Poorly Differentiated Hepatocellular Carcinoma Cells Avoid Apoptosis by Interacting with T Cells via CD40-CD40 Ligand Linkage.

Hepatocellular carcinoma (HCC) is associated with increased soluble CD40 levels. This study aimed to investigate CD40's role in liver tumor progression. CD40 levels were examined in HCC patient tissues and various HCC cell lines, and their interaction with CD4+T cells was studied. RNA sequencing analysis was performed to explore the mechanisms of CD40 induction. Poorly differentiated HCC tumor tissues exhibited high membrane-bound CD40 expression, in contrast to nontumor areas. Poorly differentiated HCC cell lines showed high expression of membrane-bound CD40 with low CD40 promoter methylation, which was the opposite of that observed in the well-differentiated HCC cell lines. Solely modulating CD40 expression in HCC cells exerted no direct consequences on cell growth or appearance. Interestingly, the human hepatoma cell line HLF co-cultured with activated (CD40 ligand+) CD4+ T cells had increased CD40 levels and a modest 3.2% dead cells. The percentage of dead cells increased to 10.9% and underwent preneutralizing CD40 condition, whereas preblocking both CD40 and integrin α5ß1 concomitantly caused only 1.9% cell death. RNA sequencing of co-cultured HLFs with activated CD4+ T cells revealed the up-regulation of interferon and immune-response pathways. Increased interferon-γ levels in the activated T-cell media stimulated the Janus kinase/signal transducer and activator of transcription 3 pathway, resulting in increased CD40 expression in HLF. Collectively, CD40 expression in poorly differentiated HCC cells prevented cell death by interacting with CD40 ligand in activated T cells. Targeting CD40 may represent a promising anticancer therapy.

Oral Microbial Profile Analysis in Patients with Oral and Pharyngeal Cancer Reveals That Tumoral Fusobacterium nucleatum Promotes Oral Cancer Progression by Activating YAP.

The incidence of oral cancer has recently been increasing worldwide, particularly among young individuals and women. The primary risk factors for head and neck cancers, including oral and pharyngeal cancers, are smoking, alcohol consumption, poor oral hygiene, and repeated exposure to mechanical stimuli. However, approximately one-third of the patients with oral and pharyngeal cancers are neither smokers nor drinkers, which points to the existence of other mechanisms. Recently, human microbes have been linked to various diseases, including cancer. Oral pathogens, especially periodontal pathobionts, are reported to play a role in the development of colon and other types of cancer. In this study, we employed a series of bioinformatics analyses to pinpoint Fusobacterium nucleatum as the predominant oral bacterial species in oral and pharyngeal cancer tissue samples. We successfully isolated Fn. polymorphum from the saliva of patients with oral cancer and demonstrated that Fn. polymorphum indeed promoted oral squamous cell carcinoma development by activating YAP in a mouse tongue cancer model. Our research offers scientific evidence for the role of the oral microbiome in oral cancer progression and provides insights that would help in devising preventative strategies against oral cancer, potentially by altering oral bacterial profiles.

Recent updates on the role of the gut-liver axis in the pathogenesis of NAFLD/NASH, HCC, and beyond.

The gut and the liver are anatomically and physiologically connected, and this connection is called the "gut-liver axis," which exerts various influences on liver physiology and pathology. The gut microbiota has been recognized to trigger innate immunity and modulate the liver immune microenvironment. Gut microbiota influences the physiological processes in the host, such as metabolism, by acting on various signaling receptors and transcription factors through their metabolites and related molecules. The gut microbiota has also been increasingly recognized to modulate the efficacy of immune checkpoint inhibitors. In this review, we discuss recent updates on gut microbiota-associated mechanisms in the pathogenesis of chronic liver diseases such as NAFLD and NASH, as well as liver cancer, in light of the gut-liver axis. We particularly focus on gut microbial metabolites and components that are associated with these liver diseases. We also discuss the role of gut microbiota in modulating the response to immunotherapy in liver diseases.

The role of cellular senescence and SASP in tumour microenvironment.

Cellular senescence refers to a state of irreversible cell cycle arrest that can be induced by various cellular stresses and is known to play a pivotal role in tumour suppression. While senescence-associated growth arrest can inhibit the proliferation of cancer-prone cells, the altered secretory profile of senescent cells, termed the senescence-associated secretory phenotype, can contribute to the microenvironment that promotes tumour development. Although the senescence-associated secretory phenotype and its effects on tumorigenesis are both highly context dependent, mechanisms underlying such diversity are becoming better understood, thereby allowing the creation of new strategies to effectively target the senescence-associated secretory phenotype and senescent cells for cancer therapy. In this review, we discuss the current knowledge on cellular senescence and the senescence-associated secretory phenotype to develop a structural understanding of their roles in the tumour microenvironment and provide perspectives for future research, including the possibility of senotherapy for the treatment of cancer.

Cancer cells produce liver metastasis via gap formation in sinusoidal endothelial cells through proinflammatory paracrine mechanisms.

Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver.

Soluble Immune Checkpoint Protein CD27 Is a Novel Prognostic Biomarker of Hepatocellular Carcinoma Development in Hepatitis C Virus-Sustained Virological Response Patients.

Factors affecting the probability of hepatocellular carcinoma (HCC) development even after sustained virological response (SVR) following anti-hepatitis C virus (HCV) therapy remain unelucidated. This study characterized the role of 16 soluble (s) immune checkpoint proteins in 168 HCV-SVR patients, with 47 developing HCC at the study end point. At baseline, high concentrations of 10 immune checkpoint proteins were found in the sera of the HCC group. At the study end point, levels of sCD27, sCD28, sCD40, and sCD86 in the HCC group, which were depleted following SVR, returned to higher levels than those in the non-HCC group. More importantly, patients with baseline levels of sCD27 ≥ 4104 pg/mL, sCD28 ≥ 1530 pg/mL, and sCD40 ≥ 688 pg/mL predicted a significantly greater HCC cumulative rate. Although sCD27 was elevated in patient sera, its membrane-bound form, mCD27, accumulated in the tumor and peritumor area, mainly localized in T cells. Interestingly, T-cell activation time dependently induced sCD27. Furthermore, CD70, the ligand of CD27, was robustly expressed in HCC area in which CD70 promoter methylation analysis indicated the hypomethylation compared with the nontumor pairs. Recombinant human CD27 treatment induced the proliferation of CD70-bearing HepG2 cells via the mitogen-activated protein kinase (MEK)-extracellular signal-regulated kinase pathway, but not NF-κB or p38 pathway. In conclusion, these data indicate that baseline sCD27, sCD28, and sCD40 levels could be used as HCC prognostic markers in HCV-SVR patients. sCD27 likely promotes HepG2 cell growth via the CD27-CD70 axis.

Gasdermin D-mediated release of IL-33 from senescent hepatic stellate cells promotes obesity-associated hepatocellular carcinoma.

Long-term senescent cells exhibit a secretome termed the senescence-associated secretory phenotype (SASP). Although the mechanisms of SASP factor induction have been intensively studied, the release mechanism and how SASP factors influence tumorigenesis in the biological context remain unclear. In this study, using a mouse model of obesity-induced hepatocellular carcinoma (HCC), we identified the release mechanism of SASP factors, which include interleukin-1ß (IL-1ß)- and IL-1ß-dependent IL-33, from senescent hepatic stellate cells (HSCs) via gasdermin D (GSDMD) amino-terminal-mediated pore. We found that IL-33 was highly induced in senescent HSCs in an IL-1ß-dependent manner in the tumor microenvironment. The release of both IL-33 and IL-1ß was triggered by lipoteichoic acid (LTA), a cell wall component of gut microbiota that was transferred and accumulated in the liver tissue of high-fat diet-fed mice, and the release of these factors was mediated through cell membrane pores formed by the GSDMD amino terminus, which was cleaved by LTA-induced caspase-11. We demonstrated that IL-33 release from HSCs promoted HCC development via the activation of ST2-positive Treg cells in the liver tumor microenvironment. The accumulation of GSDMD amino terminus was also detected in HSCs from human NASH-associated HCC patients, suggesting that similar mechanism could be involved in a certain type of human HCC. These results uncover a release mechanism for SASP factors from sensitized senescent HSCs in the tumor microenvironment, thereby facilitating obesity-associated HCC progression. Furthermore, our findings highlight the therapeutic potential of inhibitors of GSDMD-mediated pore formation for HCC treatment.

Cellular senescence and the tumour microenvironment.

The senescence-associated secretory phenotype (SASP), where senescent cells produce a variety of secreted proteins including inflammatory cytokines, chemokines, matrix remodelling factors, growth factors and so on, plays pivotal but varying roles in the tumour microenvironment. The effects of SASP on the surrounding microenvironment depend on the cell type and process of cellular senescence induction, which is often associated with innate immunity. Via SASP-mediated paracrine effects, senescent cells can remodel the surrounding tissues by modulating the character of adjacent cells, such as stromal, immune cells, as well as cancer cells. The SASP is associated with both tumour-suppressive and tumour-promoting effects, as observed in senescence surveillance effects (tumour-suppressive) and suppression of anti-tumour immunity in most senescent cancer-associated fibroblasts and senescent T cells (tumour-promoting). In this review, we discuss the features and roles of senescent cells in tumour microenvironment with emphasis on their context-dependency that determines whether they promote or suppress cancer development. Potential usage of recently developed drugs that suppress the SASP (senomorphics) or selectively kill senescence cells (senolytics) in cancer therapy are also discussed.

The role of immune cells in the liver tumor microenvironment: an involvement of gut microbiota-derived factors.

More than 500 species of microbiota reside in the human intestine and coexist with humans, their host. Gut microbial metabolites and components are absorbed from the intestine and influence cells in the liver, including hepatocytes and stromal cells, such as liver sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, natural killer (NK) cells, NK T cells and other immune cells. This gut-originated axis to the liver is called the "gut-liver axis", which underscores the importance of the link between the gut and the liver. In this review, we discuss the gut microbial components and metabolites that affect cells in the liver, particularly in association with immune cells, and the related responses. We also highlight the mechanisms underlying gut microbiota-mediated liver carcinogenesis and discuss cancer prevention, including the recently clarified modulation of immune checkpoint inhibitor efficacy by the gut microbiota.

Cytoglobin attenuates pancreatic cancer growth via scavenging reactive oxygen species.

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

The roles and mechanisms of senescence-associated secretory phenotype (SASP): can it be controlled by senolysis?

Cellular senescence is a state of irreversible cell cycle arrest that can be induced by a variety of potentially oncogenic stimuli, including DNA damage. Hence, senescence has long been considered to suppress tumorigenesis, acting as a guardian of homeostasis. However, recent studies have revealed that senescent cells exhibit the secretion of a series of inflammatory cytokines, chemokines, growth factors, and matrix remodeling factors that alter the local tissue environment and contribute to chronic inflammation and cancer. This senescence phenotype is termed as senescence-associated secretory phenotype (SASP) and is observed not only in cultured cells in vitro but also in vivo. Recently, the physiological and pathological roles of SASP have been increasingly clarified. Notably, several studies have reported that the intrinsic mechanism of SASP factor production is predominantly mediated through the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway by aberrantly accumulated DNA fragments from the nucleus of senescent cells. In contrast, various extrinsic triggers of SASP also exist in vivo, for example, the SASP induction in hepatic stellate cells in the tumor microenvironment of obesity-associated liver cancer by the translocated gut microbial metabolites. Recently, the strategy for the elimination of senescent cells (senolysis) has attracted increasing attention. Thus, the role of SASP and the effects and outcomes of senolysis in vivo will be also discussed in this review.

Development and evaluation of a colorectal cancer screening method using machine learning-based gut microbiota analysis.

Accumulating evidence indicates that alterations of gut microbiota are associated with colorectal cancer (CRC). Therefore, the use of gut microbiota for the diagnosis of CRC has received attention. Recently, several studies have been conducted to detect the differences in the gut microbiota between healthy individuals and CRC patients using machine learning-based gut bacterial DNA meta-sequencing analysis, and to use this information for the development of CRC diagnostic model. However, to date, most studies had small sample sizes and/or only cross-validated using the training dataset that was used to create the diagnostic model, rather than validated using an independent test dataset. Since machine learning-based diagnostic models cause overfitting if the sample size is small and/or an independent test dataset is not used for validation, the reliability of these diagnostic models needs to be interpreted with caution. To circumvent these problems, here we have established a new machine learning-based CRC diagnostic model using the gut microbiota as an indicator. Validation using independent test datasets showed that the true positive rate of our CRC diagnostic model increased substantially as CRC progressed from Stage I to more than 60% for CRC patients more advanced than Stage II when the false positive rate was set around 8%. Moreover, there was no statistically significant difference in the true positive rate between samples collected in different cities or in any part of the colorectum. These results reveal the possibility of the practical application of gut microbiota-based CRC screening tests.

Galectin-3 promotes the adipogenic differentiation of PDGFRα+ cells and ectopic fat formation in regenerating muscle.

Worldwide prevalence of obesity is associated with the increase of lifestyle-related diseases. The accumulation of intermuscular adipose tissue (IMAT) is considered a major problem whereby obesity leads to sarcopenia and metabolic disorders and thus is a promising target for treating these pathological conditions. However, whereas obesity-associated IMAT is suggested to originate from PDGFRα+ mesenchymal progenitors, the processes underlying this adipogenesis remain largely unexplored. Here, we comprehensively investigated intra- and extracellular changes associated with these processes using single-cell RNA sequencing and mass spectrometry. Our single-cell RNA sequencing analysis identified a small PDGFRα+ cell population in obese mice directed strongly toward adipogenesis. Proteomic analysis showed that the appearance of this cell population is accompanied by an increase in galectin-3 in interstitial environments, which was found to activate adipogenic PPARγ signals in PDGFRα+ cells. Moreover, IMAT formation during muscle regeneration was significantly suppressed in galectin-3 knockout mice. Our findings, together with these multi-omics datasets, could unravel microenvironmental networks during muscle regeneration highlighting possible therapeutic targets against IMAT formation in obesity.

Pharmacological CDK4/6 inhibition reveals a p53-dependent senescent state with restricted toxicity.

Cellular senescence is a state of stable growth arrest and a desired outcome of tumor suppressive interventions. Treatment with many anti-cancer drugs can cause premature senescence of non-malignant cells. These therapy-induced senescent cells can have pro-tumorigenic and pro-disease functions via activation of an inflammatory secretory phenotype (SASP). Inhibitors of cyclin-dependent kinases 4/6 (CDK4/6i) have recently proven to restrain tumor growth by activating a senescence-like program in cancer cells. However, the physiological consequence of exposing the whole organism to pharmacological CDK4/6i remains poorly characterized. Here, we show that exposure to CDK4/6i induces non-malignant cells to enter a premature state of senescence dependent on p53. We observe in mice and breast cancer patients that the CDK4/6i-induced senescent program activates only a partial SASP enriched in p53 targets but lacking pro-inflammatory and NF-κB-driven components. We find that CDK4/6i-induced senescent cells do not acquire pro-tumorigenic and detrimental properties but retain the ability to promote paracrine senescence and undergo clearance. Our results demonstrate that SASP composition is exquisitely stress-dependent and a predictor for the biological functions of different senescence subsets.

Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate secretion.

Emerging evidence is revealing that alterations in gut microbiota are associated with colorectal cancer (CRC). However, very little is currently known about whether and how gut microbiota alterations are causally associated with CRC development. Here we show that 12 faecal bacterial taxa are enriched in CRC patients in two independent cohort studies. Among them, 2 Porphyromonas species are capable of inducing cellular senescence, an oncogenic stress response, through the secretion of the bacterial metabolite, butyrate. Notably, the invasion of these bacteria is observed in the CRC tissues, coinciding with the elevation of butyrate levels and signs of senescence-associated inflammatory phenotypes. Moreover, although the administration of these bacteria into ApcΔ14/+ mice accelerate the onset of colorectal tumours, this is not the case when bacterial butyrate-synthesis genes are disrupted. These results suggest a causal relationship between Porphyromonas species overgrowth and colorectal tumourigenesis which may be due to butyrate-induced senescence.

Gut-liver axis-mediated mechanism of liver cancer: A special focus on the role of gut microbiota.

Gut microbiota and the mammalian host share a symbiotic relationship, in which the host provides a suitable ecosystem for the gut bacteria to digest indigestible nutrients and produce useful metabolites. Although gut microbiota primarily reside in and influence the intestine, they also regulate liver function via absorption and subsequent transfer of microbial components and metabolites through the portal vein to the liver. Due to this transfer, the liver may be continuously exposed to gut-derived metabolites and components. For example, short-chain fatty acids (SCFA) produced by gut microbiota, through the fermentation of dietary fiber, can suppress inflammation via regulatory T cell induction through SCFA-induced epigenetic mechanisms. Additionally, secondary bile acids (BA), such as deoxycholic acid, produced by gut bacteria through the 7α-dehydroxylation of primary BAs, are thought to induce DNA damage and contribute to the remodeling of tumor microenvironments. Other substances that are also thought to influence liver function include lipopolysaccharides (components of the outer membrane of gram-negative bacteria) and lipoteichoic acid (cell wall component of Gram-positive bacteria), which are ligands of innate immune receptors, Toll-like receptor-4, and Toll-like receptor-2, respectively, through which inflammatory signaling is elicited. In this review, we focus on the role of gut microbiota in the liver microenvironment, describing the anatomy of the gut-liver axis, the role of gut microbial metabolites, and the relationships that exist between gut microbiota and liver diseases, including liver cancer.

Visualization of quantitative lipid distribution in mouse liver through near-infrared hyperspectral imaging.

Lipid distribution in the liver provides crucial information for diagnosing the severity of fatty liver and fatty liver-associated liver cancer. Therefore, a noninvasive, label-free, and quantitative modality is eagerly anticipated. We report near-infrared hyperspectral imaging for the quantitative visualization of lipid content in mouse liver based on partial least square regression (PLSR) and support vector regression (SVR). Analysis results indicate that SVR with standard normal variate pretreatment outperforms PLSR by achieving better root mean square error (15.3 mg/g) and higher determination coefficient (0.97). The quantitative mapping of lipid content in the mouse liver is realized using SVR.

A BET family protein degrader provokes senolysis by targeting NHEJ and autophagy in senescent cells.

Although cellular senescence acts primarily as a tumour suppression mechanism, the accumulation of senescent cells in vivo eventually exerts deleterious side effects through inflammatory/tumour-promoting factor secretion. Thus, the development of new drugs that cause the specific elimination of senescent cells, termed senolysis, is anticipated. Here, by an unbiased high-throughput screening of chemical compounds and a bio-functional analysis, we identify BET family protein degrader (BETd) as a promising senolytic drug. BETd provokes senolysis through two independent but integrated pathways; the attenuation of non-homologous end joining (NHEJ), and the up-regulation of autophagic gene expression. BETd treatment eliminates senescent hepatic stellate cells in obese mouse livers, accompanied by the reduction of liver cancer development. Furthermore, the elimination of chemotherapy-induced senescent cells by BETd increases the efficacy of chemotherapy against xenograft tumours in immunocompromised mice. These results reveal the vulnerability of senescent cells and open up possibilities for its control.

Deciphering the mechanism for induction of senescence-associated secretory phenotype (SASP) and its role in ageing and cancer development.

Cellular senescence is an irreversible form of cell cycle arrest that can be induced by persistent DNA damage, and is well known to function as an important tumour suppression mechanism. Cellular senescence is detected in aged organisms; thus, it is also recognized as a hallmark of organismal ageing. Unlike apoptotic cells, senescent cells can survive for long periods of time. Recently, it has been shown that the late stage of senescent cells are capable of expressing a variety of secreted proteins such as cytokines, chemokines and proteases, and this condition is now known as senescence-associated secretory phenotype (SASP). These secreted factors are involved in myriad of physiological functions including tissue repair and clearance of damaged cells. Alternatively, these factors may promote detrimental effects, such as chronic inflammation or cancer progression, should the SASP persist. Recent scientific advances have indicated that innate immune responses, particularly involving the cGAS-STING pathway, trigger SASP induction. Therefore, developing a strategy to regulate SASP may provide scientific insights for the management of age-associated diseases and the implementation of healthy ageing in the future.