Protein kinase C delta enhances the diagnostic performance of hepatocellular carcinoma.

BACKGROUND: The conventional markers for hepatocellular carcinoma (HCC), α-fetoprotein (AFP) and des-γ-carboxy prothrombin (DCP), have several limitations; both have low sensitivity in patients with early-stage HCC; low sensitivity for AFP with HCC after eliminating hepatitis C virus (HCV); low specificity for DCP in patients with non-viral HCC, which is increasing worldwide; low specificity for AFP in patients with liver injury; and low specificity for DCP in patients treated with warfarin. To overcome these issues, the identification of novel biomarkers is an unmet need. OBJECTIVE: This study aimed to assess the usefulness of serum protein kinase C delta (PKCδ) for detecting these HCCs. METHODS: PKCδ levels were measured using a sandwich enzyme-linked immunosorbent assay in 363 chronic liver disease (CLD) patients with and without HCC. RESULTS: In both viral and non-viral CLD, PKCδ can detect HCCs with high sensitivity and specificity, particularly in the very early stages. Notably, the value and sensitivity of PKCδ were not modified by HCV elimination status. Liver injury and warfarin administration, which are known to cause false-positive results for conventional markers, did not modify PKCδ levels. CONCLUSIONS: PKCδ is an enhanced biomarker for the diagnosis of HCC that compensates for the drawbacks of conventional markers.

Sugar coordinates plant defense signaling.

Pathogen recognition triggers energy-intensive defense systems. Although successful defense should depend on energy availability, how metabolic information is communicated to defense remains unclear. We show that sugar, especially glucose-6-phosphate (G6P), is critical in coordinating defense in Arabidopsis. Under sugar-sufficient conditions, phosphorylation levels of calcium-dependent protein kinase 5 (CPK5) are elevated by G6P-mediated suppression of protein phosphatases, enhancing defense responses before pathogen invasion. Subsequently, recognition of bacterial flagellin activates sugar transporters, leading to increased cellular G6P, which elicits CPK5-independent signaling promoting synthesis of the phytohormone salicylic acid (SA) for antibacterial defense. In contrast, while perception of fungal chitin does not promote sugar influx or SA accumulation, chitin-induced synthesis of the antifungal compound camalexin requires basal sugar influx activity. By monitoring sugar levels, plants determine defense levels and execute appropriate outputs against bacterial and fungal pathogens. Together, our findings provide a comprehensive view of the roles of sugar in defense.

Extended-Synaptotagmin 1 Enhances Liver Cancer Progression Mediated by the Unconventional Secretion of Cytosolic Proteins.

Extended-synaptotagmin 1 (E-Syt1) is an endoplasmic reticulum membrane protein that is involved in cellular lipid transport. Our previous study identified E-Syt1 as a key factor for the unconventional protein secretion of cytoplasmic proteins in liver cancer, such as protein kinase C delta (PKCδ); however, it is unclear whether E-Syt1 is involved in tumorigenesis. Here, we showed that E-Syt1 contributes to the tumorigenic potential of liver cancer cells. E-Syt1 depletion significantly suppressed the proliferation of liver cancer cell lines. Database analysis revealed that E-Syt1 expression is a prognostic factor for hepatocellular carcinoma (HCC). Immunoblot analysis and cell-based extracellular HiBiT assays showed that E-Syt1 was required for the unconventional secretion of PKCδ in liver cancer cells. Furthermore, deficiency of E-Syt1 suppressed the activation of insulin-like growth factor 1 receptor (IGF1R) and extracellular-signal-related kinase 1/2 (Erk1/2), both of which are signaling pathways mediated by extracellular PKCδ. Three-dimensional sphere formation and xenograft model analysis revealed that E-Syt1 knockout significantly decreased tumorigenesis in liver cancer cells. These results provide evidence that E-Syt1 is critical for oncogenesis and is a therapeutic target for liver cancer.

Selective deployment of virulence effectors correlates with host specificity in a fungal plant pathogen.

The hemibiotrophic fungal plant pathogen Colletotrichum orbiculare is predicted to secrete hundreds of effector proteins when the pathogen infects cucurbit crops, such as cucumber and melon, and tobacco (Nicotiana benthamiana), a distantly related Solanaceae species. Here, we report the identification of sets of C. orbiculare effector genes that are differentially required for fungal virulence to two phylogenetically distant host species. Through targeted gene knockout screening of C. orbiculare 'core' effector candidates defined based on in planta gene expression, we identified: four host-specific virulence effectors (named effector proteins for cucurbit infection, or EPCs) that are required for full virulence of C. orbiculare to cucurbit hosts, but not to the Solanaceae host N. benthamiana; and five host-nonspecific virulence effectors, which collectively contribute to fungal virulence to both hosts. During host infection, only a small subset of genes, including the host-specific EPC effector genes, showed preferential expression on one of the hosts, while gene expression profiles of the majority of other genes, including the five host-nonspecific effector genes, were common to both hosts. This work suggests that C. orbiculare adopts a host-specific effector deployment strategy, in addition to general host-blind virulence mechanisms, for adaptation to cucurbit hosts.

Cancer-Related Unconventional Protein Secretion: A New Role of the Endoplasmic Reticulum.

Unconventional protein secretion (UPS) is a crucial mechanism controlling the localization of cytosolic proteins lacking signal peptides and is implicated in inflammation, neurodegenerative diseases, and cancer. Several previous studies on immune cells have demonstrated the mechanisms of UPS. In cancer, the active secretion of several cytosolic proteins, including PKCδ and nucleolin, has been described. Moreover, we have recently demonstrated that extended synaptotagmin 1, one of the membrane proteins of the endoplasmic reticulum, plays a critical role in UPS in liver cancer cells. Importantly, UPS in cancer cells shows characteristics that are markedly different from those of the previously known UPS, and therefore, we categorize them as cancer-related UPS (CUPS). In this article, we provide an overview of UPS mechanisms and discuss the process that leads to the naming of cancer-specific UPS as CUPS.

Inhibition of protein kinase C delta leads to cellular senescence to induce anti-tumor effects in colorectal cancer.

Protein kinase C delta (PKCδ) is a multifunctional serine-threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, the molecular mechanism of PKCδ in colorectal cancer (CRC) remains unclear. In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild-type (wt-p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt-p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53-independent pathway and that PKCδ-kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence-like phenotypes, including increased senescence-associated ß-galactosidase (SA-ß-gal) staining, low LaminB1 expression, large nucleus size, and senescence-associated secretory phenotype (SASP) detection. Finally, a kinase inhibitor of PKCδ suppressed senescence-dependent tumorigenicity in a dose-dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis. In addition, a novel therapeutic strategy for wt-p53 CRC is proposed.

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR-Cas9 and Cre-loxP systems.

Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to generate multiple knockout mutants of C. orbiculare efficiently. We first found that CRISPR-Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR-Cas9 vector, more than 90% of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre-loxP marker recycling system for C. orbiculare because a limited availability of desired selective markers hampers sequential gene disruption. In this system, the integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling the reuse of the same selective marker for the next transformation. Using our CRISPR-Cas9 and Cre-loxP systems, we attempted to identify functional sugar transporters involved in fungal virulence. Multiple disruptions of putative quinate transporter genes restricted fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. However, our analyses showed that quinate acquisition was dispensable for infection to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants that we established in this study, we inferred that repetitive rounds of gene disruption using CRISPR-Cas9 and Cre-loxP systems do not cause adverse effects on fungal virulence and growth. Therefore, these systems will be powerful tools to perform a systematic loss-of-function approach for C. orbiculare.

Extended-synaptotagmin 1 engages in unconventional protein secretion mediated via SEC22B+ vesicle pathway in liver cancer.

Protein secretion in cancer cells defines tumor survival and progression by orchestrating the microenvironment. Studies suggest the occurrence of active secretion of cytosolic proteins in liver cancer and their involvement in tumorigenesis. Here, we investigated the identification of extended-synaptotagmin 1 (E-Syt1), an endoplasmic reticulum (ER)-bound protein, as a key mediator for cytosolic protein secretion at the ER-plasma membrane (PM) contact sites. Cytosolic proteins interacted with E-Syt1 on the ER, and then localized spatially inside SEC22B+ vesicles of liver cancer cells. Consequently, SEC22B on the vesicle tethered to the PM via Q-SNAREs (SNAP23, SNX3, and SNX4) for their secretion. Furthermore, inhibiting the interaction of protein kinase Cδ (PKCδ), a liver cancer-specific secretory cytosolic protein, with E-Syt1 by a PKCδ antibody, decreased in both PKCδ secretion and tumorigenicity. Results reveal the role of ER-PM contact sites in cytosolic protein secretion and provide a basis for ER-targeting therapy for liver cancer.

Extracellular PKCδ signals to epidermal growth factor receptor for tumor proliferation in liver cancer cells.

Protein kinase C delta (PKCδ) is a multifunctional PKC family member and has been implicated in many types of cancers, including liver cancer. Recently, we have reported that PKCδ is secreted from liver cancer cells, and involved in cell proliferation and tumor growth. However, it remains unclear whether the extracellular PKCδ directly regulates cell surface growth factor receptors. Here, we identify epidermal growth factor receptor (EGFR) as a novel interacting protein of the cell surface PKCδ in liver cancer cells. Imaging studies showed that secreted PKCδ interacted with EGFR-expressing cells in both autocrine and paracrine manners. Biochemical analysis revealed that PKCδ bound to the extracellular domain of EGFR. We further found that a part of the amino acid sequence on the C-terminal region of PKCδ was similar to the putative EGFR binding site of EGF. In this regard, the point mutant of PKCδ in the binding site lacked the ability to bind to the extracellular domain of EGFR. Upon an extracellular PKCδ-EGFR association, ERK1/2 activation, downstream of EGFR signaling, was apparently induced in liver cancer cells. This study indicates that extracellular PKCδ behaves as a growth factor and provides a molecular basis for extracellular PKCδ-targeting therapy for liver cancer.

DYRK2 maintains genome stability via neddylation of cullins in response to DNA damage.

Neural precursor cell-expressed developmentally down-regulated 8 (NEDD8), an ubiquitin-like protein, is an essential regulator of the DNA damage response. Numerous studies have shown that neddylation (conjugation of NEDD8 to target proteins) dysfunction causes several human diseases, such as cancer. Hence clarifying the regulatory mechanism of neddylation could provide insight into the mechanism of genome stability underlying the DNA damage response (DDR) and carcinogenesis. Here, we demonstrate that dual-specificity tyrosine-regulated kinase 2 (DYRK2) is a novel regulator of neddylation and maintains genome stability. Deletion of DYRK2 leads to persistent DNA double-strand breaks (DSBs) and subsequent genome instability. Mechanistically, DYRK2 promotes neddylation through forming a complex with NAE1, which is a component of NEDD8-activating enzyme E1, and maintaining its protein level by suppressing polyubiquitylation. The present study is the first to demonstrate that DYRK2 controls neddylation and is necessary for maintaining genome stability. This article has an associated First Person interview with the first author of the paper.

Multiple subcellular localizations and functions of protein kinase Cδ in liver cancer.

Protein kinase Cδ (PKCδ) is a member of the PKC family, and its implications have been reported in various biological and cancerous processes, including cell proliferation, cell death, tumor suppression, and tumor progression. In liver cancer cells, accumulating reports show the bi-functional regulation of PKCδ in cell death and survival. PKCδ function is defined by various factors, such as phosphorylation, catalytic domain cleavage, and subcellular localization. PKCδ has multiple intracellular distribution patterns, ranging from the cytosol to the nucleus. We recently found a unique extracellular localization of PKCδ in liver cancer and its growth factor-like function in liver cancer cells. In this review, we first discuss the structural features of PKCδ and then focus on the functional diversity of PKCδ based on its subcellular localization, such as the nucleus, cell surface, and extracellular space. These findings improve our knowledge of PKCδ involvement in the progression of liver cancer.

Recognition of Microbe- and Damage-Associated Molecular Patterns by Leucine-Rich Repeat Pattern Recognition Receptor Kinases Confers Salt Tolerance in Plants.

In plants, a first layer of inducible immunity is conferred by pattern recognition receptors (PRRs) that bind microbe- and damage-associated molecular patterns to activate pattern-triggered immunity (PTI). PTI is strengthened or followed by another potent form of immunity when intracellular receptors recognize pathogen effectors, termed effector-triggered immunity. Immunity signaling regulators have been reported to influence abiotic stress responses as well, yet the governing principles and mechanisms remain ambiguous. Here, we report that PRRs of a leucine-rich repeat ectodomain also confer salt tolerance in Arabidopsis thaliana, following recognition of cognate ligands such as bacterial flagellin (flg22 epitope) and elongation factor Tu (elf18 epitope), and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires authentic PTI signaling components; namely, the PRR-associated kinases BAK1 and BIK1 and the NADPH oxidase RBOHD. Exposure to salt stress induces the release of Pep precursors, pointing to the involvement of the endogenous immunogenic peptides in developing plant tolerance to high salinity. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following a preexposure to immunogenic patterns. In good accordance, plants challenged with nonpathogenic bacteria also acquired salt tolerance in a manner dependent on PRRs. Our findings provide insight into signaling plasticity underlying biotic or abiotic stress cross-tolerance in plants conferred by PRRs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effects of the sliaa9 Mutation on Shoot Elongation Growth of Tomato Cultivars.

Tomato INDOLE-3-ACETIC ACID9 (SlIAA9) is a transcriptional repressor in auxin signal transduction, and SlIAA9 knockout tomato plants develop parthenocarpic fruits without fertilization. We generated sliaa9 mutants with parthenocarpy in several commercial tomato cultivars (Moneymaker, Rio Grande, and Ailsa Craig) using CRISPR-Cas9, and null-segregant lines in the T1 generation were isolated by self-pollination, which was confirmed by PCR and Southern blot analysis. We then estimated shoot growth phenotypes of the mutant plants under different light (low and normal) conditions. The shoot length of sliaa9 plants in Moneymaker and Rio Grande was smaller than those of wild-type cultivars in low light conditions, whereas there was not clear difference between the mutant of Ailsa Craig and the wild-type under both light conditions. Furthermore, young seedlings in Rio Grande exhibited shade avoidance response in hypocotyl growth, in which the hypocotyl lengths were increased in low light conditions, and sliaa9 mutant seedlings of Ailsa Craig exhibited enhanced responses in this phenotype. Fruit production and growth rates were similar among the sliaa9 mutant tomato cultivars. These results suggest that control mechanisms involved in the interaction of AUX/IAA9 and lights condition in elongation growth differ among commercial tomato cultivars.

Unconventional Secretion of PKCδ Exerts Tumorigenic Function via Stimulation of ERK1/2 Signaling in Liver Cancer.

Expression of human protein kinase C delta (PKCδ) protein has been linked to many types of cancers. PKCδ is known to be a multifunctional PKC family member and has been rigorously studied as an intracellular signaling molecule. Here we show that PKCδ is a secretory protein that regulates cell growth of liver cancer. Full-length PKCδ was secreted to the extracellular space in living liver cancer cells under normal cell culture conditions and in xenograft mouse models. Patients with liver cancer showed higher levels of serum PKCδ than patients with chronic hepatitis or liver cirrhosis or healthy individuals. In liver cancer cells, PKCδ secretion was executed in an endoplasmic reticulum (ER)-Golgi-independent manner, and the inactivation status of cytosolic PKCδ was required for its secretion. Furthermore, colocalization studies showed that extracellular PKCδ was anchored on the cell surface of liver cancer cells via association with glypican 3, a liver cancer-related heparan sulfate proteoglycan. Addition of exogenous PKCδ activated IGF-1 receptor (IGF1R) activation and subsequently enhanced activation of ERK1/2, which led to accelerated cell growth in liver cancer cells. Conversely, treatment with anti-PKCδ antibody attenuated activation of both IGF1R and ERK1/2 and reduced cell proliferation and spheroid formation of liver cancer cells and tumor growth in xenograft mouse models. This study demonstrates the presence of PKCδ at the extracellular space and the function of PKCδ as a growth factor and provides a rationale for the extracellular PKCδ-targeting therapy of liver cancer. SIGNIFICANCE: PKCδ secretion from liver cancer cells behaves as a humoral growth factor that contributes to cell growth via activation of proliferative signaling molecules, which may be potential diagnostic or therapeutic targets.

The novel ciliogenesis regulator DYRK2 governs Hedgehog signaling during mouse embryogenesis.

Mammalian Hedgehog (Hh) signaling plays key roles in embryogenesis and uniquely requires primary cilia. Functional analyses of several ciliogenesis-related genes led to the discovery of the developmental diseases known as ciliopathies. Hence, identification of mammalian factors that regulate ciliogenesis can provide insight into the molecular mechanisms of embryogenesis and ciliopathy. Here, we demonstrate that DYRK2 acts as a novel mammalian ciliogenesis-related protein kinase. Loss of Dyrk2 in mice causes suppression of Hh signaling and results in skeletal abnormalities during in vivo embryogenesis. Deletion of Dyrk2 induces abnormal ciliary morphology and trafficking of Hh pathway components. Mechanistically, transcriptome analyses demonstrate down-regulation of Aurka and other disassembly genes following Dyrk2 deletion. Taken together, the present study demonstrates for the first time that DYRK2 controls ciliogenesis and is necessary for Hh signaling during mammalian development.

Impairment of DYRK2 by DNMT1­mediated transcription augments carcinogenesis in human colorectal cancer.

Dual specificity tyrosine­phosphorylation­regulated kinase 2 (DYRK2) is a protein kinase that functions as a novel tumor suppressor. Previous studies have reported that DYRK2 expression is decreased in colorectal cancer compared with adjacent non­tumor tissues. However, the regulatory mechanisms by which the expression of DYRK2 is diminished remain unknown. The aim of the present study was to determine the regulatory mechanisms of DYRK2 expression. The present study identified the promoter regions of the DYRK2 gene and demonstrated that they contained CpG islands in human cancer cells. In addition, the DYRK2 promoter region exhibited a higher level of methylation in colorectal cancer tissues compared with healthy tissues from clinical samples. DYRK2 expression was increased at the mRNA and protein level in colorectal cancer cell lines by treatment with 5­Azacytidine, a demethylating agent. The results further demonstrated that knockdown of DNA methyltransferase (DNMT) 1 elevated DYRK2 expression in colorectal cancer cell lines. A colitis­related mouse carcinogenesis model also exhibited a lower DYRK2 level in colorectal cancer tissues compared with adjacent non­tumor tissues. In this model, nuclear staining of DNMT1 was detected in colorectal cancer cells, whereas a cytoplastic distribution pattern of DNMT1 staining was exhibited in healthy tissue. Overall, these findings suggested that DYRK2 expression was downregulated via transcriptional regulation by DNMT1 to elevate the proliferation of colorectal cancer cells.

Mechanical insights into the regulation of programmed cell death by p53 via mitochondria.

All organisms end with their death, and many parts of cells die through intrinsic suicide machineries in response to diverse stimuli. These intrinsic cell death pathways are often termed as programmed cell deaths (PCDs), and are critical for organism development, tissue homeostasis and various diseases. Recent evidence has revealed that most of PCDs involve a tumor suppressor p53 and components of the intra-mitochondria. Furthermore, the movement and positioning of p53 in cells affect the induction of each PCD pathway. Here we provide a comprehensive review on p53-related PCD mechanisms via the mitochondria, namely classical apoptosis, non-classical apoptosis, autophagic cell death, ferroptosis, necroptosis. In addition, we discuss the roles of p53 in each PCD pathway by focusing its altered intracellular localization in response to diverse cellular stresses.

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.

Plant pathogens have optimized their own effector sets to adapt to their hosts. However, certain effectors, regarded as core effectors, are conserved among various pathogens, and may therefore play an important and common role in pathogen virulence. We report here that the widely distributed fungal effector NIS1 targets host immune components that transmit signaling from pattern recognition receptors (PRRs) in plants. NIS1 from two Colletotrichum spp. suppressed the hypersensitive response and oxidative burst, both of which are induced by pathogen-derived molecules, in Nicotiana benthamianaMagnaporthe oryzae NIS1 also suppressed the two defense responses, although this pathogen likely acquired the NIS1 gene via horizontal transfer from Basidiomycota. Interestingly, the root endophyte Colletotrichum tofieldiae also possesses a NIS1 homolog that can suppress the oxidative burst in N. benthamiana We show that NIS1 of multiple pathogens commonly interacts with the PRR-associated kinases BAK1 and BIK1, thereby inhibiting their kinase activities and the BIK1-NADPH oxidase interaction. Furthermore, mutations in the NIS1-targeting proteins, i.e., BAK1 and BIK1, in Arabidopsis thaliana also resulted in reduced immunity to Colletotrichum fungi. Finally, M. oryzae lacking NIS1 displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.

Sugar compartmentation as an environmental stress adaptation strategy in plants.

The sessile nature of plants has driven their evolution to cope flexibly with ever-changing surrounding environments. The development of stress tolerance traits is complex, and a broad range of cellular processes are involved. Recent studies have revealed that sugar transporters contribute to environmental stress tolerance in plants, suggesting that sugar flow is dynamically fluctuated towards optimization of cellular conditions in adverse environments. Here, we highlight sugar compartmentation mediated by sugar transporters as an adaptation strategy against biotic and abiotic stresses. Competition for sugars between host plants and pathogens shapes their evolutionary arms race. Pathogens, which rely on host-derived carbon, manipulate plant sugar transporters to access sugars easily, while plants sequester sugars from pathogens by enhancing sugar uptake activity. Furthermore, we discuss pathogen tactics to circumvent sugar competition with host plants. Sugar transporters also play a role in abiotic stress tolerance. Exposure to abiotic stresses such as cold or drought stress induces sugar accumulation in various plants. We also discuss how plants allocate sugars under such conditions. Collectively, these findings are relevant to basic plant biology as well as potential applications in agriculture, and provide opportunities to improve crop yield for a growing population.