Fungal‐induced glycolysis in macrophages promotes colon cancer by enhancing innate lymphoid cell secretion of IL‐22

Incorporation of microbiome data has recently become important for prevention, diagnosis, and treatment of colorectal cancer, and several species of bacteria were shown to be associated with carcinogenesis. However, the role of commensal fungi in colon cancer remains poorly understood. Here, we report that mice lacking the c‐type lectin Dectin‐3 (Dectin‐3 ^−/−) show increased tumorigenesis and Candida albicans burden upon chemical induction. Elevated C. albicans load triggered glycolysis in macrophages and interleukin‐7 (IL‐7) secretion. IL‐7 induced IL‐22 production in RORγt⁺ (group 3) innate lymphoid cells (ILC3s) via aryl hydrocarbon receptor and STAT3. Consistently, IL‐22 frequency in tumor tissues of colon cancer patients positively correlated with fungal burden, indicating the relevance of this regulatory axis in human disease. These results establish a C. albicans‐driven crosstalk between macrophages and innate lymphoid cells in the intestine and expand our understanding on how commensal mycobiota regulate host immunity and promote tumorigenesis.     

Nucleolar c‐Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood‐borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein–Barr virus nuclear antigen 1‐binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpA^L10A) did not alter its translocation to the nucleus but abolished the effector’s capacity to interact with EBP2. VgpA‐EBP2 interaction led to the re‐localization of c‐Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA‐EBP2 interaction elevated EBP2’s affinity for c‐Myc and prolonged the oncoprotein’s half‐life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re‐localization of c‐Myc. Moreover, the in vivo VgpA‐EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus’ colonization and virulence. These observations suggest that direct effector stimulation of a c‐Myc controlled host cell growth program can contribute to pathogenesis.     

Cryptic association of B7‐2 molecules and its implication for clustering

T‐cell co‐stimulation through CD28/CTLA4:B7‐1/B7‐2 axis is one of the extensively studied pathways that resulted in the discovery of several FDA‐approved drugs for autoimmunity and cancer. However, many aspects of the signaling mechanism remain elusive, including oligomeric association and clustering of B7‐2 on the cell surface. Here, we describe the structure of the IgV domain of B7‐2 and its cryptic association into 1D arrays that appear to represent the pre‐signaling state of B7‐2 on the cell membrane. Super‐resolution microscopy experiments on heterologous cells expressing B7‐2 and B7‐1 suggest, B7‐2 form relatively elongated and larger clusters compared to B7‐1. The sequence and structural comparison of other B7 family members, B7‐1:CTLA4 and B7‐2:CTLA‐4 complex structures, support our view that the observed B7‐2 1D zipper array is physiologically important. This observed 1D zipper‐like array also provides an explanation for its clustering, and upright orientation on the cell surface, and avoidance of spurious signaling.     

Sorafenib attenuates liver fibrosis by triggering hepatic stellate cell ferroptosis via HIF‐1α/SLC7A11 pathway

ObjectivesEvidences demonstrate that sorafenib alleviates liver fibrosis via inhibiting HSC activation and ECM accumulation. The underlying mechanism remains unclear. Ferroptosis, a novel programmed cell death, regulates diverse physiological/pathological processes. In this study, we aim to investigate the functional role of HSC ferroptosis in the anti‐fibrotic effect of sorafenib.Materials and MethodsThe effects of sorafenib on HSC ferroptosis and ECM expression were assessed in mouse model of liver fibrosis induced by CCl₄. In vitro, Fer‐1 and DFO were used to block ferroptosis and then explored the anti‐fibrotic effect of sorafenib by detecting α‐SMA, COL1α1 and fibronectin proteins. Finally, HIF‐1α siRNA, plasmid and stabilizers were applied to assess related signalling pathway.ResultsSorafenib attenuated liver injury and ECM accumulation in CCl₄‐induced fibrotic livers, accompanied by reduction of SLC7A11 and GPX4 proteins. In sorafenib‐treated HSC‐T6 cells, ferroptotic events (depletion of SLC7A11, GPX4 and GSH; accumulation iron, ROS and MDA) were discovered. Intriguingly, these ferroptotic events were not appeared in hepatocytes or macrophages. Sorafenib‐elicited HSC ferroptosis and ECM reduction were abrogated by Fer‐1 and DFO. Additionally, both HIF‐1α and SLC7A11 proteins were reduced in sorafenib‐treated HSC‐T6 cells. SLC7A11 was positively regulated by HIF‐1α, inactivation of HIF‐1α/SLC7A11 pathway was required for sorafenib‐induced HSC ferroptosis, and elevation of HIF‐1α could inhibit ferroptosis, ultimately limited the anti‐fibrotic effect.ConclusionsSorafenib triggers HSC ferroptosis via HIF‐1α/SLC7A11 signalling, which in turn attenuates liver injury and fibrosis.     

Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non‐cell‐autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF‐κB signaling upregulation is central to elicit this immune response. Inactivating NF‐κB abolishes NK cell‐mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF‐κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell‐mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF‐κB‐mediated immunogenicity.     

Down‐regulated HDAC3 elevates microRNA‐495‐3p to restrain epithelial‐mesenchymal transition and oncogenicity of melanoma cells via reducing TRAF5

MicroRNAs (miRNAs) are emerging biomarkers in biological processes and the role of miR‐495‐3p has been identified in melanoma, while the detailed molecular mechanisms remain to be further explored. We aim to explore the effect of histone deacetylase 3 (HDAC3) and miR‐495‐3p on epithelial‐mesenchymal transition (EMT) and oncogenicity of melanoma cells by regulating tumour necrosis factor receptor‐associated factor 5 (TRAF5). Levels of HDAC3, miR‐495‐3p and TRAF5 in melanoma tissues and pigmented nevus tissues were determined, and the predictive roles of HDAC3 and miR‐495‐3p in prognosis of melanoma patients were measured. The melanoma cells were screened and transfected with relative oligonucleotides and plasmids, and the expression of HDAC3, miR‐495‐3p and TRAF5, and phenotypes of melanoma cells were gauged by a series of assays. The relations between HDAC3 and miR‐495‐3p, and between miR‐495‐3p and TRAF5 were confirmed. HDAC3 and TRAF5 were increased while miR‐495‐3p was decreased in melanoma cells and tissues, and the low expression of miR‐495‐3p as well as high expression of HDAC3 indicated a poor prognosis of melanoma patients. Inhibited HDAC3 elevated miR‐495‐3p to suppress EMT and oncogenicity of melanoma cells by reducing TRAF5. HDAC3 particularly bound to miR‐495‐3p and TRAF5 was the target gene of miR‐495‐3p. Our results revealed that down‐regulated HDAC3 elevates miR‐495‐3p to suppress malignant phenotypes of melanoma cells by inhibiting TRAF5, thereby repressing EMT progression of melanoma cells. This study may provide novel targets for melanoma treatment.     

Airway secretory cell fate conversion via YAP‐mTORC1‐dependent essential amino acid metabolism

Tissue homeostasis requires lineage fidelity of stem cells. Dysregulation of cell fate specification and differentiation leads to various diseases, yet the cellular and molecular mechanisms governing these processes remain elusive. We demonstrate that YAP/TAZ activation reprograms airway secretory cells, which subsequently lose their cellular identity and acquire squamous alveolar type 1 (AT1) fate in the lung. This cell fate conversion is mediated via distinctive transitional cell states of damage‐associated transient progenitors (DATPs), recently shown to emerge during injury repair in mouse and human lungs. We further describe a YAP/TAZ signaling cascade to be integral for the fate conversion of secretory cells into AT1 fate, by modulating mTORC1/ATF4‐mediated amino acid metabolism in vivo. Importantly, we observed aberrant activation of the YAP/TAZ‐mTORC1‐ATF4 axis in the altered airway epithelium of bronchiolitis obliterans syndrome, including substantial emergence of DATPs and AT1 cells with severe pulmonary fibrosis. Genetic and pharmacologic inhibition of mTORC1 activity suppresses lineage alteration and subepithelial fibrosis driven by YAP/TAZ activation, proposing a potential therapeutic target for human fibrotic lung diseases.     

Ouabain and chloroquine trigger senolysis of BRAF‐V600E‐induced senescent cells by targeting autophagy

The expression of BRAF‐V600E triggers oncogene‐induced senescence in normal cells and is implicated in the development of several cancers including melanoma. Here, we report that cardioglycosides such as ouabain are potent senolytics in BRAF senescence. Sensitization by ATP1A1 knockdown and protection by supplemental potassium showed that senolysis by ouabain was mediated by the Na,K‐ATPase pump. Both ion transport inhibition and signal transduction result from cardioglycosides binding to Na,K‐ATPase. An inhibitor of the pump that does not trigger signaling was not senolytic despite blocking ion transport, demonstrating that signal transduction is required for senolysis. Ouabain triggered the activation of Src, p38, Akt, and Erk in BRAF‐senescent cells, and signaling inhibitors prevented cell death. The expression of BRAF‐V600E increased ER stress and autophagy in BRAF‐senescent cells and sensitized the cell to senolysis by ouabain. Ouabain inhibited autophagy flux, which was restored by signaling inhibitors. Consequently, we identified autophagy inhibitor chloroquine as a novel senolytic in BRAF senescence based on the mode of action of cardioglycosides. Our work underlies the interest of characterizing the mechanisms of senolytics to discover novel compounds and identifies the endoplasmic reticulum stress‐autophagy tandem as a new vulnerability in BRAF senescence that can be exploited for the development of further senolytic strategies.     

BNIP3 promotes HIF‐1α‐driven melanoma growth by curbing intracellular iron homeostasis

BNIP3 is a mitophagy receptor with context‐dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient’s survival and depletion of BNIP3 in B16‐F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2‐mediated downregulation of HIF‐1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3‐deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4‐mediated ferritinophagy, which fostered PHD2‐mediated HIF‐1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF‐1α levels in BNIP3‐depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF‐1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro‐tumorigenic HIF‐1α glycolytic program in melanoma cells.     

Amino acid starvation‐induced LDLR trafficking accelerates lipoprotein endocytosis and LDL clearance

Mammalian cells utilize Akt‐dependent signaling to deploy intracellular Glut4 toward cell surface to facilitate glucose uptake. Low‐density lipoprotein receptor (LDLR) is the cargo receptor mediating endocytosis of apolipoprotein B‐containing lipoproteins. However, signaling‐controlled regulation of intracellular LDLR trafficking remains elusive. Here, we describe a unique amino acid stress response, which directs the deployment of intracellular LDLRs, causing enhanced LDL endocytosis, likely via Ca²⁺ and calcium/calmodulin‐dependent protein kinase II‐mediated signalings. This response is independent of induction of autophagy. Amino acid stress‐induced increase in LDL uptake in vitro is comparable to that by pravastatin. In vivo, acute AAS challenge for up to 72 h enhanced the rate of hepatic LDL uptake without changing the total expression level of LDLR. Reducing dietary amino acids by 50% for 2 to 4 weeks ameliorated high fat diet‐induced hypercholesterolemia in heterozygous LDLR‐deficient mice, with reductions in both LDL and VLDL fractions. We suggest that identification of signaling‐controlled regulation of intracellular LDLR trafficking has advanced our understanding of the LDLR biology, and may benefit future development of additional therapeutic strategies for treating hypercholesterolemia.     

A non‐helical region in transmembrane helix 6 of hydrophobic amino acid transporter MhsT mediates substrate recognition

MhsT of Bacillus halodurans is a transporter of hydrophobic amino acids and a homologue of the eukaryotic SLC6 family of Na⁺‐dependent symporters for amino acids, neurotransmitters, osmolytes, or creatine. The broad range of transported amino acids by MhsT prompted the investigation of the substrate recognition mechanism. Here, we report six new substrate‐bound structures of MhsT, which, in conjunction with functional studies, reveal how the flexibility of a Gly‐Met‐Gly (GMG) motif in the unwound region of transmembrane segment 6 (TM6) is central for the recognition of substrates of different size by tailoring the binding site shape and volume. MhsT mutants, harboring substitutions within the unwound GMG loop and substrate binding pocket that mimick the binding sites of eukaryotic SLC6A18/B0AT3 and SLC6A19/B0AT1 transporters of neutral amino acids, exhibited impaired transport of aromatic amino acids that require a large binding site volume. Conservation of a general (G/A/C)ΦG motif among eukaryotic members of SLC6 family suggests a role for this loop in a common mechanism for substrate recognition and translocation by SLC6 transporters of broad substrate specificity.     

Hsa_circ_0003945 promotes progression of hepatocellular carcinoma by mediating miR‐34c‐5p/LGR4/β‐catenin axis activity

Accumulating evidence suggests that circular RNAs (circRNAs) play essential roles in regulating cancer progression, but many circRNAs in hepatocellular carcinoma (HCC) remain unknown. Dysregulated circRNAs in HCC were identified through bioinformatics analysis of Gene Expression Omnibus data sets. Quantitative real‐time PCR (qRT‐PCR), Sanger sequencing, RNase R digestion and actinomycin D treatment were conducted to confirm the characterization of circRNAs. CCK‐8, wound‐healing and Transwell assays were performed to assess the functional roles of Hsa_circ_0003945 (Circ_0003945) in HCC cell lines. Subcellular fractionation and fluorescence in situ hybridization (FISH) were performed to locate Circ_0003945 in HCC cells. Dual‐luciferase reporter assay was executed to verify the binding of Circ_0003945 to microRNAs (miRNAs) or the miRNAs to their target genes. In this study, we found that Circ_0003945 was upregulated in HCC tissue, and higher Circ_0003945 expression was positively correlated with tumour size and tumour stage. Furthermore, high plasma levels of circulating Circ_0003945 were confirmed in HCC patients compared with those in non‐HCC groups. The functional experiments revealed that overexpression or knockdown of Circ_0003945 promoted or attenuated tumour growth and migration, respectively. Mechanistically, Circ_0003945 might exert as a miR‐34c‐5p sponge to upregulate the expression of leucine‐rich repeat‐containing G protein‐coupled receptor 4 (LGR4), activating the β‐catenin pathway, and finally facilitating HCC progression. Additionally, a β‐catenin activator could reverse the effect of Circ_0003945 knockdown. In conclusion, Circ_0003945 exerts a tumour‐promoting role in HCC cells by regulating the miR‐34c‐5p/LGR4/β‐catenin axis, which may be a potential target for HCC therapy.     

Role of the CCL2‐CCR2 signalling axis in cancer: Mechanisms and therapeutic targeting

The chemokine ligand CCL2 and its receptor CCR2 are implicated in the initiation and progression of various cancers. CCL2 can activate tumour cell growth and proliferation through a variety of mechanisms. By interacting with CCR2, CCL2 promotes cancer cell migration and recruits immunosuppressive cells to the tumour microenvironment, favouring cancer development. Over the last several decades, a series of studies have been conducted to explore the CCL2‐CCR2 signalling axis function in malignancies. Therapeutic strategies targeting the CCL2‐ CCR2 axis have also shown promising effects, enriching our approaches for fighting against cancer. In this review, we summarize the role of the CCL2‐CCR2 signalling axis in tumorigenesis and highlight recent studies on CCL2‐CCR2 targeted therapy, focusing on preclinical studies and clinical trials.

The chemokine ligand CCL2 and its receptor CCR2 are implicated in the initiation and progression of various cancers. The CCL2‐CCR2 signalling axis plays a critical role in the promotion of pathological angiogenesis, the survival and invasion of tumour cells, and the recruitment of immune inhibitory cells. Therefore, CCL2 and CCR2 enable us to explore the sophisticated mechanisms underlying cancer development and provide potential options for treating malignant tumours.     

Opposing activities of IFITM proteins in SARS‐CoV‐2 infection

Interferon‐induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS‐CoV‐2 Spike‐pseudotyped virus and genuine SARS‐CoV‐2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain‐ and loss‐of‐function approaches. Mechanistically, SARS‐CoV‐2 restriction occurred independently of IFITM3 S‐palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis‐promoting YxxФ motif converted human IFITM3 into an enhancer of SARS‐CoV‐2 infection, and cell‐to‐cell fusion assays confirmed the ability of endocytic mutants to enhance Spike‐mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS‐CoV‐2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro‐ and anti‐viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity‐based mechanisms used for endosomal SARS‐CoV‐2 restriction.     

The role of E2A in ATPR‐induced cell differentiation and cycle arrest in acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) is a biologically heterogeneous disease with an overall poor prognosis; thus, novel therapeutic approaches are needed. Our previous studies showed that 4‐amino‐2‐trifluoromethyl‐phenyl retinate (ATPR), a new derivative of all‐trans retinoic acid (ATRA), could induce AML cell differentiation and cycle arrest. The current study aimed to determine the potential pharmacological mechanisms of ATPR therapies against AML. Our findings showed that E2A was overexpressed in AML specimens and cell lines, and mediate AML development by inactivating the P53 pathway. The findings indicated that E2A expression and activity decreased with ATPR treatment. Furthermore, we determined that E2A inhibition could enhance the effect of ATPR‐induced AML cell differentiation and cycle arrest, whereas E2A overexpression could reverse this effect, suggesting that the E2A gene plays a crucial role in AML. We identified P53 and c‐Myc were downstream pathways and targets for silencing E2A cells using RNA sequencing, which are involved in the progression of AML. Taken together, these results confirmed that ATPR inhibited the expression of E2A/c‐Myc, which led to the activation of the P53 pathway, and induced cell differentiation and cycle arrest in AML.     

Activation of angiotensin‐converting enzyme 2/angiotensin (1–7)/mas receptor axis triggers autophagy and suppresses microglia proinflammatory polarization via forkhead box class O1 signaling

Brain renin‐angiotensin (Ang) system (RAS) is implicated in neuroinflammation, a major characteristic of aging process. Angiotensin (Ang) II, produced by angiotensin‐converting enzyme (ACE), activates immune system via angiotensin type 1 receptor (AT1), whereas Ang(1–7), generated by ACE2, binds with Mas receptor (MasR) to restrain excessive inflammatory response. Therefore, the present study aims to explore the relationship between RAS and neuroinflammation. We found that repeated lipopolysaccharide (LPS) treatment shifted the balance between ACE/Ang II/AT1 and ACE2/Ang(1–7)/MasR axis to the deleterious side and treatment with either MasR agonist, AVE0991 (AVE) or ACE2 activator, diminazene aceturate, exhibited strong neuroprotective actions. Mechanically, activation of ACE2/Ang(1–7)/MasR axis triggered the Forkhead box class O1 (FOXO1)‐autophagy pathway and induced superoxide dismutase (SOD) and catalase (CAT), the FOXO1‐targeted antioxidant enzymes. Meanwhile, knockdown of MasR or FOXO1 in BV2 cells, or using the selective FOXO1 inhibitor, AS1842856, in animals, suppressed FOXO1 translocation and compromised the autophagic process induced by MasR activation. We further used chloroquine (CQ) to block autophagy and showed that suppressing either FOXO1 or autophagy abrogated the anti‐inflammatory action of AVE. Likewise, Ang(1–7) also induced FOXO1 signaling and autophagic flux following LPS treatment in BV2 cells. Cotreatment with AS1842856 or CQ all led to autophagic inhibition and thereby abolished Ang(1–7)‐induced remission on NLRP3 inflammasome activation caused by LPS exposure, shifting the microglial polarization from M1 to M2 phenotype. Collectively, these results firstly illustrated the mechanism of ACE2/Ang(1–7)/MasR axis in neuroinflammation, strongly indicating the involvement of FOXO1‐mediated autophagy in the neuroimmune‐modulating effects triggered by MasR activation.