NAMPT-Driven M2 Polarization of Tumor-Associated Macrophages Leads to an Immunosuppressive Microenvironment in Colorectal Cancer.

Nicotinamide phosphoribosyltransferase (NAMPT) is a metabolic enzyme with key roles in inflammation. Previous studies have examined the consequences of its upregulated expression in cancer cells themselves, but studies are limited with respect to its role in the other cells within the tumor microenvironment (TME) during colorectal cancer (CRC) progression. Using single-cell RNA sequencing (scRNA-seq) data, it is founded that NAMPT is highly expressed in SPP1+ tumor-associated macrophages (TAMs), a unique subset of TAMs associated with immunosuppressive activity. A NAMPThigh gene signature in SPP1+ TAMs correlated with worse prognostic outcomes in CRC patients. The effect of Nampt deletion in the myeloid compartment of mice during CRC development is explored. NAMPT deficiency in macrophages resulted in HIF-1α destabilization, leading to reduction in M2-like TAM polarization. NAMPT deficiency caused significant decreases in the efferocytosis activity of macrophages, which enhanced STING signaling and the induction of type I IFN-response genes. Expression of these genes contributed to anti-tumoral immunity via potentiation of cytotoxic T cell activity in the TME. Overall, these findings suggest that NAMPT-initiated TAM-specific genes can be useful in predicting poor CRC patient outcomes; strategies aimed at targeting NAMPT may provide a promising therapeutic approach for building an immunostimulatory TME in CRC progression.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

NAMPT mitigates colitis severity by supporting redox-sensitive activation of phagocytosis in inflammatory macrophages.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway and plays a crucial role in the maintenance of the NAD+ pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment (Namptf/fLysMCre+/-, Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt-deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD+ abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD+, effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD+ biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases.

RIPK3 activation induces TRIM28 derepression in cancer cells and enhances the anti-tumor microenvironment.

BACKGROUND: Necroptosis is emerging as a new target for cancer immunotherapy as it is now recognized as a form of cell death that increases tumor immunogenicity, which would be especially helpful in treating immune-desert tumors. De novo synthesis of inflammatory proteins during necroptosis appears especially important in facilitating increased anti-tumor immune responses. While late-stage transcription mediated by NF-κB during cell death is believed to play a role in this process, it is otherwise unclear what cell signaling events initiate this transactivation of inflammatory genes. METHODS: We employed tandem-affinity purification linked to mass spectrometry (TAP-MS), in combination with the analysis of RNA-sequencing (RNA-Seq) datasets to identify the Tripartite Motif Protein 28 (TRIM28) as a candidate co-repressor. Comprehensive biochemical and molecular biology techniques were used to characterize the role of TRIM28 in RIPK3 activation-induced transcriptional and immunomodulatory events. The cell composition estimation module was used to evaluate the correlation between RIPK3/TRIM28 levels and CD8+ T cells or dendritic cells (DC) in all TCGA tumors. RESULTS: We identified TRIM28 as a co-repressor that regulates transcriptional activity during necroptosis. Activated RIPK3 phosphorylates TRIM28 on serine 473, inhibiting its chromatin binding activity, thereby contributing to the transactivation of NF-κB and other transcription factors, such as SOX9. This leads to elevated cytokine expression, which then potentiates immunoregulatory processes, such as DC maturation. The expression of RIPK3 has a significant positive association with the tumor-infiltrating immune cells populations in various tumor type, thereby activating anti-cancer responses. CONCLUSION: Our data suggest that RIPK3 activation-dependent derepression of TRIM28 in cancer cells leads to increased immunostimulatory cytokine production in the tumor microenvironment, which then contributes to robust cytotoxic anti-tumor immunity.

Global spliceosome activity regulates entry into cellular senescence.

Cellular senescence is a state of permanent growth arrest that can ultimately contribute to aging. Senescence can be induced by various stressors and is associated with a myriad of cellular functions and phenotypic markers. Alternative splicing is emerging as a critical contributor to senescence and aging. However, it is unclear how the composition and function of the spliceosome are involved in senescence. Here, using replicative and oxidative stress-induced senescence models in primary human fibroblasts, we report a common shift in the expression of 58 spliceosomal genes at the pre-senescence stage, prior to the detection of senescence-associated ß-galactosidase (SA-ß-gal) activity. Spliceosomal perturbation, induced by pharmacologic and genetic inhibition of splicesomal genes, triggered cells to enter senescence, suggesting a key role as a gatekeeper. Association analysis of transcription factors based on the 58 splicesomal genes revealed Sp1 as a key regulator of senescence entry. Indeed, Sp1 depletion suppressed the expression of downstream spliceosomal genes (HNRNPA3, SRSF7, and SRSF4) and effectively induced senescence. These results indicate that spliceosomal gene sets, rather than a single spliceosomal gene, regulate the early transition into senescence prior to SA-ß-gal expression. Furthermore, our study provides a spliceosome signature that may be used as an early senescence marker.

Reduction in MLKL-mediated endosomal trafficking enhances the TRAIL-DR4/5 signal to increase cancer cell death.

Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.

Mitochondrial Respiratory Defect Enhances Hepatoma Cell Invasiveness via STAT3/NFE2L1/STX12 Axis.

Mitochondrial respiratory defects have been implicated in cancer progression and metastasis, but how they control tumor cell aggressiveness remains unclear. Here, we demonstrate that a mitochondrial respiratory defect induces nuclear factor-erythroid 2 like 1 (NFE2L1) expression at the transcriptional level via reactive oxygen species (ROS)-mediated STAT3 activation. We identified syntaxin 12 (STX12) as an effective downstream target of NFE2L1 by performing cDNA microarray analysis after the overexpression and depletion of NFE2L1 in hepatoma cells. Bioinformatics analysis of The Cancer Genome Atlas Liver Hepatocellular carcinoma (TCGA-LIHC) open database (n = 371) also revealed a significant positive association (r = 0.3, p = 2.49 × 10-9) between NFE2L1 and STX12 expression. We further demonstrated that STX12 is upregulated through the ROS/STAT3/NFE2L1 axis and is a key downstream effector of NFE2L1 in modulating hepatoma cell invasiveness. In addition, gene enrichment analysis of TCGA-LIHC also showed that epithelial-mesenchymal transition (EMT)-related core genes are significantly upregulated in tumors co-expressing NFE2L1 and STX12. The positive association between NFE2L1 and STX12 expression was validated by immunohistochemistry of the hepatocellular carcinoma tissue array. Finally, higher EMT gene enrichment and worse overall survival (p = 0.043) were observed in the NFE2L1 and STX12 co-expression group with mitochondrial defect, as indicated by low NDUFA9 expression. Collectively, our results indicate that NFE2L1 is a key mitochondrial retrograde signaling-mediated primary gene product enhancing hepatoma cell invasiveness via STX12 expression and promoting liver cancer progression.

Lactic acidosis caused by repressed lactate dehydrogenase subunit B expression down-regulates mitochondrial oxidative phosphorylation via the pyruvate dehydrogenase (PDH)-PDH kinase axis.

Aerobic glycolysis and mitochondrial dysfunction are key metabolic features of cancer cells, but their interplay during cancer development remains unclear. We previously reported that human hepatoma cells with mitochondrial defects exhibit down-regulated lactate dehydrogenase subunit B (LDHB) expression. Here, using several molecular and biochemical assays and informatics analyses, we investigated how LDHB suppression regulates mitochondrial respiratory activity and contributes to liver cancer progression. We found that transcriptional LDHB down-regulation is an upstream event during suppressed oxidative phosphorylation. We also observed that LDHB knockdown increases inhibitory phosphorylation of pyruvate dehydrogenase (PDH) via lactate-mediated PDH kinase (PDK) activation and thereby attenuates oxidative phosphorylation activity. Interestingly, monocarboxylate transporter 1 was the major lactate transporter in hepatoma cells, and its expression was essential for PDH phosphorylation by modulating intracellular lactate levels. Finally, bioinformatics analysis of the hepatocellular carcinoma cohort from The Cancer Genome Atlas revealed that a low LDHB/LDHA ratio is statistically significantly associated with poor prognostic outcomes. A low ratio was also associated with a significant enrichment in glycolysis genes and negatively correlated with PDK1 and 2 expression, supporting a close link between LDHB suppression and the PDK-PDH axis. These results suggest that LDHB suppression is a key mechanism that enhances glycolysis and is critically involved in the maintenance and propagation of mitochondrial dysfunction via lactate release in liver cancer progression.

Metabolic features and regulation in cell senescence.

Organismal aging is accompanied by a host of progressive metabolic alterations and an accumulation of senescent cells, along with functional decline and the appearance of multiple diseases. This implies that the metabolic features of cell senescence may contribute to the organism's metabolic changes and be closely linked to age-associated diseases, especially metabolic syndromes. However, there is no clear understanding of senescent metabolic characteristics. Here, we review key metabolic features and regulators of cellular senescence, focusing on mitochondrial dysfunction and anabolic deregulation, and their link to other senescence phenotypes and aging. We further discuss the mechanistic involvement of the metabolic regulators mTOR, AMPK, and GSK3, proposing them as key metabolic switches for modulating senescence. [BMB Reports 2019; 52(1): 5-12].

NNMT depletion contributes to liver cancer cell survival by enhancing autophagy under nutrient starvation.

Nicotinamide N-methyl transferase (NNMT) transfers a methyl group from S-adenosyl-L-methionine (SAM) to nicotinamide (NAM), producing 1-methylnicotinamide (1MNA). NNMT has been implicated in several cancer types and recently in metabolism, but its role in autophagy regulation has not yet been investigated. In this study, we determined that NNMT negatively regulated autophagy at the stage of ULK1 activation through protein phosphatase 2A (PP2A) activity. Specifically, NNMT knockdown increased PP2A methylation and subsequently enhanced phosphatase activity. Consequent p-ULK1 (S638) dephosphorylation derepressed ULK1 activity, resulting in autophagy induction. Accordingly, NNMT downregulation rescued tumor cells under nutrient deficiency in vivo, which was alleviated by ULK1 inhibitor treatment. In summary, our results suggest a novel mechanism by which tumor cells protect themselves against nutrient deprivation through NNMT suppression to accelerate autophagy.

Identification of Caveolin-1 as an Invasion-Associated Gene in Liver Cancer Cells Using Dendron-Coated DNA Microarrays.

The conventional gene expression profiling approaches have been replaced with DNA microarrays with exhibiting a powerful high-throughput capacity. Most solid surfaces of DNA microarrays contain such a high area density of functional groups to immobilize capture DNAs to the surface that the hybridization of capture DNAs with cDNA can be hindered, resulting in low intensity and reproducibility. Since our previous works showed that the 9-acid dendron was able to increase the hybridization efficiency, we aimed to demonstrate the feasibility of 9-acid dendron-coated glass slides as an advanced microarray platform for gene expression profiling. The 9-acid dendron-coated DNA microarray could reproducibly obtain the expression levels of 2800 human cancer-associated genes in the two liver cancer lines: Hep3B and SK-Hep1. Among the differentially expressed genes, Caveolin-1 (Cav-1) was identified as the most highly up-regulated gene in invasive SK-Hep1 in comparison to non-motile Hep3B. The overexpression of Cav-1 in Hep3B promoted the cell invasion, whereas its knockdown in SK-Hep1 suppressed the invasive feature, which confirms that the overexpression of Cav-1 is closely associated with cell invasion of liver carcinoma. Collectively, the 9-acid dendron-coated surface could successfully detect the transcript levels of cells, demonstrating its feasible potential to identify the candidate genes for further functional studies or diagnosis of diseases.

BNIP3 is degraded by ULK1-dependent autophagy via MTORC1 and AMPK.

BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) is an atypical BH3-only protein that is induced by hypoxia-inducible factor 1 (HIF1) under hypoxia. BNIP3 is primarily regulated at the transcriptional level. However, little is known about the underlying mechanism of BNIP3 degradation. In this study, we found that BNIP3 was downregulated when hypoxia was accompanied by amino acid starvation. The BNIP3 downregulation did not occur at the transcription level and was independent of HIF1A. BNIP3 was primarily degraded by the proteasome, but BNIP3 was subjected to both proteasomal and autophagic degradation in response to starvation. The autophagic degradation of BNIP3 was dependent on ATG7 and MAP1LC3. We determined that autophagic degradation of BNIP3 was specifically regulated by ULK1 via the MTOR-AMPK pathway. Moreover, we confirmed that BNIP3 could play a protective role in tumor cells under hypoxia, and the treatment with Torin1, an MTOR inhibitor, decreased the BNIP3 level and enhanced the death of hypoxic tumor cells.

REST-dependent expression of TRF2 renders non-neuronal cancer cells resistant to DNA damage during oxidative stress.

REST is a neuronal gene silencing factor ubiquitously expressed in non-neuronal tissues. REST is additionally believed to serve as a tumor suppressor in non-neuronal cancers. Conversely, recent findings on REST-dependent tumorigenesis in non-neuronal cells consistently suggest a potential role of REST as a tumor promoter. Here, we have uncovered for the first time the mechanism by which REST contributes to cancer cell survival in non-neuronal cancers. We observed abundant expression of REST in various types of non-neuronal cancer cells compared to normal tissues. The delicate roles of REST were further evaluated in HCT116 and HeLa, non-neuronal cancer cell lines expressing REST. REST silencing resulted in decreased cell survival and activation of the DNA damage response (DDR) through a decrease in the level of TRF2, a telomere-binding protein. These responses were correlated with reduced colony formation ability and accelerated telomere shortening in cancer cells upon the stable knockdown of REST. Interestingly, REST was down-regulated under oxidative stress conditions via ubiquitin proteasome system, suggesting that sustainability of REST expression is critical to determine cell survival during oxidative stress in a tumor microenvironment. Our results collectively indicate that REST-dependent TRF2 expression renders cancer cells resistant to DNA damage during oxidative stress, and mechanisms to overcome oxidative stress, such as high levels of REST or the stress-resistant REST mutants found in specific human cancers, may account for REST-dependent tumorigenesis.

Rapamycin inhibits both motility through down-regulation of p-STAT3 (S727) by disrupting the mTORC2 assembly and peritoneal dissemination in sarcomatoid cholangiocarcinoma.

Cholangiocarcinoma (CC) is a malignant epithelium neoplasm that originates from the bile epithelium and for which there are few therapeutic strategies. The mTOR pathway involved in many cellular processes was reported to be up-regulated in various cancers. We investigated the activation of the AKT/mTOR pathway in CC cell lines with different degrees of dedifferentiation and found that rapamycin could suppress the motility and the peritoneal dissemination of sarcomatoid SCK cells. Inhibition of the mTOR pathway with rapamycin decreased significantly the number of tumor nodules and prolonged the survival rates of nude mice inoculated with sarcomatoid CC cells. Prolonged treatments with rapamycin were found to disrupt the mTORC2 assembly and to reduce the phosphorylation of STAT3 at Ser 727. Rapamycin decreased both mRNA and protein levels of MMP2 and Twist1, which are regulated by STAT3 and associated with cancer metastasis. The overexpression of STAT3 S727A lacking the phosphorylation site resulted in significantly less sensitivity to rapamycin than the overexpression of STAT3 WT. Taken together, our results suggest that rapamycin could suppress the motility of sarcomatoid CC by down-regulating p-STAT3 (S727) through the impairment of mTORC2 assembly.

Enhanced percutaneous delivery of recombinant human epidermal growth factor employing nano-liposome system.

We encapsulated recombinant human epidermal growth factor (rhEGF) into nano-liposomes (NLs) system for topical delivery. The rhEGF-loaded NLs were prepared using a high pressure homogenization method. Morphology and overall particle distribution of NLs were investigated using transmission electron microscopy (TEM) and high resolution microscope (CytoViva™). Particle size, zeta (ζ) potential and encapsulation efficiency were measured and the percutaneous delivery of NLs was evaluated using Franz diffusion cells and immunofluorescence confocal laser scanning microscopy (CLSM). The mean particle size, zeta potential and encapsulation efficiency of the NLs were 155.57 ± 2.59 nm, -57.92 ± 4.35 mV and 9.00 ± 0.39%, respectively. TEM and microscopic analysis showed spherical, very even-sized vesicles approximately 150 nm. The skin permeation and localization of rhEGF were enhanced by NLs. CLSM image analysis provided that the NLs enhanced the permeation and localization of rhEGF in rat skin by facilitating entry through pores of skin.

Overexpression of renal tumor antigen is associated with tumor invasion and poor prognosis of hepatocellular carcinoma.

PURPOSE: The aim of this study was to investigate the roles of renal tumor antigen (RAGE) in the progression and clinical outcome of hepatocellular carcinoma (HCC). METHODS: RAGE mRNA levels in 350 cases of HCC were investigated by quantitative real-time reverse transcription polymerase chain reaction. We analyzed the relationship of RAGE mRNA level with clinicopathologic parameters and clinical outcome. To identify the possible role of RAGE on cellular invasion, we performed in vitro analyses using small interfering RNAs (siRNAs). RESULTS: RAGE mRNA level was significantly higher in HCC than in noncancerous hepatic tissues (P < 0.001). Overexpression of RAGE was significantly correlated with the presence of multiple tumors (P = 0.021), high alfa-fetoprotein level (P = 0.042), and advanced tumor stage (P = 0.016). Higher levels of RAGE expression were associated with significantly shorter overall survival time (P = 0.029). Knockdown of RAGE expression by siRNAs suppressed the invasive ability of HCC cells and the expression and secretion of matrix metalloproteinase-9 (MMP-9). We found that RAGE and MMP-9 expressions were correlated in HCCs, and furthermore, the combination of RAGE and MMP-9 expression was associated with the survival of patients (P = 0.0066). CONCLUSIONS: Our results suggest that RAGE may be important in tumor invasion and could be a potential predictor for the prognosis of HCC patients.

Cultured cambial meristematic cells as a source of plant natural products.

A plethora of important, chemically diverse natural products are derived from plants. In principle, plant cell culture offers an attractive option for producing many of these compounds. However, it is often not commercially viable because of difficulties associated with culturing dedifferentiated plant cells (DDCs) on an industrial scale. To bypass the dedifferentiation step, we isolated and cultured innately undifferentiated cambial meristematic cells (CMCs). Using a combination of deep sequencing technologies, we identified marker genes and transcriptional programs consistent with a stem cell identity. This notion was further supported by the morphology of CMCs, their hypersensitivity to γ-irradiation and radiomimetic drugs and their ability to differentiate at high frequency. Suspension culture of CMCs derived from Taxus cuspidata, the source of the key anticancer drug, paclitaxel (Taxol), circumvented obstacles routinely associated with the commercial growth of DDCs. These cells may provide a cost-effective and environmentally friendly platform for sustainable production of a variety of important plant natural products.

Overexpression of high-mobility group box 2 is associated with tumor aggressiveness and prognosis of hepatocellular carcinoma.

PURPOSE: We investigated the expression of high-mobility group box 2 (HMGB2) in patients with hepatocellular carcinoma (HCC) and its clinical effects with underlying mechanisms. EXPERIMENTAL DESIGN: HMGB2 mRNA levels were measured in 334 HCC patients by real-time reverse transcription-PCR and HMGB2 protein levels in 173 HCC patients by immunohistochemical studies. The HMGB2 expression level was measured by Western blotting for three HCC cell lines. To clarify the precise role of HMGB2 on cell proliferation, we did in vitro analysis with expression vectors and small interfering RNAs. RESULTS: HMGB2 mRNA and protein expression were significantly higher in HCC than in noncancerous surrounding tissues (P < 0.0001) and showed a positive correlation (ρ = 0.35, P < 0.001). HMGB2 overexpression was significantly correlated with shorter overall survival time, both at mRNA (P = 0.0054) and protein level (P = 0.023). Moreover, HMGB2 mRNA level was an independent prognostic factor for overall survival in a multivariate analysis (P = 0.0037). HMGB2 knockdown by small interfering RNAs decreased cell proliferation, and overexpression of HMGB2 by expression vectors diminished cisplatin- and etoposide-induced cell death. CONCLUSIONS: Our clinical and in vitro data suggest that HMGB2 plays a significant role in tumor development and prognosis of HCC. These results can partly be explained by altered cell proliferations by HMGB2 associated with the antiapoptotic pathway.