Characterization of Cellulose-Degrading Bacteria Isolated from Silkworm Excrement and Optimization of Its Cellulase Production.

An abundance of refractory cellulose is the key limiting factor restricting the resource utilization efficiency of silkworm (Bombyx mori) excrement via composting. Screening for cellulose-degrading bacteria is likely to provide high-quality strains for the safe and rapid decomposition of silkworm excrement. In this study, bacteria capable of degrading cellulose with a high efficiency were isolated from silkworm excrement and the conditions for cellulase production were optimized. The strains were preliminarily screened via sodium carboxymethyl cellulose culture and staining with Congo red, rescreened via a filter paper enzyme activity test, and identified via morphological observation, physiological and biochemical tests, and phylogenetic analysis of the 16S rDNA sequence. Enzyme activity assay was performed using the 3,5-dinitrosalicylic acid method. DC-11, a highly cellulolytic strain, was identified as Bacillus subtilis. The optimum temperature and pH of this strain were 55 °C and 6, respectively, and the filter paper enzyme activity (FPase), endoglucanase activity (CMCase), and exoglucanase activity (CXase) reached 15.40 U/mL, 11.91 U/mL, and 20.61 U/mL. In addition, the cellulose degradation rate of the treatment group treated with DC-11 was 39.57% in the bioaugmentation test, which was significantly higher than that of the control group without DC-11 (10.01%). Strain DC-11 was shown to be an acid-resistant and heat-resistant cellulose-degrading strain, with high cellulase activity. This strain can exert a bioaugmentation effect on cellulose degradation and has the potential for use in preparing microbial inocula that can be applied for the safe and rapid composting of silkworm excrement.

Integrated analysis reveals common DNA methylation patterns of alcohol-associated cancers: A pan-cancer analysis.

Background: The role of alcohol in carcinogenesis has received increasing attention in recent years. Evidence shows its impacts on various aspects, including epigenetics alteration. The DNA methylation patterns underlying alcohol-associated cancers are not fully understood. Methods: We investigated the aberrant DNA methylation patterns in four alcohol-associated cancers based on the Illumina HumanMethylation450 BeadChip. Pearson coefficient correlations were identified between differential methylated CpG probes and annotated genes. Transcriptional factor motifs were enriched and clustered using MEME Suite, and a regulatory network was constructed. Results: In each cancer, differential methylated probes (DMPs) were identified, and 172 hypermethylated and 21 hypomethylated pan-cancer DMPs (PDMPs) were examined further. Annotated genes significantly regulated by PDMPs were investigated and enriched in transcriptional misregulation in cancers. The CpG island chr19:58220189-58220517 was hypermethylated in all four cancers and silenced in the transcription factor ZNF154. Various biological effects were exerted by 33 hypermethylated and seven hypomethylated transcriptional factor motifs grouped into five clusters. Eleven pan-cancer DMPs were identified to be associated with clinical outcomes in the four alcohol-associated cancers, which might provide a potential point of view for clinical outcome prediction. Conclusion: This study provides an integrated insight into DNA methylation patterns in alcohol-associated cancers and reveals the corresponding features, influences, and potential mechanisms.

Studies on 1-deoxynojirimycin biosynthesis in mulberry (Morus alba L.) seeds through comparative transcriptomics.

Mulberry (Morus alba L.) plants are rich in 1-deoxynojirimycin (DNJ), which is a potential α-glucosidase inhibitor exhibiting various physiological activities. Compared to other tissues, Morus alba L. seeds contain the highest DNJ content, however, the DNJ biosynthesis mechanisms are unclear. In this study, we examined fruits of 27 mulberry varieties and found that variety MS02 had the highest DNJ levels (22.28 mg/g), whereas variety MS15 contained the lowest DNJ levels (0.37 mg/g). Through comparative transcriptomics, 1,719 differentially expressed genes (DEGs) were identified, 1,170 of which were upregulated, and 549 were downregulated in MS02 compared to MS15. DEGs were associated with cellular processes, metabolic processes, and catalytic activity. Specifically, nine DEGs were identified to be involved in alkaloid biosynthesis pathways, according to Kyoto Encyclopaedia of Genes and Genomes enrichment analysis, and four enzymes, i.e. polyphenol oxidase, tyrosine aminotransferase, aromatic-L-amino-acid decarboxylase, and tropinone reductase, are proposed to play important roles in DNJ biosynthesis. In conclusion, DNJ biosynthesis in mulberry seeds appears to be mediated by upregulation of polyphenol oxidase, tyrosine aminotransferase, aromatic-L-amino-acid decarboxylase, and tropinone reductase.

Differential effects of WRAP53 transcript variants on non-small cell lung cancer cell behaviors.

BACKGROUND: The WD40-encoding RNA antisense to p53 (WRAP53) is an antisense gene of TP53 with three transcriptional start sites producing three transcript variants involved in the progression of non-small cell lung cancer. However, the mechanism by which these different transcript variants regulate non-small cell lung cancer cell behaviors is to be elucidated. METHODS: Two non-small cell lung cancer cell lines, A549 cells with wild-type p53 and H1975 with mutated p53, were transfected with WRAP53-1α and WRAP53-1ß siRNA. The biological effects were assessed via colony formation, cell viability, apoptosis, cell cycle, wound healing and cell invasion assays, as well as immunoblotting. RESULTS: Knockdown of WRAP53-1α increased the mRNA and protein levels of p53; suppressed colony formation and proliferation of A549 cells but promoted them in H1975 cells; increased the proportion of cells in the G0/G1 phase in A549 cells but decreased that in H1975 cells; and suppressed migration and invasion in A549 cells but not in H1975 cells. Conversely, knockdown of WRAP53-1ß had no effect on p53 expression; promoted the growth of A549 cells but not of H1975 cells; decreased the proportion of cells in the G0/G1 phase in A549 cells but not in H1975 cells; and promoted migration and invasion in A549 cells but not in H1975 cells. Knockdown of both WRAP53-1α and WRAP53-1ß promoted apoptosis in A549 cells but not in H1975 cells. CONCLUSIONS: WRAP53 transcript variants exerted different functions in non-small cell lung cancer cells and regulated non-small cell lung cancer cell behaviors depending on the p53 expression.

Efficacy and safety of docetaxel plus S-1-based therapy in gastric cancer: a quantitative evidence synthesis of randomized controlled trials.

Objective: To systematically evaluate the safety and efficacy of docetaxel plus S-1-based therapy in gastric cancer treatment. Methods: PubMed, Embase, The Cochrane Library, and Web of Science electronic databases were searched for randomized controlled trials on docetaxel plus S-1-based therapy in the treatment of gastric cancer from the establishment of the database to 1 September 2022. Relevant studies were included per pre-defined eligibility criteria, and two researchers independently screened and assessed the included literature using Review Manager v5. Outcome measures and statistics related with efficacy and safety profiles were extracted from the included studies, and Stata v15.1 was used for pooled analysis. Results: Objective response rate (odds ratio = 2.34, 95% CI = [1.32, 4.13], p = 0.003), relapse-free survival (HR = 0.68, 95% CI = [0.58, 0.79], p < 0.001), progression-free survival (HR = 0.81, 95% CI = [0.68, 0.96], p = 0.016), and overall survival (HR = 0.86, 95% CI = [0.79, 0.95], p = 0.002) of docetaxel plus S-1-based therapy (DS-based therapy) in gastric cancer treatment were better than those of the non-DS-based therapy. However, DS-based therapy was associated with increased risk of certain adverse drug effects, such as alopecia, leukopenia, and oral mucositis. Further studies are warranted to validate the efficacy superiority of DS-based versus non-DS-based regimens as per our trial sequential analysis findings. Conclusion: DS-based therapy significantly improves patients' clinical outcomes in gastric cancer, albeit at the cost of increased toxicity. Further RCTs are needed to confirm the efficacy superiority of DS-based regimens.

SAMHD1 silencing cooperates with radiotherapy to enhance anti-tumor immunity through IFI16-STING pathway in lung adenocarcinoma.

BACKGROUND: Sterile alpha motif domain and histidine-aspartate domain-containing protein 1 (SAMHD1) is a DNA end resection factor, which is involved in DNA damage repair and innate immunity. However, the role of SAMHD1 in anti-tumor immunity is still unknown. This study investigated the effects of SAMHD1 on stimulator of interferon genes (STING)-type I interferon (IFN) pathway and radiation-induced immune responses. METHODS: The roles of SAMHD1 in the activation of cytosolic DNA sensing STING pathway in lung adenocarcinoma (LUAD) cells were investigated with flow cytometry, immunofluorescence, immunoblotting and qPCR. The combined effects of SAMHD1 silencing and radiation on tumor cell growth and STING pathway activation were also evaluated with colony formation and CCK8 assay. The Lewis lung cancer mouse model was used to evaluate the combined efficiency of SAMHD1 silencing and radiotherapy in vivo. Macrophage M1 polarization and cytotoxic T cell infiltration were evaluated with flow cytometry. RESULTS: The single-stranded DNA (ssDNA) accumulated in the cytosol of SAMHD1-deficient lung adenocarcinoma (LUAD) cells, accompanied by upregulated DNA sensor IFN-γ-inducible protein 16 (IFI16) and activated STING pathway. The translocation of IFI16 from nucleus to cytosol was detected in SAMHD1-deficient cells. IFI16 and STING were acquired in the activation of STING-IFN-I pathway in SAMHD1-deficient cells. SAMHD1 silencing in LUAD cells promoted macrophage M1 polarization in vitro. SAMHD1 silencing synergized with radiation to activate ssDNA-STING-IFN-I pathway, inhibit proliferation, promote apoptosis and regulate cell cycle. SAMHD1 silencing cooperated with radiotherapy to inhibit tumor growth and increase CD86+MHC-IIhigh M1 proportion and CD8+ T cell infiltration in vivo. CONCLUSIONS: SAMHD1 deficiency induced IFN-I production through cytosolic IFI16-STING pathway in LUAD cells. Moreover, SAMHD1 downregulation and radiation cooperated to inhibit tumor growth and enhance anti-tumor immune responses through macrophage M1 polarization and CD8+ T cell infiltration. Combination of SAMHD1 inhibition and radiotherapy may be a potentially therapeutic strategy for LUAD patients.

Accelerated degradation of cellulose in silkworm excrement by the interaction of housefly larvae and cellulose-degrading bacteria.

The environmental pollution caused by silkworm (Bombyx mori) excrement is prominent, and rich in refractory cellulose is the bottleneck restricting the efficient recycling of silkworm excrement. This study was performed to investigate the effects of housefly larvae vermicomposting on the biodegradation of cellulose in silkworm excrement. After six days, a 58.90% reduction of cellulose content in treatment groups was observed, which was significantly higher than 11.5% of the control groups without housefly larvae. Three cellulose-degrading bacterial strains were isolated from silkworm excrement, which were identified as Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis based on 16S rRNA gene sequence analysis. These three bacterial stains had a high cellulose degradation index (HC value ranged to between 1.86 and 5.97 and FPase ranged from 5.07 U/mL to 7.31 U/mL). It was found that housefly larvae increased the abundance of cellulose-degrading bacterial genus (Bacillus and Pseudomonas) by regulating the external environmental conditions (temperature and pH). Carbohydrate metabolism was the bacterial communities' primary function during vermicomposting based on the PICRUSt. The results of Tax4Fun indicated that the abundance of endo-ß-1,4-glucanase and exo-ß-1,4-glucanase increased rapidly and maintained at a higher level in silkworm excrement due to the addition of housefly larvae, which contributed to the accelerated degradation of cellulose in silkworm excrement. The finding of this investigation showed that housefly larvae can significantly accelerate the degradation of cellulose in silkworm excrement by increasing the abundance of cellulose-degrading bacterial genera and cellulase.

PARP inhibitor plus radiotherapy reshapes an inflamed tumor microenvironment that sensitizes small cell lung cancer to the anti-PD-1 immunotherapy.

Small cell lung cancer (SCLC) is a highly malignant tumor with extremely poor prognosis. The treatment strategy is very limited, and patient outcomes remain dismal with the 5-year survival rate being mere 3-6%. Thus, novel therapeutic strategies for SCLC patients are urgently needed. In this study, we found that the triple-therapy of poly (ADP-ribose) polymerase (PARP) inhibitor, radiotherapy (RT) and anti-PD-1 treatment significantly inhibited tumor growth and prolonged survival in the syngeneic SCLC models in immunocompetent C57BL/6 mice. Mechanistically, we demonstrated that the combination of PARP inhibitor niraparib and RT reshaped an inflamed tumor microenvironment, including activation of the cGAS/STING immune response pathway, induction of immunogenic cell death, and upregulation of PD-L1 on tumor cells. Furthermore, this triple-therapy substantially augmented CD8+ T cell infiltration and activation, and enhanced anti-tumor effects as revealed by increased median survival time and reduced tumor volume without additional myelosuppression or hepatic injury. Together, our studies demonstrated that PARP inhibitor combined with RT potentiated anti-tumor immunity and enhanced the efficacy of anti-PD-1 immunotherapy in preclinical study, which provided a promising therapeutic strategy for SCLC patients in clinic.

LncRNA PCAT1 activates SOX2 and suppresses radioimmune responses via regulating cGAS/STING signalling in non-small cell lung cancer.

BACKGROUND: The expression of long non-coding RNA (lncRNA) prostate cancer-associated ncRNA transcripts 1 (PCAT1) is increased in non-small cell lung cancer (NSCLC). It stimulates tumour growth and metastasis, but its role in the radioimmune responses remain unknown. We aimed to explore the impacts of PCAT1 on tumorigenesis and radioimmune responses and the underlying molecular mechanisms in NSCLC. METHODS: Comprehensive bioinformatics analysis was performed to identify immunosuppressive lncRNAs involved with tumour invasion in NSCLC. The expression levels of PCAT1 were analysed by in situ hybridisation in 55 paired NSCLC tissues and adjacent normal tissues. Both loss- and gain-of-function assays were performed to examine the effects of PCAT1 and SOX2 on NSCLC cell behaviours in vivo and in vitro. Bioinformatic analyses, chromatin isolation by RNA purification (ChIRP) and dual-luciferase reporter assays were applied to validate the regulatory effects of PCAT1 on SOX2 expression. Chromatin immunoprecipitation, luciferase and rescue assays were utilised to identify the relationship between SOX2 and the cGAS/stimulator of interferon genes (STING) signalling. RESULTS: PCAT1 was immunosuppressive and related with NSCLC invasion. Increased PCAT1 was negatively correlated with immune cell infiltration in NSCLC. PCAT1 knockdown restrained proliferation, increased apoptosis, and repressed cell metastasis in vivo and in vitro. PCAT1 activated SOX2 that accelerated tumorigenesis and immunosuppression. SOX2 promoted tumour growth through inhibiting cytotoxic T-cell immunity. Moreover, SOX2 restrained cGAS transcription and hampered downstream type I interferon (IFN)-induced immune responses. Inhibition of PCAT1/SOX2 in collaboration with radiation further inhibited tumour growth, and initiated the cGAS/STING signalling pathway, which enhanced the immune responses of radiotherapy in NSCLC. CONCLUSIONS: PCAT1/SOX2 axis promoted tumorigenesis and immunosuppression through inhibition of cGAS/STING signalling-mediated T-cell activation. Inhibition of PCAT1 and SOX2 synergised with radiotherapy to activate the immune response and could serve as potential therapeutic targets.

The miR-23b/27b/24-1 Cluster Inhibits Hepatic Fibrosis by Inactivating Hepatic Stellate Cells.

BACKGROUND & AIMS: Hepatic fibrosis is characterized by hepatic stellate cell (HSC) activation and transdifferentiation-mediated extracellular matrix (ECM) deposition, which both contribute to cirrhosis. However, no antifibrotic regimen is available in the clinic. microRNA-23b/27b/24-1 cluster inhibition of transforming growth factor-ß (TGF-ß) signaling during hepatic development prompted us to explore whether this cluster inhibits HSC activation and hepatic fibrosis. METHODS: Experimental fibrosis was studied in carbon tetrachloride (CCl4)-treated C57BL/6 mice. After administration of miR-23b/27b/24-1 lentivirus or vehicle, animals were euthanized for liver histology. In primary rat HSC and HSC-T6, the anti-fibrotic effect of miR-23b/27b/24-1 cluster was furtherly investigated by RNA-sequencing, luciferase reporter assay, western blotting and bioinformatic means. RESULTS: In this study, we showed that increasing the miR-23b/27b/24-1 level through intravenous delivery of miR-23b/27b/24-1 lentivirus ameliorated mouse hepatic fibrosis. Mechanistically, the miR-23b/27b/24-1 cluster directly targeted messenger RNAs, which reduced the protein expression of 5 secretory profibrotic genes (TGF-ß2, Gremlin1, LOX, Itgα2, and Itgα5) in HSCs. Suppression of the TGF-ß signaling pathway by down-regulation of TGF-ß2, Itgα2, and Itgα5, and activation of the bone morphogenetic protein signaling pathway by inhibition of Gremlin1, decreased extracellular matrix secretion of HSCs. Furthermore, down-regulation of LOX expression softened the ECM. Moreover, a reduction in tissue inhibitors of metalloproteinase 1 expression owing to weakened TGF-ß signaling increased ECM degradation. CONCLUSIONS: Hepatic overexpression of the miR-23b/27b/24-1 cluster blocked hepatic fibrosis and may be a novel therapeutic regimen for patients with hepatic fibrosis.

Effects of MicroRNA-195-5p on Biological Behaviors and Radiosensitivity of Lung Adenocarcinoma Cells via Targeting HOXA10.

OBJECTIVE: To explore the effects of miR-195-5p and its target gene HOXA10 on the biological behaviors and radiosensitivity of lung adenocarcinoma (LUAD) cells. METHODS: The effects of miR-195-5p on LUAD cell proliferation, migration, invasion, cycle arrest, apoptosis, and radiosensitivity were investigated by in vitro experiments. The bioinformatics analysis was used to assess its clinical value and predict target genes. Double-luciferase experiments were used to verify whether the miR-195-5p directly targeted HOXA10. A xenograft tumor-bearing mouse model was used to examine its effects on the radiosensitivity of LUAD in vivo. RESULTS: Both gain- and loss-of-function assays demonstrated that miR-195-5p inhibited LUAD cell proliferation, invasion, and migration, induced G1 phase arrest and apoptosis, and enhanced radiosensitivity. Double-luciferase experiments confirmed that miR-195-5p directly targeted HOXA10. Downregulation of HOXA10 also inhibited LUAD cell proliferation, migration, and invasion, induced G1 phase arrest and apoptosis, and enhanced radiosensitivity. The protein levels of ß-catenin, c-myc, and Wnt1 were decreased by miR-195-5p and increased by its inhibitor. Moreover, the effects of the miR-195-5p inhibitor could be eliminated by HOXA10-siRNA. Furthermore, miR-195-5p improved radiosensitivity of LUAD cells in vivo. CONCLUSION: miR-195-5p has excellent antitumor effects via inhibiting cancer cell growth, invasion, and migration, arresting the cell cycle, promoting apoptosis, and sensitizing LUAD cells to X-ray irradiation by targeting HOXA10. Thus, miR-195-5p may serve as a potential candidate for the treatment of LUAD.

Systematic Analyses of a Chemokine Family-Based Risk Model Predicting Clinical Outcome and Immunotherapy Response in Lung Adenocarcinoma.

Chemokines exhibited complicated functions in antitumor immunity, with their expression profile and clinical importance of lung adenocarcinoma (LUAD) patients remaining largely undetermined. This study aimed to explore the expression patterns of chemokine family in LUAD and construct a predictive chemokine family-based signature. A total of 497 samples were downloaded from the Cancer Genome Atlas (TCGA) data portal as the training set, and the combination of 4 representative Gene Expression Omnibus (GEO) datasets, including GSE30219, GSE50081, GSE37745, and GSE31210, were utilized as the validation set. A three gene-based signature was constructed using univariate and stepwise multivariate Cox regression analysis, classifying patients into high and low risk groups according to the overall survival. The independent GEO datasets were utilized to validate this signature. Another multivariate analysis revealed that this signature remained an independent prognostic factor in LUAD patients. Furthermore, patients in the low risk group featured immunoactive tumor microenvironment (TME), higher IPS scores and lower TIDE scores, and was regarded as the potential beneficiaries of immunotherapy. Finally, the role of risky CCL20 was validated by immunohistochemistry (IHC), and patients possessed higher CCL20 expression presented shorter overall survival (P = 0.011).

Comprehensive analysis reveals common DNA methylation patterns of tobacco-associated cancers: A pan-cancer analysis.

The role of tobacco in carcinogenesis has received increasing attention across a number of disciplines in recent years. Accumulating evidences reveal that tobacco consumption affects various epigenetic modifications, especially DNA methylation. However, the genetic modifications of methylation patterns involved in tobacco-attributable cancers remain poorly understood. In this manuscript, aberrant DNA methylation patterns were investigated in 9 tobacco-attributable cancers. Differential methylated probes (DMPs) were identified in each cancer type and a total of 2,392 hyper- and 736 hypomethylated pan-cancer DMPs (PDMPs) were screened out for further analysis. PDMP-associated genes were mostly enriched in metabolism-associated pathways, suggesting the potential roles of methylation alternation in reprogramming cancer cell metabolism. Hypomethylated PDMPs cg12422154, cg02772121 and cg06051311 constituted an enhancer region, significantly downregulating TRIM15, TRIM26 and RPP21, which serve as epigenetically therapeutic biomarkers. Forty-three hypermethylated and 13 hypomethylated transcription factor motifs were clustered into 6 groups, and exhibited various biological functions. Forty-nine PDMPs were reported to be associated with prognosis, providing effective tools to predict clinical outcomes. In summary, our studies revealed the characteristics, influences and potential mechanisms of DNA methylation patterns of tobacco-attributable cancer.

HHLA2 deficiency inhibits non-small cell lung cancer progression and THP-1 macrophage M2 polarization.

BACKGROUND: Human endogenous retrovirus-H long terminal repeat-associating protein 2 (HHLA2) is a member of B7 family, which is upregulated in multiple tumors. However, its exact functions in non-small cell lung cancer (NSCLC) have not been fully understood. This study aimed to investigate the biological roles of HHLA2 in human NSCLC and the relevant mechanisms. In addition, the effects of tumor cell-derived HHLA2 on tumor-associated macrophage (TAM) polarization were explored. METHODS: NSCLC cell growth, migration, and invasion were assessed by colony formation and modified Boyden chamber assays. Cell cycle and the CD163+ TAMs were examined by flow cytometry. A co-culture model of THP-1 macrophages and NSCLC cells was conducted to investigate the impacts of tumor cell-derived HHLA2 on THP-1 macrophage polarization. Moreover, a xenograft nude mouse model was established to explore the effects of HHLA2 on tumorigenesis in vivo. RESULTS: HHLA2 was upregulated in A549 and H1299 cells compared with the normal lung epithelial BEAS-2B cells. HHLA2 deficiency inhibited NSCLC cell proliferation, migration, invasion, and induced G0/G1 phase arrest partially via inhibiting EGFR/MAPK/ERK signaling pathway. Furthermore, HHLA2 knockdown inhibited M2 polarization of TAMs via downregulating IL-10. In addition, knockdown of HHLA2 inhibited tumor growth in vivo. CONCLUSION: HHLA2 downregulation inhibited NSCLC growth and TAM M2 polarization. HHLA2 may serve as a therapeutic target and promising prognostic biomarker in NSCLC.

Hollow PtCo alloy nanospheres as a high-Z and oxygen generating nanozyme for radiotherapy enhancement in non-small cell lung cancer.

Radiotherapy, as well as chemotherapy and surgery, occupies an essential position in tumor treatment. Nonetheless, insufficient radiation deposition and hypoxia-related radioresistance of cancer cells still are serious challenges in radiotherapy. Herein, we proposed a hollow PtCo nanosphere (PtCo NS)-based novel radiosensitizer with three advantages to sensitize tumor radiotherapy: (i) the high-Z element Pt ensured higher radiation absorption to cause more DNA damage, (ii) the platinum (Pt) and cobalt (Co) elements exhibited a dual catalase-like enzymatic activity to convert endogenic H2O2 to O2 efficiently, and (iii) the unique hollow nature of the PtCo NS provided a large specific surface area, which could amplify the catalytic reaction of H2O2 to induce reactive oxygen species and cancer cell apoptosis upon combination with radiation. Both in vivo and in vitro studies showed that the hollow PtCo NS could significantly inhibit tumor growth, simultaneously relieving tumor hypoxia with good biocompatibility and biosafety. This work presents a simple but multifunctional radiosensitizer with a unique hollow structure for radiotherapy enhancement.

RRM2 silencing suppresses malignant phenotype and enhances radiosensitivity via activating cGAS/STING signaling pathway in lung adenocarcinoma.

BACKGROUND: As one of the most common malignancy, lung adenocarcinoma (LUAD) is characterized by low 5-year survival rate. This research aimed to investigate the effects of ribonucleotide reductase regulatory subunit M2 (RRM2) on malignant biological behaviors and activation of cGAS/STING pathway. We also explored the synergistic sensitization mechanisms of RRM2 and radiotherapy. METHODS: Bioinformatic tools were used to evaluate the clinical significance of RRM2 in LUAD patients. The roles of RRM2 in malignant phenotype and DNA damage in LUAD cells were investigated with cell proliferation, colony formation, immunofluorescence, modified Boyden chamber and comet assays. The mouse models were used to evaluate the biological significance of RRM2 in vivo. Cytotoxic T cell infiltration was evaluated via flow cytometric analysis and immunohistochemistry staining in C57BL/6 mice. We also explored the synergistic effects of RRM2 silencing and radiation on LUAD cells with apoptosis assay and immunoblotting in vitro. RESULTS: Bioinformatic analysis revealed that RRM2 had diagnostic values for LUAD patients. Higher levels of RRM2 predicted worse prognosis. RRM2 silencing inhibited LUAD cell proliferation, invasion and migration. RRM2 knockdown induced S phase arrest and DNA damage. RRM2 silencing induced cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway, and the downstream targets were regulated in a STING-dependent manner. Knockdown of RRM2 suppressed tumor growth in the xenograft tumor models. RRM2 deficiency increased CD8 + T cells in the tumor tissues and spleens. Furthermore, RRM2 silencing had synergistic effects with radiation on inhibiting cell proliferation and promoting apoptosis. Meanwhile, this combination promoted the activation of cGAS/STING signaling pathway synergistically, and simultaneously increased expression of IFNß, CCL5 and CXCL10. CONCLUSION: Our results demonstrated that RRM2 silencing had anti-tumor values and activated the cGAS/STING signaling pathway. RRM2 silencing increased CD8 + T cells infiltration. RRM2 silencing cooperated with radiation to inhibit LUAD cell proliferation, promote apoptosis and enhance the activation of cGAS/STING signaling pathway. RRM2 could be a promising target for tumor regression through cancer immunotherapy in LUAD.

Agrin Promotes Non-Small Cell Lung Cancer Progression and Stimulates Regulatory T Cells via Increasing IL-6 Secretion Through PI3K/AKT Pathway.

Non-small cell lung cancer (NSCLC) has high mortality rates worldwide. Agrin contributes to immune synapse information and is involved in tumor metastasis. However, its roles in NSCLC and tumor immune microenvironment remain unclear. This study examined the effects and the underlying mechanisms of Agrin in NSCLC and tumor-infiltrated immune cells. Clinical tissue samples were used to confirm the bioinformatic predictions. NSCLC cells were used to investigate the effects of Agrin on cell cycle and proliferation, as well as invasion and migration. Tumor xenograft mouse model was used to confirm the effects of Agrin on NSCLC growth and tumor-infiltrated regulatory T cells (Tregs) in vivo. Agrin levels in NSCLC cells were closely related to tumor progression and metastasis, and its function was enriched in the PI3K/AKT pathway. In vitro assays demonstrated that Agrin knockdown suppressed NSCLC cell proliferation and metastasis, while PI3K/AKT activators reversed the inhibitory effects of Agrin deficiency on NSCLC cell behaviors. Agrin expression was negatively associated with immunotherapy responses in NSCLC patients. Agrin knockdown suppressed Tregs, as well as interleukin (IL)-6 expression and secretion, while PI3K/AKT activators and exogenous IL-6 rescued the inhibitory effects. In the mouse model, Agrin downregulation alleviated NSCLC cell growth and Treg infiltration in vivo. Our results indicated that Agrin promotes tumor cell growth and Treg infiltration via increasing IL-6 expression and secretion through PI3K/AKT pathway in NSCLC. Our studies suggested Agrin as a therapeutically potential target to increase the efficacy of immunotherapy in NSCLC patients.

Establishment of Immune-related Gene Pair Signature to Predict Lung Adenocarcinoma Prognosis.

Tumor microenvironment (TME) has critical impacts on the pathogenesis of lung adenocarcinoma (LUAD). However, the molecular mechanism of TME effects on the prognosis of LUAD patients remains unclear. Our study aimed to establish an immune-related gene pair (IRGP) model for prognosis prediction and internal mechanism investigation. Based on 702 TME-related differentially expressed genes (DEGs) extracted from The Cancer Genome Atlas (TCGA) training cohort using the ESTIMATE algorithm, a 10-IRGP signature was established to predict LUAD patient prognosis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that DEGs were significantly associated with tumor immune response. In both TCGA training and Gene Expression Omnibus validation datasets, the risk score was an independent prognostic factor for LUAD patients using Lasso-Cox analysis, and patients in the high-risk group had poorer prognosis than those in the low-risk one. In the high-risk group, M2 macrophage and neutrophil infiltrations were higher, while the levels of T cell follicular helpers were significantly lower. The gene set enrichment analysis results showed that DNA repair signaling pathways were involved. In summary, we established an IRGP signature as a potential biomarker to predict the prognosis of LUAD patients.

Systematic Profiling of Immune Risk Model to Predict Survival and Immunotherapy Response in Head and Neck Squamous Cell Carcinoma.

BACKGROUND AND PURPOSE: Head and neck squamous carcinoma (HNSCC), characterized by immunosuppression, is a group of highly heterogeneous cancers. Although immunotherapy exerts a promising influence on HNSCC, the response rate remains low and varies in assorted primary sites. Immunological mechanisms underlying HNSCC pathogenesis and treatment response are not fully understood. This study aimed to develop a differentially expressed genes (DEGs)-based risk model to predict immunotherapy efficacy and stratify prognosis of HNSCC patients. MATERIALS AND METHODS: The expression profiles of HNSCC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The tumor microenvironment and immune response were estimated by cell type identification via estimating relative subset of known RNA transcripts (CIBERSORT) and immunophenoscore (IPS). The differential expression pattern based on human papillomavirus status was identified. A DEGs-based prognostic risk model was developed and validated. All statistical analyses were performed with R software (version 3.6.3). RESULTS: By using the TCGA database, we identified DKK1, HBEGF, RNASE7, TNFRSF12A, INHBA, and IPIK3R3 as DEGs that were associated with patients' overall survival (OS). Patients were stratified into the high- and low-risk subgroups according to a DEGs-based prognostic risk model. Significant difference in OS was found between the high- and low-risk patients (1.64 vs. 2.18 years, P = 0.0017). In multivariate Cox analysis, the risk model was an independent prognostic factor for OS (hazard radio = 1.06, 95% confidence interval [1.02-1.10], P = 0.004). More CD8+ T cells and regulatory T cells were observed in the low-risk group and associated with a favorable prognosis. The IPS analysis suggested that the low-risk patients possessed a higher IPS score and a higher immunoreactivity phenotype, which were correlated with better immunotherapy response. CONCLUSION: Collectively, we established a reliable DEGs-based risk model with potential prognostic value and capacity to predict the immunophenotype of HNSCC patients.