inhibited proliferation of STK11-mutated lung cancer lines. Furthermore, challenge with a panel of PDE4-specific inhibitors was shown to selectively reduce the growth of STK11-mutated lung cancer lines. Thus, we show that multidimensional analysis of a well-characterized large-scale panel of cancer cell lines provides unprecedented opportunities for the identification of unexpected oncogenic mechanisms and mutation-specific drug targets.