MAPK4 facilitates angiogenesis by inhibiting the ERK pathway in non-small cell lung cancer.

Background: Angiogenesis plays an important role in the occurrence and development of non-small cell lung cancer (NSCLC). The atypical mitogen-activated protein kinase 4 (MAPK4) has been shown to be involved in the pathogenesis of various diseases. However, the potential role of MAPK4 in the tumor angiogenesis of NSCLC remains unclear. Methods: Adult male C57BL/6 wild-type mice were randomly divided into the control group and p-siMAPK4 intervention group, respectively. The cell proliferation was analyzed with flow cytometry and immunofluorescence staining. The vascular density in tumor mass was analyzed by immunofluorescence staining. The expressions of MAPK4 and related signaling molecules were detected by western blot analysis and immunofluorescence staining, and so on. Results: We found that the expression of MAPK4, which was dominantly expressed in local endothelial cells (ECs), was correlated with tumor angiogenesis of NSCLC. Furthermore, MAPK4 silencing inhibited the proliferation and migration abilities of human umbilical vein ECs (HUVECs). Global gene analysis showed that MAPK4 silencing altered the expression of multiple genes related to cell cycle and angiogenesis pathways, and that MAPK4 silencing increased transduction of the extracellular regulated protein kinases 1/2 (ERK1/2) pathway but not Akt and c-Jun n-terminal kinase pathways. Further analysis showed that MAPK4 silencing inhibited the proliferation and migration abilities of HUVECs cultured in tumor cell supernatant, which was accompanied with increased transduction of the ERK1/2 pathway. Clinical data analysis suggested that the higher expression of MAPK4 and CD34 were associated with poor prognosis of patients with NSCLC. Targeted silencing of MAPK4 in ECs using small interfering RNA driven by the CD34 promoter effectively inhibited tumor angiogenesis and growth of NSCLC in vivo. Conclusion: Our results reveal that MAPK4 plays an important role in the angiogenesis and development of NSCLC. MAPK4 may thus represent a new target for NSCLC.

Tumor-secreted FGF21 acts as an immune suppressor by rewiring cholesterol metabolism of CD8+T cells.

Tumors employ diverse strategies for immune evasion. Unraveling the mechanisms by which tumors suppress anti-tumor immunity facilitates the development of immunotherapies. Here, we have identified tumor-secreted fibroblast growth factor 21 (FGF21) as a pivotal immune suppressor. FGF21 is upregulated in multiple types of tumors and promotes tumor progression. Tumor-secreted FGF21 significantly disrupts anti-tumor immunity by rewiring cholesterol metabolism of CD8+T cells. Mechanistically, FGF21 sustains the hyperactivation of AKT-mTORC1-sterol regulatory-element-binding protein 1 (SREBP1) signal axis in the activated CD8+T cells, resulting in the augment of cholesterol biosynthesis and T cell exhaustion. FGF21 knockdown or blockade using a neutralizing antibody normalizes AKT-mTORC1 signaling and reduces excessive cholesterol accumulation in CD8+T cells, thus restoring CD8+T cytotoxic function and robustly suppressing tumor growth. Our findings reveal FGF21 as a "secreted immune checkpoint" that hampers anti-tumor immunity, suggesting that inhibiting FGF21 could be a valuable strategy to enhance the cancer immunotherapy efficacy.

Cancer CD39 drives metabolic adaption and mal-differentiation of CD4+ T cells in patients with non-small-cell lung cancer.

While ectonucleotidase CD39 is a cancer therapeutic target in clinical trials, its direct effect on T-cell differentiation in human non-small-cell lung cancer (NSCLC) remains unclear. Herein, we demonstrate that human NSCLC cells, including tumor cell lines and primary tumor cells from clinical patients, efficiently drive the metabolic adaption of human CD4+ T cells, instructing differentiation of regulatory T cells while inhibiting effector T cells. Of importance, NSCLC-induced T-cell mal-differentiation primarily depends on cancer CD39, as this can be fundamentally blocked by genetic depletion of CD39 in NSCLC. Mechanistically, NSCLC cells package CD39 into their exosomes and transfer such CD39-containing exosomes into interacting T cells, resulting in ATP insufficiency and AMPK hyperactivation. Such CD39-dependent NSCLC-T cell interaction holds well in patients-derived primary tumor cells and patient-derived organoids (PDOs). Accordingly, genetic depletion of CD39 alone or in combination with the anti-PD-1 immunotherapy efficiently rescues effector T cell differentiation, instigates anti-tumor T cell immunity, and inhibits tumor growth of PDOs. Together, targeting cancer CD39 can correct the mal-differentiation of CD4+ T cells in human NSCLC, providing in-depth insight into therapeutic CD39 inhibitors.

Myocardial Mitochondrial DNA Drives Macrophage Inflammatory Response through STING Signaling in Coxsackievirus B3-Induced Viral Myocarditis.

Coxsackievirus B3 (CVB3), a single-stranded positive RNA virus, primarily infects cardiac myocytes and is a major causative pathogen for viral myocarditis (VMC), driving cardiac inflammation and organ dysfunction. However, whether and how myocardial damage is involved in CVB3-induced VMC remains unclear. Herein, we demonstrate that the CVB3 infection of cardiac myocytes results in the release of mitochondrial DNA (mtDNA), which functions as an important driver of cardiac macrophage inflammation through the stimulator of interferon genes (STING) dependent mechanism. More specifically, the CVB3 infection of cardiac myocytes promotes the accumulation of extracellular mtDNA. Such myocardial mtDNA is indispensable for CVB3-infected myocytes in that it induces a macrophage inflammatory response. Mechanistically, a CVB3 infection upregulates the expression of the classical DNA sensor STING, which is predominantly localized within cardiac macrophages in VMC murine models. Myocardial mtDNA efficiently triggers STING signaling in those macrophages, resulting in strong NF-kB activation when inducing the inflammatory response. Accordingly, STING-deficient mice are able to resist CVB3-induced cardiac inflammation, exhibiting minimal inflammation with regard to their functional cardiac capacities, and they exhibit higher survival rates. Moreover, our findings pinpoint myocardial mtDNA as a central element driving the cardiac inflammation of CVB3-induced VMC, and we consider the DNA sensor, STING, to be a promising therapeutic target for protecting against RNA viral infections.

Akkermansia muciniphila: A potential target and pending issues for oncotherapy.

In the wake of the development of metagenomic, metabolomic, and metatranscriptomic approaches, the intricate interactions between the host and various microbes are now being progressively understood. Numerous studies have demonstrated evident changes in gut microbiota during the process of a variety of diseases, such as diabetes, obesity, aging, and cancers. Notably, gut microbiota is viewed as a potential source of novel therapeutics. Currently, Next-generation probiotics (NGPs) are gaining popularity as therapeutic agents that alter the gut microbiota and affect cancer development. Akkermansia muciniphila (A. muciniphila), a representative commensal bacterium, has received substantial attention over the past decade as a promising NGP. The components and metabolites of A. muciniphila can directly or indirectly affect tumorigenesis, in particular through its effects on antitumor immunosurveillance, including the stimulation of pattern recognition receptors (PRRs), which also leads to better outcomes in a variety of situations, including the prevention and curation of cancers. In this article, we systematically summarize the role of A. muciniphila in tumorigenesis (involving gastrointestinal and non-gastrointestinal cancers) and in tumor therapy. In particular, we carefully discuss some critical scientific issues that need to be solved for the future using A. muciniphila as a representative beneficial bacterium in tumor treatment, which might provide bright clues and assistance for the application of drugs targeting A. muciniphila in clinical oncotherapy.

Low-dose metronomic chemotherapy triggers oxidized mtDNA sensing inside tumor cells to potentiate CD8+T anti-tumor immunity.

Low-dose metronomic (LDM) chemotherapy, the frequent and continuous use of low doses of conventional chemotherapeutics, is emerging as a promising form of chemotherapy utilization. LDM chemotherapy exerts immunomodulatory effects. However, the underlying mechanism is not fully understood. Here we found that suppressing tumor growth by LDM chemotherapy was dependent on the activation of CD8+T cells. LDM chemotherapy potentiated the cytotoxic function of CD8+T cells by stimulating cancer-cell autonomous type I interferon (IFN) induction. Mechanistically, LDM chemotherapy evoked mitochondrial dysfunction and increased reactive oxygen species (ROS) production. ROS triggered the oxidation of cytosolic mtDNA, which was sensed by cGAS-STING, consequently inducing type I IFN production in the cancer cells. Moreover, the cGAS-STING-IFN axis increased PD-L1 expression and predicted favorable clinical responses to chemoimmunotherapy. Antioxidant N-acetylcysteine inhibited oxidized mtDNA-induced type I IFN production and attenuated the efficacy of combination therapy with LDM chemotherapy and PD-L1 blockade. This study elucidates the critical role of intratumoral oxidized mtDNA sensing in LDM chemotherapy-mediated activation of CD8+T cell immune response. These findings may provide new insights for designing combinatorial immunotherapy for cancer patients.

Activated Lymphocyte-Derived DNA Drives Glucose Metabolic Adaptation for Inducing Macrophage Inflammatory Response in Systemic Lupus Erythematosus.

Activated lymphocyte-derived DNA (ALD-DNA) has been reported to drive the polarization of macrophages toward M2b, producing inflammatory cytokines and inducing inflammation, correspondingly playing an essential role in the development of systemic lupus erythematosus (SLE). Recently, accumulating evidence has pinpointed metabolic adaptation as the crucial cell-intrinsic determinant for inflammatory response, in which glucose metabolism is the key event. However, whether and how glucose metabolism was involved in ALD-DNA-induced macrophage inflammatory response and SLE development remains unclear. Herein, we performed glucose metabolomic analyses of ALD-DNA-stimulated macrophages and uncovered increased glycolysis and diminished pentose phosphate pathway (PPP), as well as enhanced glycogenesis. In ALD-DNA-stimulated macrophages, increased glycolysis resulted in higher lactate production, whereas diminished PPP efficiently led to lower levels of nicotinamide adenine dinucleotide phosphate (NADPH) with higher levels of reactive oxygen species (ROS). While blockade of lactate generation exerted no significant effect on macrophage inflammation in response to ALD-DNA, scavenging ROS fundamentally inhibited the inflammatory response of ALD-DNA-stimulated macrophages. Further, cyclic adenosine monophosphate (cAMP), a master for regulating glycogen metabolism, was downregulated by ALD-DNA in macrophages, which subsequently imbalanced glycogen metabolism toward glycogenesis but not glycogenolysis. Administration of cAMP effectively restored glycogenolysis and enhanced PPP, which correspondingly reduced ROS levels and inhibited the inflammatory response of ALD-DNA-stimulated macrophages. Finally, blocking glucose metabolism using 2-deoxy-D-glucose (2-DG) efficiently restricted macrophage inflammatory response and alleviated ALD-DNA-induced lupus disease. Together, our findings demonstrate that ALD-DNA drives the adaptation of glucose metabolism for inducing macrophage inflammatory response in SLE, which might further our understanding of disease pathogenesis and provide clues for interventive explorations.

MiRNA-Based Therapies for Lung Cancer: Opportunities and Challenges?

Lung cancer is a commonly diagnosed cancer and the leading cause of cancer-related deaths, posing a serious health risk. Despite new advances in immune checkpoint and targeted therapies in recent years, the prognosis for lung cancer patients, especially those in advanced stages, remains poor. MicroRNAs (miRNAs) have been shown to modulate tumor development at multiple levels, and as such, miRNA mimics and molecules aimed at regulating miRNAs have shown promise in preclinical development. More importantly, miRNA-based therapies can also complement conventional chemoradiotherapy, immunotherapy, and targeted therapies to reverse drug resistance and increase the sensitivity of lung cancer cells. Furthermore, small interfering RNA (siRNA) and miRNA-based therapies have entered clinical trials and have shown favorable development prospects. Therefore, in this paper, we review recent advances in miRNA-based therapies in lung cancer treatment as well as adjuvant therapy and present the current state of clinical lung cancer treatment. We also discuss the challenges facing miRNA-based therapies in the clinical application of lung cancer treatment to provide new ideas for the development of novel lung cancer therapies.

Lipogenesis promotes mitochondrial fusion and maintains cancer stemness in human NSCLC.

Cancer stem-like cells (CSCs) are critically involved in cancer metastasis and chemoresistance, acting as one major obstacle in clinical practice. While accumulating studies have implicated the metabolic reprogramming of CSCs, mitochondrial dynamics in such cells remain poorly understood. Here we pinpointed OPA1hi with mitochondrial fusion as a metabolic feature of human lung CSCs, licensing their stem-like properties. Specifically, human lung CSCs exerted enhanced lipogenesis, inducing OPA1 expression via transcription factor SAM Pointed Domain containing ETS transcription Factor (SPDEF). In consequence, OPA1hi promoted mitochondrial fusion and stemness of CSCs. Such lipogenesishi, SPDEFhi, and OPA1hi metabolic adaptions were verified with primary CSCs from lung cancer patients. Accordingly, blocking lipogenesis and mitochondrial fusion efficiently impeded CSC expansion and growth of organoids derived from patients with lung cancer. Together, lipogenesis regulates mitochondrial dynamics via OPA1 for controlling CSCs in human lung cancer.

Mitophagy bridges DNA sensing with metabolic adaption to expand lung cancer stem-like cells.

While previous studies have identified cancer stem-like cells (CSCs) as a crucial driver for chemoresistance and tumor recurrence, the underlying mechanisms for populating the CSC pool remain unclear. Here, we identify hypermitophagy as a feature of human lung CSCs, promoting metabolic adaption via the Notch1-AMPK axis to drive CSC expansion. Specifically, mitophagy is highly active in CSCs, resulting in increased mitochondrial DNA (mtDNA) content in the lysosome. Lysosomal mtDNA acts as an endogenous ligand for Toll-like receptor 9 (TLR9) that promotes Notch1 activity. Notch1 interacts with AMPK to drive lysosomal AMPK activation by inducing metabolic stress and LKB1 phosphorylation. This TLR9-Notch1-AMPK axis supports mitochondrial metabolism to fuel CSC expansion. In patient-derived xenograft chimeras, targeting mitophagy and TLR9-dependent Notch1-AMPK pathway restricts tumor growth and CSC expansion. Taken together, mitochondrial hemostasis is interlinked with innate immune sensing and Notch1-AMPK activity to increase the CSC pool of human lung cancer.

NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis.

The aorta and the large conductive arteries are immunoprivileged tissues and are protected against inflammatory attack. A breakdown of immunoprivilege leads to autoimmune vasculitis, such as giant cell arteritis, in which CD8+ Treg cells fail to contain CD4+ T cells and macrophages, resulting in the formation of tissue-destructive granulomatous lesions. Here, we report that the molecular defect of malfunctioning CD8+ Treg cells lies in aberrant NOTCH4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, NOTCH4 signaling restricted vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). Specifically, NOTCH4hiCD8+ Treg cells increased RAB5A and RAB11A expression and suppressed RAB7A, culminating in the accumulation of early and recycling endosomes and sequestering of NOX2 in an intracellular compartment. RAB7AloCD8+ Treg cells failed in the surface translocation and exosomal release of NOX2. NOTCH4hiRAB5AhiRAB7AloRAB11AhiCD8+ Treg cells left adaptive immunity unopposed, enabling a breakdown in tissue tolerance and aggressive vessel wall inflammation. Inhibiting NOTCH4 signaling corrected the defect and protected arteries from inflammatory insult. This study implicates NOTCH4-dependent transcriptional control of RAB proteins and intracellular vesicle trafficking in autoimmune disease and in vascular inflammation.

The DNA Repair Nuclease MRE11A Functions as a Mitochondrial Protector and Prevents T Cell Pyroptosis and Tissue Inflammation.

In the autoimmune disease rheumatoid arthritis (RA), CD4+ T cells promote pro-inflammatory effector functions by shunting glucose away from glycolysis and ATP production. Underlying mechanisms remain unknown, and here we implicate the DNA repair nuclease MRE11A in the cells' bioenergetic failure. MRE11A deficiency in RA T cells disrupted mitochondrial oxygen consumption and suppressed ATP generation. Also, MRE11A loss of function caused leakage of mitochondrial DNA (mtDNA) into the cytosol, triggering inflammasome assembly, caspase-1 activation, and pyroptotic cell death. Caspase-1 activation was frequent in lymph-node-residing T cells in RA patients. In vivo, pharmacologic and genetic inhibition of MRE11A resulted in tissue deposition of mtDNA, caspase-1 proteolysis, and aggressive tissue inflammation. Conversely, MRE11A overexpression restored mitochondrial fitness and shielded tissue from inflammatory attack. Thus, the nuclease MRE11A regulates a mitochondrial protection program, and MRE11A deficiency leads to DNA repair defects, energy production, and failure and loss of tissue homeostasis.

N-myristoyltransferase deficiency impairs activation of kinase AMPK and promotes synovial tissue inflammation.

N-myristoyltransferase (NMT) attaches the fatty acid myristate to the N-terminal glycine of proteins to sort them into soluble and membrane-bound fractions. Function of the energy-sensing AMP-activated protein kinase, AMPK, is myristoylation dependent. In rheumatoid arthritis (RA), pathogenic T cells shift glucose away from adenosine tri-phosphate production toward synthetic and proliferative programs, promoting proliferation, cytokine production, and tissue invasion. We found that RA T cells had a defect in NMT1 function, which prevented AMPK activation and enabled unopposed mTORC1 signaling. Lack of the myristate lipid tail disrupted the lysosomal translocation and activation of AMPK. Instead, myristoylation-incompetent RA T cells hyperactivated the mTORC1 pathway and differentiated into pro-inflammatory TH1 and TH17 helper T cells. In vivo, NMT1 loss caused robust synovial tissue inflammation, whereas forced NMT1 overexpression rescued AMPK activation and suppressed synovitis. Thus, NMT1 has tissue-protective functions by facilitating lysosomal recruitment of AMPK and dampening mTORC1 signaling.

The microvascular niche instructs T cells in large vessel vasculitis via the VEGF-Jagged1-Notch pathway.

Microvascular networks in the adventitia of large arteries control access of inflammatory cells to the inner wall layers (media and intima) and thus protect the immune privilege of the aorta and its major branches. In autoimmune vasculitis giant cell arteritis (GCA), CD4 T helper 1 (TH1) and TH17 cells invade into the wall of the aorta and large elastic arteries to form tissue-destructive granulomas. Whether the disease microenvironment provides instructive cues for vasculitogenic T cells is unknown. We report that adventitial microvascular endothelial cells (mvECs) perform immunoregulatory functions by up-regulating the expression of the Notch ligand Jagged1. Vascular endothelial growth factor (VEGF), abundantly present in GCA patients' blood, induced Jagged1 expression, allowing mvECs to regulate effector T cell induction via the Notch-mTORC1 (mammalian target of rapamycin complex 1) pathway. We found that circulating CD4 T cells in GCA patients have left the quiescent state, actively signal through the Notch pathway, and differentiate into TH1 and TH17 effector cells. In an in vivo model of large vessel vasculitis, exogenous VEGF functioned as an effective amplifier to recruit and activate vasculitogenic T cells. Thus, systemic VEGF co-opts endothelial Jagged1 to trigger aberrant Notch signaling, biases responsiveness of CD4 T cells, and induces pathogenic effector functions. Adventitial microvascular networks function as an instructive tissue niche, which can be exploited to target vasculitogenic immunity in large vessel vasculitis.

Metabolic control of the scaffold protein TKS5 in tissue-invasive, proinflammatory T cells.

Pathogenic T cells in individuals with rheumatoid arthritis (RA) infiltrate non-lymphoid tissue sites, maneuver through extracellular matrix and form lasting inflammatory microstructures. Here we found that RA T cells abundantly express the podosome scaffolding protein TKS5, which enables them to form tissue-invasive membrane structures. TKS5 overexpression was regulated by the intracellular metabolic environment of RA T cells-specifically, by reduced glycolytic flux that led to deficiencies in ATP and pyruvate. ATPlopyruvatelo conditions triggered fatty acid biosynthesis and the formation of cytoplasmic lipid droplets. Restoration of pyruvate production or inhibition of fatty acid synthesis corrected the tissue-invasiveness of RA T cells in vivo and reversed their proarthritogenic behavior. Thus, metabolic control of T cell locomotion provides new opportunities to interfere with T cell invasion into specific tissue sites.

Pro-inflammatory and anti-inflammatory T cells in giant cell arteritis.

Giant cell arteritis is an autoimmune disease defined by explicit tissue tropism to the walls of medium and large arteries. Pathognomic inflammatory lesions are granulomatous in nature, emphasizing the functional role of CD4T cells and macrophages. Evidence for a pathogenic role of antibodies and immune complexes is missing. Analysis of T cell populations in giant cell arteritis, both in the tissue lesions and in the circulation, has supported a model of broad, polyclonal T cell activation, involving an array of functional T cell lineages. The signature of T cell cytokines produced by vasculitic lesions is typically multifunctional, including IL-2, IFN-γ, IL-17, IL-21, and GM-CSF, supportive for a general defect in T cell regulation. Recent data describing the lack of a lymph node-based population of anti-inflammatory T cells in giant cell arteritis patients offers a fresh look at the immunopathology of this vasculitis. Due to defective CD8+NOX2+ regulatory T cells, giant cell arteritis patients appear unable to curtail clonal expansion within the CD4T cell compartment, resulting in widespread CD4T cell hyperimmunity. Why unopposed expansion of committed CD4 effector T cells would lead to invasion of the walls of medium and large arteries needs to be explored in further investigations.

NADPH oxidase deficiency underlies dysfunction of aged CD8+ Tregs.

Immune aging results in progressive loss of both protective immunity and T cell-mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis.

MiR-21 controls in situ expansion of CCR6⁺ regulatory T cells through PTEN/AKT pathway in breast cancer.

Our recent evidence showed that prior expansion of CCR6(+) Foxp3(+) regulatory T cells (Tregs) was important for their dominant enrichment in tumor tissue, which was closely related to poor prognosis of breast cancer patients. However, the underlying regulation mechanism of expansion of CCR6(+) Tregs in situ remains largely unknown. In this study, we reported that miR-21 was highly expressed in CCR6(+) Tregs in tumor tissues from a murine breast cancer model. And silencing of miR-21 could significantly reduce the proliferation of CCR6(+) Tregs in vitro. Adoptive cell-transfer assay further showed that silencing of miR-21 could alter the enrichment of CCR6(+) Tregs in the tumor mass and endow effectively antitumor effect of CD8(+) T cells using a murine breast cancer model. Mechanistic evidence showed that silencing of miR-21 enhanced the expression of its target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and subsequently altered the activation of Akt pathway, which was ultimately responsible for reduced proliferation activity of CCR6(+) Tregs. Finally, we further revealed that miR-21 was also highly expressed on CCR6(+) Tregs in clinical breast cancer patients. Therefore, miR-21 can act as a fine tuner in the regulation of PTEN/Akt pathway transduction in the expansion of CCR6(+) Tregs in tumor sites and provided a novel insight into the development of therapeutic strategies for promoting T-cell immunity by regulating distinct subset of Tregs through targeting specific miRNAs.