PD-L2 mediates tobacco smoking-induced recruitment of regulatory T cells via the RGMB/NFκB/CCL20 cascade.

Programmed cell death ligand 2 (PD-L2), a ligand for the receptor programmed cell death 1 (PD-1), has an identity of 34% with its twin ligand PD-L1 and exhibits higher binding affinity with PD-1 than PD-L1. However, the role of PD-L2 in non-small cell lung cancer (NSCLC) progression, especially tobacco-induced cancer progression, has not been fully understood. Here, we found that PD-L2 promoted tumor growth in murine models with recruitment of regulatory T cells (Tregs). In patients with NSCLC, PD-L2 expression level in tumor samples was higher than in counterpart normal controls and was positively associated with patients' response to anti-PD-1 treatment. Mechanismly, PD-L2 bound its receptor Repulsive guidance molecule B (RGMB) on cancer cells and activated extracellular signal-regulated kinase (Erk) and nuclear factor κB (NFκB), leading to increased production of chemokine CCL20, which recruited Tregs and contributed to NSCLC progression. Consistently, knockdown of RGMB or NFκB p65 inhibited PD-L2-induced CCL20 production, and silencing of PD-L2 repressed Treg recruitment by NSCLC cells. Furthermore, cigarette smoke and carcinogen benzo(a)pyrene (BaP) upregulated PD-L2 in lung epithelial cells via aryl hydrocarbon receptor (AhR)-mediated transcription activation, whose deficiency markedly suppressed BaP-induced PD-L2 upregulation. These results suggest that PD-L2 mediates tobacco-induced recruitment of Tregs via the RGMB/NFκB/CCL20 cascade, and targeting this pathway might have therapeutic potentials in NSCLC.

Oncogenic functions and therapeutic potentials of targeted inhibition of SMARCAL1 in small cell lung cancer.

Small cell lung cancer (SCLC) is a recalcitrant cancer characterized by high frequency loss-of-function mutations in tumor suppressors with a lack of targeted therapy due to absence of high frequency gain-of-function abnormalities in oncogenes. SMARCAL1 is a member of the ATP-dependent chromatin remodeling protein SNF2 family that plays critical roles in DNA damage repair and genome stability maintenance. Here, we showed that SMARCAL1 was overexpressed in SCLC patient samples and was inversely associated with overall survival of the patients. SMARCAL1 was required for SCLC cell proliferation and genome integrity. Mass spectrometry revealed that PAR6B was a downstream SMARCAL1 signal molecule which rescued inhibitory effects caused by silencing of SMARCAL1. By screening of 36 FDA-approved clinically available agents related to DNA damage repair, we found that an aza-anthracenedione, pixantrone, was a potent SMARCAL1 inhibitor which suppressed the expression of SMARCAL1 and PAR6B at protein level. Pixantrone caused DNA damage and exhibited inhibitory effects on SCLC cells in vitro and in a patient-derived xenograft mouse model. These results indicated that SMARCAL1 functions as an oncogene in SCLC, and pixantrone as a SMARCAL1 inhibitor bears therapeutic potentials in this deadly disease.

CIP2A induces PKM2 tetramer formation and oxidative phosphorylation in non-small cell lung cancer.

Tumor cells are usually considered defective in mitochondrial respiration, but human non-small cell lung cancer (NSCLC) tumor tissues are shown to have enhanced glucose oxidation relative to adjacent benign lung. Here, we reported that oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) inhibited glycolysis and promoted oxidative metabolism in NSCLC cells. CIP2A bound to pyruvate kinase M2 (PKM2) and induced the formation of PKM2 tetramer, with serine 287 as a novel phosphorylation site essential for PKM2 dimer-tetramer switching. CIP2A redirected PKM2 to mitochondrion, leading to upregulation of Bcl2 via phosphorylating Bcl2 at threonine 69. Clinically, CIP2A level in tumor tissues was positively correlated with the level of phosphorylated PKM2 S287. CIP2A-targeting compounds synergized with glycolysis inhibitor in suppressing cell proliferation in vitro and in vivo. These results indicated that CIP2A facilitates oxidative phosphorylation by promoting tetrameric PKM2 formation, and targeting CIP2A and glycolysis exhibits therapeutic potentials in NSCLC.

Tobacco carcinogen induces tryptophan metabolism and immune suppression via induction of indoleamine 2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1), the enzyme that catabolizes tryptophan (Trp) metabolism to promote regulatory T cells (Tregs) and suppress CD8+ T cells, is regulated by several intrinsic signaling pathways. Here, we found that tobacco smoke, a major public health concern that kills 8 million people each year worldwide, induced IDO1 in normal and malignant lung epithelial cells in vitro and in vivo. The carcinogen nicotine-derived nitrosaminoketone (NNK) was the tobacco compound that upregulated IDO1 via activation of the transcription factor c-Jun, which has a binding site for the IDO1 promoter. The NNK receptor α7 nicotinic acetylcholine receptor (α7nAChR) was required for NNK-induced c-Jun activation and IDO1 upregulation. In A/J mice, NNK reduced CD8+ T cells and increased Tregs. Clinically, smoker patients with non-small-cell lung cancer (NSCLC) exhibited high IDO1 levels and low Trp/kynurenine (Kyn) ratios. In NSCLC patients, smokers with lower IDO1 responded better to anti-PD1 antibody treatment than those with higher IDO1. These data indicate that tobacco smoke induces IDO1 to catabolize Trp metabolism and immune suppression to promote carcinogenesis, and lower IDO1 might be a potential biomarker for anti-PD1 antibodies in smoker patients, whereas IDO1-high smoker patients might benefit from IDO1 inhibitors in combination with anti-PD1 antibodies.

Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of COVID-19 pathogen SARS-CoV-2, but the transcription factors (TFs) that regulate the expression of the gene encoding ACE2 (ACE2) have not been systematically dissected. In this study we evaluated TFs that control ACE2 expression, and screened for small molecule compounds that could modulate ACE2 expression to block SARS-CoV-2 from entry into lung epithelial cells. By searching the online datasets we found that 24 TFs might be ACE2 regulators with signal transducer and activator of transcription 3 (Stat3) as the most significant one. In human normal lung tissues, the expression of ACE2 was positively correlated with phosphorylated Stat3 (p-Stat3). We demonstrated that Stat3 bound ACE2 promoter, and controlled its expression in 16HBE cells stimulated with interleukin 6 (IL-6). To screen for medicinal compounds that could modulate ACE2 expression, we conducted luciferase assay using HLF cells transfected with ACE2 promoter-luciferase constructs. Among the 64 compounds tested, 6-O-angeloylplenolin (6-OAP), a sesquiterpene lactone in Chinese medicinal herb Centipeda minima (CM), represented the most potent ACE2 repressor. 6-OAP (2.5 µM) inhibited the interaction between Stat3 protein and ACE2 promoter, thus suppressed ACE2 transcription. 6-OAP (1.25-5 µM) and its parental medicinal herb CM (0.125%-0.5%) dose-dependently downregulated ACE2 in 16HBE and Beas-2B cells; similar results were observed in the lung tissues of mice following administration of 6-OAP or CM for one month. In addition, 6-OAP/CM dose-dependently reduced IL-6 production and downregulated chemokines including CXCL13 and CX3CL1 in 16HBE cells. Moreover, we found that 6-OAP/CM inhibited the entry of SARS-CoV-2 S protein pseudovirus into target cells. These results suggest that 6-OAP/CM are ACE2 inhibitors that may potentially protect lung epithelial cells from SARS-CoV-2 infection.

Mutations and clinical significance of calcium voltage-gated channel subunit alpha 1E (CACNA1E) in non-small cell lung cancer.

CACNA1E is a gene encoding the ion-conducting α1 subunit of R-type voltage-dependent calcium channels, whose roles in tumorigenesis remain to be determined. We previously showed that CACNA1E was significantly mutated in patients with non-small cell lung cancer (NSCLC) who were long-term exposed to household air pollution, with a mutation rate of 19% (15 of 79 cases). Here we showed that CACNA1E was also mutated in 207 (12.8%) of the 1616 patients with NSCLC in The Cancer Genome Atlas (TCGA) datasets. At mRNA and protein levels, CACNA1E was elevated in tumor tissues compared to counterpart non-tumoral lung tissues in NSCLCs of the public datasets and our settings, and its expression level was inversely associated with clinical outcome of the patients. Overexpression of wild type (WT) or A275S or R249G mutant CACNA1E transcripts promoted NSCLC cell proliferation with activation of epidermal growth factor receptor (EGFR) signaling pathway, whereas knockdown of this gene exerted inhibitory effects on NSCLC cells in vitro and in vivo. CACNA1E increased current density and Ca2+ entrance, whereas calcium channel blockers inhibited NSCLC cell proliferation. These data indicate that CACNA1E is required for NSCLC cell proliferation, and blockade of this oncoprotein may have therapeutic potentials for this deadly disease.

Comprehensive analysis of BTN3A1 in cancers: mining of omics data and validation in patient samples and cellular models.

Butyrophilin 3A1 (BTN3A1), a major histocompatibility complex-associated gene that encodes a membrane protein with two extracellular immunoglobulin domains and an intracellular B30.2 domain, is critical in T-cell activation and adaptive immune response. Here, the expression of BTN3A1 in cancers was analyzed in eight databases comprising 86 733 patients of 33 cancers, and the findings were validated in patient samples and cell models. We showed that BTN3A1 was expressed in most cancers, and its expression level was strongly correlated with clinical outcome of 13 cancers. Mutations of BTN3A1 were detected, and the mutations were distributed throughout the entire gene. Gene set enrichment analysis showed that BTN3A1 co-expression genes and interacting proteins were enriched in immune regulation-related pathways. BTN3A1 was associated with tumor-infiltrating immune cells and was co-expressed with multiple immune checkpoints in patients with breast cancer (BRCA) and non-small cell lung cancer (NSCLC). We reported that BTN3A1 was downregulated in 46 of 65 (70.8%) NSCLCs, and its expression level was inversely associated with clinical outcome of the patients. BTN3A1 in tumor samples was lower than in counterpart normal tissues in 31 of 38 (81.6%) BRCAs. Bioinformatics analyses showed that BTN3A1 could be a target gene of transcription factor Spi-1 proto-oncogene (SPI1), and our 'wet' experiments showed that ectopic expression of SPI1 upregulated, whereas silencing of SPI1 downregulated, BTN3A1 expression in cells. These results suggest that BTN3A1 may function as a tumor suppressor and may serve as a potential prognostic biomarker in NSCLCs and BRCAs.

[Pharmacokinetics of SN-38 in rats and tissue distribution of 7-ethyl-10-hydroxycamptothecin in mice after intravenous injection of irinotecan hydrochloride nanoparticles].

The paper reported an investigation of the pharmacokinetics of SN-38 (7-ethyl-10-hydroxy-camptothecin) in rats and the tissue distribution in mice after injection of irinotecan hydrochloride nanoparticles (CPT-11) via tail veins. An LC-MS/MS method was established to determine the concentrations of SN-38 in whole blood of rats and in different tissues of mice. The pharmacokinetics and tissue distribution of SN-38 were compared after the intravenous injection of CPT-11 NPs and CPT-11 solution. Compared with irinotecan solution, the elimination half-life of SN-38 was prolonged from 2.17 h to 2.67 h after the intravenous injection of CPT-11 NPs, but its AUC had little change. After the injection of CPT-11 NPs in mice, over time, the concentrations of CPT-11-metabolized SN-38 in CPT-11 NPs were significantly higher in the whole blood, colon and lungs than those in CPT-11 solution, followed by in the spleen and liver, but those in the heart and brain had no change. However, the amount of SN-38 in the kidneys was reduced with time. CPT-11 NPs could prolong SN-38's (one of its metabolites) blood circulation time in rats and significantly increased the concentration of CPT-11-metabolized SN-38 in the whole blood, colon and lungs of mice. CPT-11 NPs made SN-38 efficiently target-bind to the colon and lungs of mice.

[Pharmacokinetics and tissue distribution of irinotecan hydrochloride nanoparticles].

To investigate the pharmacokinetics of irinotecan hydrochloride (CPT-11) in rats and the tissue distribution of CPT-11 in mice after injection of irinotecan hydrochloride nanoparticles (CPT-11 NPs) via tail veins, separately, a LC-MS/MS method was established to determine the concentration of CPT-11 in whole blood of rats and in different tissues of mice. The pharmacokinetics and tissue distribution of CPT-11 were compared after the intravenous injection of CPT-11 NPs and CPT-11 solution. Compared with CPT-11 solution, the elimination half-life of CPT-11 was prolonged from 2.28 h to 3.95 h after the intravenous injection of CPT-11 NPs, and its AUC was 1.47 times than that of CPT-11 solution. After the injection of CPT-11 NPs in mice, the concentrations of CPT-11 loaded in CPT-11 NPs were significantly higher in the whole blood, colon and lungs than those in CPT-11 solution, but lower in the spleen, liver, kidney and heart, but the least in brain. CPT-11 NPs could improve CPT-11 's AUC, and help CPT-11 to reach long circulation activity.

[Pharmacokinetic interaction of pioglitazone hydrochloride and atorvastatin calcium in Beagle dogs].

The object of this study is to investigate the pharmacokinetic interaction of pioglitazone hydrochloride and atorvastatin calcium in healthy adult Beagle dogs following single and multiple oral dose administration. A randomized, cross-over study was conducted with nine healthy adult Beagle dogs assigned to three groups. Each group was arranged to take atorvastatin calcium (A), pioglitazone hydrochloride (B), atorvastatin calcium and pioglitazone hydrochloride (C) orally in the first period, to take B, C, A in the second period, and to take C, A, B in the third period for 6 days respectively. The blood samples were collected at the first and the sixth day after the administration, plasma drug concentrations were determined by LC-MS/MS, a one-week wash-out period was needed between each period. The pharmacokinetic parameters of drug combination group and the drug alone group were calculated by statistical moment method, calculation of C(max) and AUC(0-t) was done by using 90% confidence interval method of the bioequivalence and bioavailability degree module DAS 3.2.1 software statistics. Compared with the separate administration, the main pharmacokinetic parameters (C(max) and AUC(0-t)) of joint use of pioglitazone hydrochloride and atorvastatin calcium within 90% confidence intervals for bioequivalence statistics were unqualified, the mean t(max) with standard deviation used paired Wilcoxon test resulted P > 0.05. There was no significant difference within t1/2, CL(int), MRT, V/F. Pioglitazone hydrochloride and atorvastatin calcium had pharmacokinetic interaction in healthy adult Beagle dogs.

Prevalence of subclinical hepatic encephalopathy in cirrhotic patients in China.

AIM: Subclinical hepatic encephalopathy (SHE) is a common complication of liver diseases. The aim of this study was to find out the normal value of psychometric test and to investigate the prevalence of SHE in Chinese patients with stabilized hepatic cirrhosis. METHODS: Four hundred and nine consecutive cirrhotic patients without overt clinical encephalopathy were screened for SHE by using number connection test part A (NCT-A) and symbol digit test (SDT). SHE was defined as presence of at least one abnormal psychometric test. The age-corrected normal values were defined as the mean+/-2 times standard deviation (2SD), and developed in 356 healthy persons as normal controls. Four hundred and sixteen patients with chronic viral hepatitis were tested as negative controls to assess the diagnostic validity of this test battery. RESULTS: There was no significant difference in NCT scores and SDT quotients between healthy controls and chronic hepatitis group (P>0.05). In all age subgroups, the NCT and SDT measurements of cirrhotic patients differed significantly from those of the controls (P<0.05). When mean+/-2SD of SDT and NCT measurements from healthy control group was set as the normal range, 119 cirrhotic patients (29.1%) were found to have abnormal NCT-A and SDT tests, 53 (13.0%) were abnormal only in SDT and 36 (8.8%) only in NCT-A. Taken together, SHE was diagnosed in 208 (50.9%) cirrhotic patients by this test battery. The prevalence of SHE increased from 39.9% and 55.2% in Child-Pugh's grade A and B groups to 71.8% in Child-Pugh's grade C group (P<0.05). After the adjustment of age and residential areas required from the tests, no correlation was found in the rate of SHE and causes of cirrhosis, education level and smoking habit. CONCLUSION: Psychometric tests are simple and reliable indicators for screening SHE among Chinese cirrhotic patients. By using a NCT and SDT battery, SHE could be found in 50.9% of cirrhotic patients without overt clinical encephalopathy. The prevalence of SHE is significantly correlated with the severity of liver functions.