c-Rel is a Novel Oncogene in Lung Squamous Cell Carcinoma Regulating Cell Proliferation and Migration.

Lung squamous cell carcinoma (LUSC) accounts for approximately 25% to 30% of lung cancers, but largely no targeted therapy is available against it, calling for identification of new oncogenes in LUSC growth for new therapeutic targets. In this study, REL was identified through a screening for oncogenes that are highly amplified in human LUSC. Its expression was associated with poor prognosis in LUSC patients. Furthermore, knockdown of c-Rel in LUSC cell lines lead to significant decrease in cell proliferation and migration. Mechanistically, c-Rel knockdown suppressed NFκB pathway by blocking phosphorylation of IκB. Consistently, pharmaceutic inhibition of c-Rel also. In orthotopic xenograft lung cancer mouse model, c-Rel knockdown inhibited the tumor growth. Cancer cell proliferation and epithelial-mesenchymal-transition (EMT) of the tumors were impaired by c-Rel knockdown. Finally, it's confirmed in precision-cut tumor slices of LUSC that deletion of c-Rel inhibits the NFκB pathway and cancer cell growth. Accordingly, we hypothesize that c-Rel promotes the activation of the NFκB pathway by promoting the phosphorylation of IκB in LUSC. Our study reveals REL as a novel LUSC oncogene and provides new insights into the molecular regulation of LUSC, which will provide new therapeutic targets for the treatment of squamous lung cancer.

Influence of Smoking Habits on the Efficacy of EGFR-TKI Therapy in Patients with Advanced NSCLC: A Systematic Review and Meta-Analysis.

Background: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are considered as the first-line treatment for advanced EGFR mutation-positive non-small cell lung cancer (NSCLC). We aimed to analyze the efficacy of EGFR-TKIs treatment in patients with advanced NSCLC of different smoking habits. Methods: We conducted a search for meta-analyses and systematic reviews on the PubMed, MEDLINE, Embase, and the Cochrane Library to address this knowledge gap. Patients were divided into 2 groups: (1) experimental group: treated with EGFR-TKIs or EGFR-TKIs combined with chemotherapy, immunotherapy, antiangiogenesis, radiotherapy and (2) control group: treated with chemotherapy. Progressive-free survival (PFS) and total survival (OS) were adopted for evaluating the efficacy of EGFR-TKIs between experimental group and control group. Results: Eleven studies including 6760 patients were included in the meta-analysis. The results showed that smoking (including previous and current smoking) significantly reduces the PFS and OS in comparison to non-smoking group in the treatment of NSCLC with EGFR-TKIs. In addition, EGFR-TKIs combined with anti-vascular endothelial growth factor therapy can reduce the risk of disease progression in smokers. Conclusions: Our study indicated that smoking significantly reduced the PFS and OS in comparison to non-smoking group in the treatment of NSCLC with EGFR-TKIs.

LncRNA HHIP-AS1 suppresses lung squamous cell carcinoma by stabilizing HHIP mRNA.

AIMS: Lung squamous cell carcinoma (LUSC) causes over 400,000 deaths annually, yet it lacks targeted therapy. A major antagonist of Hedgehog pathway, HHIP (Hedgehog Interacting Protein) plays an important role in LUSC; however, the regulatory mechanism remains unclear. Long non-coding RNA HHIP-AS1 plays suppressive or promotive roles in different cancers, but its role in LUSC remains unknown. This manuscript is to investigate regulatory mechanism of HHIP and the role of HHIP-AS1 in LUSC. MAIN METHODS: Precision-cut lung slices (PCLS) from human LUSC samples are cultured to mimic LUSC growth. Overexpression and knockdown in multiple LUSC cell lines and PCLS are achieved by lentivirus infection. Transcriptome profile and lung cancer activity are evaluated by RNA-sequencing, immunostaining and CCK8 assay etc. KEY FINDINGS: HHIP is regulated independently of Hh pathway in LUSC. Additionally, downregulation of HHIP-AS1 is associated with poor prognosis. Consistently, HHIP-AS1 inhibits LUSC growth by suppressing cell proliferation and migration. Transcriptome profiling of HHIP-AS1 knockdown (KD) cells uncovered HHIP downregulation. Interestingly, a comparison between the transcriptomes of HHIP-AS1 KD or HHIP KD cells manifested high similarity. Subsequently it's confirmed that HHIP-AS1 regulates HHIP in LUSC cells. Notably, HHIP-AS1 regulation on LUSC growth is achieved through stabilizing HHIP mRNA rather than regulating MIR-153-3P/PCDHGA9 or MIR-425-5P/DNYC1I2. Finally, it's confirmed in PCLS from human LUSC samples that HHIP-AS1 suppresses LUSC via regulating HHIP mRNA. SIGNIFICANCE: This study uncovers HHIP-AS1 as a novel tumor suppressor in LUSC and provides new insights into the molecular regulation of LUSC, which will help developing new therapeutic strategies.

Sex-biased molecular differences in lung adenocarcinoma are ethnic and smoking specific.

BACKGROUND: Sex-related differences in cancer epidemiology, tumor biology, immune system activity, and pharmacogenomics have been suggested to be important considerations for precision cancer control. Here we elucidated systematically sex biases in genetic variants, gene expression profiles, and immunological landscapes of lung adenocarcinoma patients (LUADs) with different ancestry and smoking status. METHODS: Somatic mutation and mRNA expression data of Asian and Non-Asian LUADs were obtained from public databases. Sex-biased genetic mutations, gene expression, biological pathways, and immune infiltration were identified in the context of smoking status and race. RESULTS: Among nonsmokers, male-biased mutations were prevalent in Asian LUADs, while few sex-biased mutations were detected in Non-Asian LUADs. EGFR was the only mutation whose frequency was significantly higher in females than males in both Asian and Non-Asian nonsmokers. More genes exhibited sex-biased expression in Non-Asian LUADs compared to Asian LUADs. Moreover, genes distinctly expressed in females were mainly related to immune-related pathways, whereas those in males were more involved in activation of DNA repair, E2F_targets, and MYC_targets pathways. We also detected sex-specific immune infiltration in the context of genetic variation. In EGFR-mutant LUADs, males had a significantly increased infiltration of CD8 + T cells, whereas resting CD4 + memory T cells were more abundant in females. Additionally, in KRAS-mutant LUADs, CD8 + and CD4 + T cells were more abundant in females than males. In addition, we detected all female patients with high SCGB3A2 expression were exclusively sensitive to immunotherapy, while this phenomenon was not observed in male patients. CONCLUSIONS: Our findings provided evidence that sex-related molecular and cellular components are involved in shaping tumor distinct genetic and immune features, which might have important impact on personalized targeted and immune therapy.

Immunogenicity of small-cell lung cancer associates with STING pathway activation and is enhanced by ATR and TOP1 inhibition.

INTRODUCTION: The activation of STING (stimulator of interferon genes) pathway enhances antitumor immunity in small-cell lung cancer (SCLC), while the DNA damage induced by non-cGAMP-based agonists is a potent inducer of STING activity. Here, we investigate the intrinsic expression of STING in cancer cells and evaluate the value of the combination of ATR and TOP1 inhibitors in enhancing antitumor immunity. METHODS: STING expression was assessed at mRNA and protein levels in SCLC and normal lung tissues. Transcriptomic subsets of SCLC were identified based on STING-related genes. Distinct mutation and immunogenomic profiles of these subsets were determined. The direct antitumor efficacy and the potential of enhancing antitumor immunity of the strategy using the ATR-TOP1-inhibitor combination were tested in SCLC cell lines. RESULTS: The intrinsic expression of STING was significantly reduced in SCLC compared to normal lung tissues (p < 0.0001). Three STING-related SCLC subtypes were identified in which the STING-high subtype was associated with (1) high immune infiltration, (2) high expression of genes related to MHC and immune checkpoints, and (3) high EMT and ferroptosis score. On the contrary, the STING-low subtype was enriched with pathways related to DNA damage response (DDR) and cell cycle progression. The association between the DDR pathway activity and the STING-IFN innate immune response was verified by in vitro experiments in which the inhibition of ATR and TOP1 triggered the expression of genes encoding type I IFN signaling and pro-inflammatory cytokines/chemokines in a STING-low SCLC cell line. CONCLUSION: Our study verifies that activation of the STING-IFN response by ATR and TOP1 inhibitors might be a therapeutic strategy to improve the response to immune checkpoint therapy in STING-low SCLC. Furthermore, the combinations of ATR and TOP1 inhibitors can augment tumor inflammation in STING-low SCLC.

Genomic features of Chinese small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is an aggressive disease with poor survival. Although molecular and clinical characteristics have been established for SCLC in western patients, limited investigation has been performed for Chinese SCLC patients. OBJECTIVE: In this study, we investigated the genomic features of Chinese SCLC patients. METHODS: A total of 75 SCLC patients were enrolled. Genomic alterations in 618 selected genes were analyzed by targeted next-generation sequencing. RESULTS: Here, we showed that TP53 (77.30%) and RB1 (30.70%) were the most prevalent genes alterations, followed by KMT2D, ALK, LRP1B, EGFR, NOTCH3, AR, CREBBP, ROS1, and BRCA2. And the most common genetic alterations were enriched in the cell cycle signaling pathway (84.00%) of Chinese SCLC patients. DNA damage repair (DDR) pathway analysis showed that the most frequently enriched DDR pathways were fanconi anaemia (FA, 29.41%) and homology recombination (HR, 21.57%). Notably, 9.33% SCLC patients in our cohort had pathogenic or likely pathogenic germline gene variants. Compared with the U Cologne cohort, a higher prevalence in EGFR, AR, BRCA2, TSC1, ATXN3, MET, MSH2, ERBB3 and FOXA1 were found in our cohort; while compared to the data from the Johns Hopkins cohort, a higher mutated frequency in TP53, KMT2D, ALK, and EGFR were found in our cohort. Moreover, a significant association was found between high tumor mutation burden (TMB) and mutations involved in TP53, CREBBP, EPHA3, KMT2D, ALK and RB1. Approximately 33.33% of patients with SCLC harbored at least one actionable alteration annotated by OncoKB, of which one patient had alterations of level 1; seventeen patients had level 3; fifteen patients possessed level 4. CONCLUSION: Our data might provide an insightful meaning in targeted therapy for Chinese SCLC patients.

Life-threatening hemoptysis accompanied by internal thoracic artery aneurysms in a 28-year-old perinatal woman: a case report.

BACKGROUND: Life-threatening hemoptysis presents an immediate diagnostic and therapeutic challenge, especially during the perinatal period. CASE PRESENTATION: A 28-year-old perinatal woman with no significant past medical or surgical history presented with repeating hemoptysis and respiratory failure. Computed tomography revealed a 2.1 × 3.2  cm2 inhomogeneous tumorous lesion in the right superior mediastinum and a right main bronchus obstruction along with atelectasis of the right lung. Bronchoscopy showed a tumorous protrusion blocking the right main bronchus with active hemorrhage, and malignancy was suspected. Bronchial artery embolization (BAE) was performed to control the bleeding. The arteriogram revealed tortuosity, dilation and hypertrophy of the right bronchial arteries and aneurysms of the internal thoracic artery (ITA). The bleeding completely stopped after BAE. Bronchoscopy was performed again to remove residual blood clots. The patient recovered soon after the procedure and was discharged. CONCLUSIONS: Life-threatening hemoptysis concomitant with ITA aneurysms, which may have a misleading clinical diagnosis and treatment options, has not been reported previously in perinatal women. BAE could be used as a first-line treatment irrespective of the underlying causes.

Knockdown of the lncRNA MALAT1 alleviates lipopolysaccharide­induced A549 cell injury by targeting the miR­17­5p/FOXA1 axis.

Long­noncoding RNAs (lncRNAs) are crucial for the pathophysiology of acute lung injury (ALI). Metastasis­associated lung adenocarcinoma transcript 1 (MALAT1) suppresses inflammatory responses via microRNA (miR)­146a in lipopolysaccharide (LPS)­induced ALI. However, the molecular mechanisms underlying the MALAT1­mediated regulation of cell proliferation and apoptosis in LPS­induced ALI remain unclear. In the present study, it was found that LPS treatment upregulated MALAT1 expression and suppressed the proliferation of A549 cells. MALAT1 knockdown significantly promoted the proliferation and G1/S phase transition and inhibited apoptosis in LPS­treated A549 cells. In addition, miR­17­5p was a direct target of MALAT1. miR­17­5p expression was downregulated and FOXA1 expression was upregulated in LPS­treated A549 cells. Further, MALAT1 knockdown promoted miR­17­5p expression and inhibited FOXA1 expression, whereas the combined suppression of MALAT1 and miR­17­5p induced FOXA1 expression. Moreover, miR­17­5p knockdown reversed the effects of MALAT1 suppression on LPS­induced A549 cell proliferation. These results indicated that MALAT1 serves as a competing endogenous lncRNA that, by sequestering miR­17­5p, stimulates FOXA1 expression and mediates LPS­induced A549 cell injury. In conclusion, the present study demonstrated that MALAT1 knockdown alleviates LPS­induced A549 cell injury by targeting the miR­17­5p/FOXA1 axis.

LncRNA MALAT1 contributes to non-small cell lung cancer progression via modulating miR-200a-3p/programmed death-ligand 1 axis.

This study was to investigate the expression correlation between long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1), miR-200a-3p and programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC), and their roles in NSCLC. Real-time polymerase chain reaction (PCR) was performed to detect the expressions of MALAT1, miR-200a-3p and PD-L1 in NSCLC tissues and cells for the correlation analysis. The starBase and Targetscan databases were used to predict the binding sites between MALAT1 and miR-200a-3p, and miR-200a-3p and PD-L1, respectively. The targeting relationship between MALAT1 and miR-200a-3p, and miR-200a-3p and PD-L1 were further verified by real-time PCR and dual luciferase reporter gene assay. Cell proliferation was monitored by CCK8 and colony formation assays. The apoptosis was detected using flow cytometry. Wound healing assay and transwell assay were conducted to determine cell migration and invasion. In this study, we demonstrated that in NSCLC tissues, the expression level of MALAT1 was negatively correlated with that of miR-200a-3p, while positively correlated with PD-L1. Besides, MALAT1 promoted proliferation, mobility, migration, and invasion of NSCLC cells via sponging miR-200a-3p. PD-L1 was validated as a target of miR-200a-3p, and indirectly modulated by MALAT1. In conclusion, LncRNA MALAT1 facilitates the progression of NSCLC by modulating miR-200a-3p/PDL1 axis.

WISP-3/CCN6 inhibits apoptosis by regulating caspase pathway after hyperoxia in lung epithelial cells.

Cell death is a normal phenomenon in the course of biological development, moreover, which is also a prominent feature in lung exposed to hyperoxia. Severe hypoxia occurs in ALI/ARDS patients, who generally require high concentration oxygen therapy assisted by mechanical ventilation. Nevertheless, high oxygen can cause excessive reactive oxygen species (ROS), leading to apoptosis in lung epithelial cells, which has been reported in our previous study. Herein, the correlation between increments of ROS and CCN6 expression was negative in CCN6-mediated the mitochondria dependent, intrinsic apoptotic pathway. Our latest research explained that CCN6 can inhibit caspase-8 mediated extrinsic apoptotic pathway to protect cells from hyperoxia-induced apoptosis. As demonstrated by Western Blot Analysis, Caspase 8 cleavage and Caspase 3 cleavage in CCN6-depleted cells exceeded the control group treated with high oxygen (48 h). And deletion of CCN6 enhanced caspase-8 activation after hyperoxia shown by Flow Cytometry. Although, it is unclear how CCN6 participated in the regulation of apoptotic pathways, the future targeted therapy drugs inhibiting CCN6 may be useful in the treatment of ALI/ARDS.

In vitro synergistic effect of amlodipine and imipenem on the expression of the AdeABC efflux pump in multidrug-resistant Acinetobacter baumannii.

INTRODUCTION: Multidrug-resistant Acinetobacter baumannii (A. baumannii) has become one of the greatest threats worldwide to the therapeutic management of infections. Our previous research confirmed an in vitro synergistic effect of amlodipine and imipenem against A. baumannii, and this study is designed to understand its mechanism. METHODS: Sixty-four non-duplicate A. baumannii isolates were collected and tested for antimicrobial susceptibility by the disk diffusion method. PCR amplification and sequencing were used to identify the presence of the adeB, adeE, adeH, adeJ, abeM and abeS efflux pump genes. The minimal inhibitory concentrations of imipenem, imipenem+amlodipine and imipenem+carbonyl cyanide m-chlorophenyl-hydrazone against these isolates were also determined by the broth microdilution method before and after siRNA silencing of the expression of the adeABC efflux pump. RESULTS: In this study, the combination of amlodipine with imipenem showed synergistic antimicrobial activity against sixty-four A. baumannii isolates when compared with the activity of imipenem alone (p<0.025). In the multidrug-resistant group, AML was more effective than carbonyl cyanide m-chlorophenyl-hydrazone (p<0.001). The efflux pump genes adeB, adeE, adeH, adeJ, abeM and abeS were detected in 100% (4/64), 75% (48/64), 0% (0/64), 100% (64/64), 96.9% (62/64) and 96.9% (62/64) of the sixty-four A. baumannii isolates, respectively. The expression of the adeABC efflux pump genes in the multidrug-resistant group (5.05±19.25) is clearly higher than in the non-multidrug-resistant group (0.17±0.20), (p = 0.01). A gene silencing test verified that the mRNA expression levels of adeABC were decreased at 12 h and increased at 24 h, while the reversal of imipenem resistance by amlodipine disappeared at 12 h and reappeared at 24 h. CONCLUSIONS: The combination of amlodipine with imipenem exhibits an in vitro synergistic antimicrobial effect on multidrug-resistant A. baumannii, which may be due to the inhibition of the AdeABC efflux pump.

Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles as photosensitizers for photodynamic therapy against ovarian cancer (SKOV-3) cells.

Magnetic iron oxide modified pyropheophorbide-a fluorescence nanoparticles, Fe3O4@SiO2@APTES@PPa (FSAP), were designed as magnetically targeted photodynamic antineoplastic agents and prepared through continuous covalent chemical modification on the surface of Fe3O4 nanoparticles. The properties of the intermediates and the final product were comprehensively characterized by transmission electron microscopy, powder X-ray diffraction analysis, Fourier transform infrared spectroscopy, vibrating sample magnetometry, zeta potential measurement, ultraviolet-visible absorption spectroscopy, fluorescence emission spectroscopy, and thermogravimetric analysis. In this work, we demonstrated the in vitro photodynamic therapy (PDT) of FSAP against ovarian cancer (SKOV-3) cells, which indicated that FSAP could be taken up successfully and showed low dark toxicity without irradiation, but remarkable phototoxicity after irradiation. Meanwhile, FSAP had showed good biocompatibility and low dark toxicity against normal cells in the biological experiments on mouse normal fibroblast cell lines (L929 cells). In addition, in the photochemical process of FSAP mediated photodynamic therapy, the Type-II photo-oxygenation process (generated singlet oxygen) played an important role in the induction of cell damage.

Flotillin-2 modulates fas signaling mediated apoptosis after hyperoxia in lung epithelial cells.

Lipid rafts are subdomains of the cell membrane with distinct protein composition and high concentrations of cholesterol and glycosphingolipids. Raft proteins are thought to mediate diverse cellular processes including signal transduction. However, its cellular mechanisms remain unclear. Caveolin-1 (cav-1, marker protein of caveolae) has been thought as a switchboard between extracellular matrix (ECM) stimuli and intracellular signals. Flotillin-2/reggie-1(Flot-2) is another ubiquitously expressed raft protein which defines non-caveolar raft microdomains (planar raft). Its cellular function is largely uncharacterized. Our novel studies demonstrated that Flot-2, in conjunction with cav-1, played important functions on controlling cell death via regulating Fas pathways. Using Beas2B epithelial cells, we found that in contrast to cav-1, Flot-2 conferred cytoprotection via preventing Fas mediated death-inducing signaling complex (DISC) formation, subsequently suppressed caspase-8 mediated extrinsic apoptosis. Moreover, Flot-2 reduced the mitochondria mediated intrinsic apoptosis by regulating the Bcl-2 family and suppressing cytochrome C release from mitochondria to cytosol. Flot-2 further modulated the common apoptosis pathway and inhibited caspase-3 activation via up-regulating the members in the inhibitor of apoptosis (IAP) family. Last, Flot-2 interacted with cav-1 and limited its expression. Taken together, we found that Flot-2 protected cells from Fas induced apoptosis and counterbalanced the pro-apoptotic effects of cav-1. Thus, Flot-2 played crucial functions in cellular homeostasis and cell survival, suggesting a differential role of individual raft proteins.

Suppression of PTRF alleviates the polymicrobial sepsis induced by cecal ligation and puncture in mice.

BACKGROUND: Sepsis and sepsis-associated organ failure are devastating conditions. Understanding the detailed cellular/molecular mechanisms involved in sepsis should lead to the identification of novel therapeutic targets. METHODS: Cecal ligation and puncture (CLP) was used as a polymicrobial sepsis model in vivo to determine mortality and end-organ damage. Macrophages were adopted as the cellular model in vitro for mechanistic studies. RESULTS: PTRF+/- mice survived longer and suffered less organ damage after CLP. Reductions in nitric oxide (NO) and iNOS biosynthesis were observed in plasma, macrophages, and vital organs in the PTRF+/- mice. Using an acute sepsis model after CLP, we found that iNOS-/- mice had a comparable level of survival as the PTRF+/- mice. Similarly, polymerase I transcript release factor (PTRF) deficiency resulted in decreased iNOS and NO/ROS production in macrophages in vitro. Mechanistically, lipopolysaccharide (LPS) enhanced the co-localization and interaction between PTRF and TLR4 in lipid rafts. Deletion of PTRF blocked formation of the TLR4/Myd88 complex after LPS. Consistent with this, lack of PTRF impaired the TLR4 signaling, as shown by the decreased p-JNK, p-ERK, and p-p38, which are upstream factors involved in iNOS transcription. CONCLUSION: PTRF is a crucial regulator of TLR4 signaling in the development of sepsis.

p62 sequestosome 1/light chain 3b complex confers cytoprotection on lung epithelial cells after hyperoxia.

Lung epithelial cell death is a prominent feature of hyperoxic lung injury, and has been considered a very important underlying mechanism of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Here we report on a novel mechanism involved in epithelial cytoprotection and homeostasis after oxidative stress. p62 (sequestosome 1; SQSTM1) is a ubiquitously expressed cellular protein. It interacts with ubiquitinated proteins and autophagic marker light chain 3b (LC3b), thus mediating the degradation of selective targets. In this study, we explored the role of p62 in mitochondria-mediated cell death after hyperoxia. Lung alveolar epithelial cells demonstrate abundant p62 expression, and p62 concentrations are up-regulated by oxidative stress at both the protein and mRNA levels. The p62/LC3b complex interacts with Fas and truncated BID (tBID) physically. These interactions abruptly diminish after hyperoxia. The deletion of p62 robustly increases tBID and cleaved caspase-3, implying an antiapoptotic effect. This antiapoptotic effect of p62 is further confirmed by measuring caspase activities, cleaved poly ADP ribose polymerase, and cell viability. The deletion of the p62 PBI domain or the ubiquitin-associated domain both lead to elevated tBID, cleaved caspase-3, and significantly more cell death after hyperoxia. Moreover, p62 traffics in an opposite direction with LC3b after hyperoxia, leading to the dissociation of the p62/Cav-1/LC3b/BID complex. Subsequently, the LC3b-mediated lysosomal degradation of tBID is eliminated. Taken together, our data suggest that the p62/LC3b complex regulates lung alveolar epithelial cell homeostasis and cytoprotection after hyperoxia.