m6 A demethylase ALKBH5 promotes proliferation of esophageal squamous cell carcinoma associated with poor prognosis.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal types of malignant tumors worldwide. Epitranscriptome, such as N6 -methyladenosine (m6 A) of mRNA, is an abundant post-transcriptional mRNA modification and has been recently implicated to play roles in several cancers, whereas the significance of m6 A modifications is virtually unknown in ESCC. Analysis of tissue microarray of the tumors in 177 ESCC patients showed that higher expression of m6 A demethylase ALKBH5 correlated with poor prognosis and that ALKBH5 was an independent prognostic factor of the survival of patients. There was no correlation between the other demethylase FTO and prognosis. siRNA knockdown of ALKBH5 but not FTO significantly suppressed proliferation and migration of human ESCC cells. ALKBH5 knockdown delayed progression of cell cycle and accumulated the cells to G0/G1 phase. Mechanistically, expression of CDKN1A (p21) was significantly up-regulated in ALKBH5-depleted cells, and m6 A modification and stability of CDKN1A mRNA were increased by ALKBH5 knockdown. Furthermore, depletion of ALKBH5 substantially suppressed tumor growth of ESCC cells subcutaneously transplanted in BALB/c nude mice. Collectively, we identify ALKBH5 as the first m6 A demethylase that accelerates cell cycle progression and promotes cell proliferation of ESCC cells, which is associated with poor prognosis of ESCC patients.

Haploinsufficiency of Cnot3 Aggravates Acid-Induced Acute Lung Injury Likely Through Transcriptional and Post-Transcriptional Upregulation of Pro-Inflammatory Genes.

Background: Acute lung injury (ALI) is caused by a variety of illnesses, including aspiration pneumonia and sepsis. The CCR4-NOT complex is a large multimeric protein complex that degrades mRNA through poly(A) tail shortening, whereas it also contributes to regulation of transcription and translation. Cnot3 is a scaffold component of the CCR4-NOT complex and is essential for the integrity of the complex; loss of Cnot3 leads to depletion of whole complex. While the significance of cytokine mRNA degradation in limiting inflammation has been established, the roles of CCR4-NOT complex-mediated in ALI remain elusive. Methods: The effects of Cnot3 haploinsufficiency in the pathology and cytokine expression were analyzed in the mouse lungs of acid aspiration-induced acute lung injury. The decay rate and transcription activity of cytokine mRNAs under Cnot3 heterozygous deletion were analyzed in lipopolysaccharide (LPS) -stimulated mouse embryonic fibroblasts (MEFs). Results: Tamoxifen-induced heterozygous deletion of Cnot3 in adult mice (Cnot3 Hetz) did not show body weight loss or any apparent abnormality. Under acid aspiration-induced acute lung injury, Cnot3 Hetz mice exhibited increased pulmonary edema, worse lung pathologies and more severe inflammation compared with wild type mice. mRNA expression of pro-inflammatory genes Il1b and Nos2 were significantly upregulated in the lungs of Cnot3 Hetz mice. Consistently, mRNA expression of Il1b and Nos2 was upregulated in LPS-stimulated Cnot3 Hetz MEFs. Mechanistically, while heterozygous depletion of Cnot3 stabilized both Il1b and Nos2 mRNAs, the nascent pre-mRNA level of Il1b was upregulated in Cnot3 Hetz MEFs, implicating Cnot3-mediated transcriptional repression of Il1b expression in addition to destabilization of Il1b and Nos2 mRNAs. PU.1 (Spi1) was identified as a causative transcription factor to promote Il1b expression under Cnot3 haploinsufficient conditions. Conclusion: CNOT3 plays a protective role in ALI by suppressing expression of pro-inflammatory genes Il1b and Nos2 through both post-transcriptional and transcriptional mechanisms, including mRNA stability control of Spi1.

ACE2-like enzyme B38-CAP suppresses abdominal sepsis and severe acute lung injury.

Angiotensin-converting enzyme 2 (ACE2) is the carboxypeptidase to degrade angiotensin II (Ang II) to angiotensin 1-7 (Ang 1-7) and improves the pathologies of cardiovascular disease and acute respiratory distress syndrome (ARDS)/acute lung injury. B38-CAP is a bacteria-derived ACE2-like carboxypeptidase as potent as human ACE2 and ameliorates hypertension, heart failure and SARS-CoV-2-induced lung injury in mice. Recombinant B38-CAP is prepared with E. coli protein expression system more efficiently than recombinant soluble human ACE2. Here we show therapeutic effects of B38-CAP on abdominal sepsis- or acid aspiration-induced acute lung injury. ACE2 expression was downregulated in the lungs of mice with cecal ligation puncture (CLP)-induced sepsis or acid-induced lung injury thereby leading to upregulation of Ang II levels. Intraperitoneal injection of B38-CAP significantly decreased Ang II levels while upregulated angiotensin 1-7 levels. B38-CAP improved survival rate of the mice under sepsis. B38-CAP suppressed the pathologies of lung inflammation, improved lung dysfunction and downregulated elevated cytokine mRNA levels in the mice with acute lung injury. Thus, systemic treatment with an ACE2-like enzyme might be a potential therapeutic strategy for the patients with severe sepsis or ARDS.

Identification of Galectin-7 as a crucial metastatic enhancer of squamous cell carcinoma associated with immunosuppression.

Metastasis predicts poor prognosis in cancer patients. It has been recognized that specific tumor microenvironment defines cancer cell metastasis, whereas the underlying mechanisms remain elusive. Here we show that Galectin-7 is a crucial mediator of metastasis associated with immunosuppression. In a syngeneic mouse squamous cell carcinoma (SCC) model of NR-S1M cells, we isolated metastasized NR-S1M cells from lymph nodes in tumor-bearing mice and established metastatic NR-S1M cells in in vitro culture. RNA-seq analysis revealed that interferon gene signature was markedly downregulated in metastatic NR-S1M cells compared with parental cells, and in vivo NR-S1M tumors heterogeneously developed focal immunosuppressive areas featured by deficiency of anti-tumor immune cells. Spatial transcriptome analysis (Visium) for the NR-S1M tumors revealed that various pro-metastatic genes were significantly upregulated in immunosuppressive areas when compared to immunocompetent areas. Notably, Galectin-7 was identified as a novel metastasis-driving factor. Galectin-7 expression was induced during tumorigenesis particularly in the microenvironment of immunosuppression, and extracellularly released at later stage of tumor progression. Deletion of Galectin-7 in NR-S1M cells significantly suppressed lymph node and lung metastasis without affecting primary tumor growth. Therefore, Galectin-7 is a crucial mediator of tumor metastasis of SCC, which is educated in the immune-suppressed tumor areas, and may be a potential target of cancer immunotherapy.

ACE2-like carboxypeptidase B38-CAP protects from SARS-CoV-2-induced lung injury.

Angiotensin-converting enzyme 2 (ACE2) is a receptor for cell entry of SARS-CoV-2, and recombinant soluble ACE2 protein inhibits SARS-CoV-2 infection as a decoy. ACE2 is a carboxypeptidase that degrades angiotensin II, thereby improving the pathologies of cardiovascular disease or acute lung injury. Here we show that B38-CAP, an ACE2-like enzyme, is protective against SARS-CoV-2-induced lung injury. Endogenous ACE2 expression is downregulated in the lungs of SARS-CoV-2-infected hamsters, leading to elevation of angiotensin II levels. Recombinant Spike also downregulates ACE2 expression and worsens the symptoms of acid-induced lung injury. B38-CAP does not neutralize cell entry of SARS-CoV-2. However, B38-CAP treatment improves the pathologies of Spike-augmented acid-induced lung injury. In SARS-CoV-2-infected hamsters or human ACE2 transgenic mice, B38-CAP significantly improves lung edema and pathologies of lung injury. These results provide the first in vivo evidence that increasing ACE2-like enzymatic activity is a potential therapeutic strategy to alleviate lung pathologies in COVID-19 patients.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Pulmonary phagocyte-derived NPY controls the pathology of severe influenza virus infection.

Crosstalk between the autonomic nervous system and the immune system by means of the sympathetic and parasympathetic pathways is a critical process in host defence. Activation of the sympathetic nervous system results in the release of catecholamines as well as neuropeptide Y (NPY). Here, we investigated whether phagocytes are capable of the de novo production of NPY, as has been described for catecholamines. We show that the synthesis of NPY and its Y1 receptor (Y1R) is increased in phagocytes in lungs following severe influenza virus infection. The genetic deletion of Npy or Y1r specifically in phagocytes greatly improves the pathology of severe influenza virus infection, which is characterized by excessive virus replication and pulmonary inflammation. Mechanistically, it is the induction of suppressor of cytokine signalling 3 (SOCS3) via NPY-Y1R activation that is responsible for impaired antiviral response and promoting pro-inflammatory cytokine production, thereby enhancing the pathology of influenza virus infection. Thus, direct regulation of the NPY-Y1R-SOCS3 pathway on phagocytes may act as a fine-tuner of an innate immune response to virus infection, which could be a therapeutic target for lethal influenza virus infection.