Machine-learning developed an iron, copper, and sulfur-metabolism associated signature predicts lung adenocarcinoma prognosis and therapy response.

BACKGROUND: Previous studies have largely neglected the role of sulfur metabolism in LUAD, and no study has combine iron, copper, and sulfur-metabolism associated genes together to create prognostic signatures. METHODS: This study encompasses 1564 LUAD patients, 1249 NSCLC patients, and over 10,000 patients with various cancer types from diverse cohorts. We employed the R package ConsensusClusterPlus to separate patients into different ICSM (Iron, Copper, and Sulfur-Metabolism) subtypes. Various machine-learning methods were utilized to develop the ICSMI. Enrichment analyses were conducted using ClusterProfiler and GSVA, while IOBR quantified immune cell infiltration. GISTIC2.0 and maftools were utilized for CNV and SNV data analysis. The Oncopredict package predicted drug information based on GDSC1. TIDE algorithm and cohorts GSE91061 and IMvigor210 evaluated patient response to immunotherapy. Single-cell data was processed using the Seurat package, AUCell package calculated cells geneset activity scores, and the Scissor algorithm identified ICSMI-associated cells. In vitro experiments was conducted to explore the role of ICSMRGs in LUAD. RESULTS: Unsupervised clustering identified two distinct ICSM subtypes of LUAD, each with unique clinical characteristics. The ICSMI, comprising 10 genes, was constructed using integrated machine-learning methods. Its prognostic power was validated in 10 independent datasets, revealing that LUAD patients with higher ICSMI levels had poorer prognoses. Furthermore, ICSMI demonstrated superior predictive abilities compared to 102 previously published signatures. A nomogram incorporating ICSMI and clinical features exhibited high predictive performance. ICSMI positively correlated with patients gene mutations, and integrated analysis of bulk and single-cell transcriptome data revealed its association with TME modulators. Cells representing the high-ICSMI phenotype exhibited more malignant features. LUAD patients with high ICSMI levels exhibited sensitivity to chemotherapy and targeted therapy but displayed resistance to immunotherapy. In a comprehensive analysis across various cancers, ICSMI retained significant prognostic value and emerged as a risk factor for the majority of cancer patients. CONCLUSIONS: ICSMI provides critical prognostic insights for LUAD patients, offering valuable insights into the tumor microenvironment and predicting treatment responsiveness.

Identification of a novel ADCC-related gene signature for predicting the prognosis and therapy response in lung adenocarcinoma.

BACKGROUND: Previous studies have largely neglected the role of ADCC in LUAD, and no study has systematically compiled ADCC-associated genes to create prognostic signatures. METHODS: In this study, 1564 LUAD patients, 2057 NSCLC patients, and more than 5000 patients with various cancer types from diverse cohorts were included. R package ConsensusClusterPlus was utilized to classify patients into different subtypes. A number of machine-learning algorithms were used to construct the ADCCRS. GSVA and ClusterProfiler were used for enrichment analyses, and IOBR was used to quantify immune cell infiltration level. GISTIC2.0 and maftools were used to analyze the CNV and SNV data. The Oncopredict package was used to predict drug information based on the GDSC1. Three immunotherapy cohorts were used to evaluate patient response to immunotherapy. The Seurat package was used to process single-cell data, the AUCell package was used to calculate cells' geneset activity scores, and the Scissor algorithm was used to identify ADCCRS-associated cells. RESULTS: Through unsupervised clustering, two distinct subtypes of LUAD were identified, each exhibiting distinct clinical characteristics. The ADCCRS, consisted of 16 genes, was constructed by integrated machine-learning methods. The prognostic power of ADCCRS was validated in 28 independent datasets. Further, ADCCRS shows better predictive abilities than 102 previously published signatures in predicting LUAD patients' survival. A nomogram incorporating ADCCRS and clinical features was constructed, demonstrating high predictive performance. ADCCRS positively correlates with patients' gene mutation, and integrated analysis of bulk and single-cell transcriptome data revealed the association of ADCCRS with TME modulators. Cells representing high-ADCCRS phenotype exhibited more malignant features. LUAD patients with high ADCCRS levels exhibited sensitivity to chemotherapy and targeted therapy, while displaying resistance to immunotherapy. In pan-cancer analysis, ADCCRS still exhibited significant prognostic value and was found to be a risk factor for most cancer patients. CONCLUSIONS: ADCCRS offers a critical prognostic insight for patients with LUAD, shedding light on the tumor microenvironment and forecasting treatment responsiveness.

Lung-specific exosomes for doxorubicin delivery in lung adenocarcinoma therapy.

Doxorubicin (DOX) could be utilized to treat lung adenocarcinoma (LUAD), while dose-limiting cardiotoxicity limits its clinical utilization. MDA-MB-231 cell-derived exosomes show lung-specific organotropism features. In this study, we aimed to explore the potential of MDA-MB-231 cell-derived exosomes in DOX specific delivery to the lung. MDA-MB-231 cell-derived exosomes were coincubated with to construct for the doxorubicin delivery system (D-EXO). Exosomes labeled with fluorescein isothiocyanate were incubated with A549 cells or 293T cells, and the engulf and the mean intensity of the fluorescence were detected with immunofluorescence and flow cytometry assay. Cell viability was detected with cell counting kit-8 (CCK-8), and cell migration was determined by scratch test. The protein expression was detected by Western blot assay. A549 cell line-derived xenograft mouse model was constructed to examine the treatment effect of D-EXO. MDA-MB-231 cell-derived exosomes could be specially taken up by A549 cells with diminished cell viability but not engulfed by 293T cells. D-EXO inhibited A549 cell migration, and upregulated the protein expression of caspase 3 and cleaved caspase 3 expression, while did not show any inhibition on 293T cells. In vivo orthotopic xenotransplantation model indicated that D-EXO inhibited tumor growth characterized by diminished tumor weight and improved survival rate. No significant change in body weight was observed after the D-EXO treatment. In conclusion, D-EXO proposed in this study could be utilized to treat LUAD with lung-specific delivery effects to improve the survival rate.

In-depth single-cell and bulk-RNA sequencing developed a NETosis-related gene signature affects non-small-cell lung cancer prognosis and tumor microenvironment: results from over 3,000 patients.

Background: Cell death caused by neutrophil extracellular traps (NETs) is known as NETosis. Despite the increasing importance of NETosis in cancer diagnosis and treatment, its role in Non-Small-Cell Lung Cancer (NSCLC) remains unclear. Methods: A total of 3298 NSCLC patients from different cohorts were included. The AUCell method was used to compute cells' NETosis scores from single-cell RNA-sequencing data. DEGs in sc-RNA dataset were obtained by the Seurat's "FindAllMarkers" function, and DEGs in bulk-RNA dataset were acquired by the DESeq2 package. ConsensusClusterPlus package was used to group patients into different NETosis subtypes, and the Enet algorithm was used to construct the NETosis-Related Riskscore (NETRS). Enrichment analyses were conducted using the GSVA and ClusterProfiler packages. Six distinct algorithms were utilized to evaluate patients' immune cell infiltration level. Patients' SNV and CNV data were analyzed by maftools and GISTIC2.0, respectively. Drug information was obtained from the GDSC1, and predicted by the Oncopredict package. Patient response to immunotherapy was evaluated by the TIDE algorithm in conjunction with the phs000452 immunotherapy cohort. Six NRGs' differential expression was verified using qRT-PCR and immunohistochemistry. Results: Among all cell types, neutrophils had the highest AUCell score. By Intersecting the DEGs between high and low NETosis classes, DEGs between normal and LUAD tissues, and prognostic related genes, 61 prognostic related NRGs were identified. Based on the 61 NRGs, all LUAD patients can be divided into two clusters, showing different prognostic and TME characteristics. Enet regression identified the NETRS composed of 18 NRGs. NETRS significantly associated with LUAD patients' clinical characteristics, and patients at different NETRS groups showed significant differences on prognosis, TME characteristics, immune-related molecules' expression levels, gene mutation frequencies, response to immunotherapy, and drug sensitivity. Besides, NETRS was more powerful than 20 published gene signatures in predicting LUAD patients' survival. Nine independent cohorts confirmed that NETRS is also valuable in predicting the prognosis of all NSCLC patients. Finally, six NRGs' expression was confirmed using three independent datasets, qRT-PCR and immunohistochemistry. Conclusion: NETRS can serves as a valuable prognostic indicator for patients with NSCLC, providing insights into the tumor microenvironment and predicting the response to cancer therapy.

Effects of SpsNAC042 transgenic Populus hopeiensis on root development, leaf morphology and stress resistance.

To identify the function of the SpsNAC042 gene and its response to salt and drought stress, the SpsNAC042 gene was transformed into Populus hopeiensis by the Agrobacterium-mediated leaf disc method, and the phenotypic, physiological changes and related genes expression of transgenic lines were analyzed. The results showed that the number and length of roots of transgenic lines increased significantly. The leaves of transgenic lines curled inward. Under salt and simulated drought stress, the transgenic lines showed improved tolerance to salt and drought. The activities of SOD, POD, CAT and proline content in the transgenic lines were significantly increased, and the reduction rates of total chlorophyll and MDA content were significantly decreased, which indicated that the transgenic lines showed strong physiological responses under stress. Meanwhile, the gene expression of MPK6, SOS1, HKT1 and P5CS1 were significantly upregulated, and the gene expression of PRODH1 was significantly downregulated, which preliminarily verified the stress regulation mechanism that SpsNAC042 might activate. The above results showed that the SpsNAC042 gene could promote root development, make leaf morphology curl, and enhance P. hopeiensis tolerance to stress.

Machine-learning and combined analysis of single-cell and bulk-RNA sequencing identified a DC gene signature to predict prognosis and immunotherapy response for patients with lung adenocarcinoma.

BACKGROUND: Innate immune effectors, dendritic cells (DCs), influence cancer prognosis and immunotherapy significantly. As such, dendritic cells are important in killing tumors and influencing tumor microenvironment, whereas their roles in lung adenocarcinoma (LUAD) are largely unknown. METHODS: In this study, 1658 LUAD patients from different cohorts were included. In addition, 724 cancer patients who received immunotherapy were also included. To identify DC marker genes in LUAD, we used single-cell RNAsequencing data for analysis and determined 83 genes as DC marker genes. Following that, integrative machine learning procedure was developed to construct a signature for DC marker genes. RESULTS: Using TCGA bulk-RNA sequencing data as the training set, we developed a signature consisting of seven genes and classified patients by their risk status. Another six independent cohorts demonstrated the signature' s prognostic power, and multivariate analysis demonstrated it was an independent prognostic factor. LUAD patients in the high-risk group displayed more advanced features, discriminatory immune-cell infiltrations and immunosuppressive states. Cell-cell communication analysis indicates that tumor cells with lower risk scores communicate more actively with the tumor microenvironment. Eight independent immunotherapy cohorts revealed that patients with low-risk had better immunotherapy responses. Drug sensitivity analysis indicated that targeted therapy agents exhibited greater sensitivity to low-risk patients, while chemotherapy agents displayed greater sensitivity to high-risk patients. In vitro experiments confirmed that CTSH is a novel protective factor for LUAD. CONCLUSIONS: An unique signature based on DC marker genes that is highly predictive of LUAD patients' prognosis and response to immunotherapy. CTSH is a new biomarker for LUAD.

SiO2/hyaluronic acid nanoparticles carry CaO2, DOX and p53 plasmid to effectively achieve ion interference/chemical/gene multimodal therapy of lung cancer.

Monotherapy of lung cancer shows limited therapeutic effects due to its poorly targeted enrichment and low bioavailability. Using nanomaterials as carriers to form drug delivery systems has become a popular method to improve the targeting of anticancer drug therapy and patients' safety. However, the uniformity of the loaded drugs and the unsatisfactory effects are still the bottleneck in this field up to now. This study aims to construct a novel nanocomposite carrying 3 different types of anticancer drugs to enhance treatment efficacy. Herein, mesoporous silica (MSN) with high loading rate was constructed by dilute sulfuric acid thermal etching as the framework. Hyaluronic acid (HA) was loaded with CaO2, p53 and DOX to construct nanoparticle complexes-SiO2@CaO2@DOX@P53-HA. First, MSN was proved to be a porous sorbent with a mesoporous structure through BET analysis. The images obtained from the uptake experiment clearly show the gradual enrichment of the DOX and Ca2+ within the target cell. For in vitro experiments, the pro-apoptotic effects of SiO2@CaO2@DOX@P53-HA significantly increased compared to that of the single-agent group at different time points. Furthermore, in the tumor-bearing mouse experiment, the tumor volume was remarkably inhibited in the SiO2@CaO2@DOX@P53-HA group compared to that in the single-agent group. By observing the pathological sections of the euthanized mice, it is obvious that the tissues of the mice treated with the nanoparticles were more intact. Based on these beneficial results, it is believed that multimodal therapy is a meaningful treatment strategy for lung cancer.

Knockdown of deoxythymidylate kinase suppresses progression and epithelial-mesenchymal-transition of lung adenocarcinoma via STAT3 signaling.

Current research has shown that inhibiting deoxythymidylate kinase (DTYMK) can significantly reduce development of lung cancer without liver kinase B1. However, its underlying regulatory mechanism is still unclear. We therefore aimed to investigate whether DTYMK inhibitors could suppress lung adenocarcinoma (LUAD) progression. In this study, human tissues, A549 cells, and xenograft tumors were used to explore the regulation and mechanism of DTYMK on LUAD cell proliferation and migration. Meanwhile, YMU1 (a DTYMK inhibitor) was applied to A549 cells and xenograft tumors to investigate its potential as a drug for LUAD. DTYMK was overexpressed in LUAD tissues and correlated with tumor stage. Knockdown of DTYMK suppressed cell viability, migration, and invasion. In addition, the activation of signal transducers and activators of transcription 3 (STAT3) was repressed upon DTYMK inhibition. YMU1 showed the same effect as DTYMK knockdown in vivo and in vitro. DTYMK plays an important role in progression of LUAD through the STAT3 signaling pathway. YMU1 may have the potential to inhibit the development of LUAD.NEW & NOTEWORTHY DTYMK plays an important role in progression of LUAD through the STAT3 signaling pathway. YMU1 may serve as a novel drug to suppress the development of LUAD.

Protein Tyrosine Phosphatase Receptor Type R (PTPRR) Reduces AChR Clustering by Dephosphorylating MuSK.

Neuromuscular junction (NMJ) formation and maintenance depend on the proper localization and concentration of various molecules at synaptic contact sites. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in NMJ formation. Muscle-specific tyrosine kinase (MuSK), which functions depending on its phosphorylation, plays an essential role in AChR clustering. In the present study, we used plasmid-based biochemical screening and determined that protein tyrosine phosphatase receptor type R (PTPRR) is responsible for dephosphorylating MuSK on tyrosine residue 754. Furthermore, we showed that PTPRR significantly reduced MuSK-dependent AChR clustering in C2C12 myotubes. Collectively, these data illustrate a negative regulation function of PTPRR in AChR clustering.

Glucose-6-phosphate dehydrogenase promotes the proliferation and migration of lung adenocarcinoma cells via the STAT3 signaling pathway.

Lung adenocarcinoma (LUAD) is the most common histological subtype of lung cancer, and the leading cause of cancer-related deaths worldwide. G6PD has been reported to enhance the progression of various tumors by regulating the intracellular redox state and mediating nucleic acid synthesis. However, the biological role and molecular mechanism of G6PD in LUAD remain largely unknown. In this study, we found that G6PD was significantly upregulated in LUAD specimens and cell lines, and that the high levels of G6PD expression were closely associated with a poor prognosis for LUAD patients. Moreover, we found that G6PD significantly promoted the proliferation and migration of LUAD cells in vitro, and overexpression of G6PD also play a role of facilitating tumorigenesis in in vivo experiments. Mechanistically, the STAT3 signaling pathway was significantly activated by G6PD-mediated LUAD progression. Overall, our results suggest that G6PD could serve as a novel prognostic marker and therapeutic target for treating LUAD.

Hypoxic tumor-derived exosomal miR-31-5p promotes lung adenocarcinoma metastasis by negatively regulating SATB2-reversed EMT and activating MEK/ERK signaling.

BACKGROUND: Exosomes have emerged as critical mediators of intercellular communication. Hypoxia is widely recognized as a key regulator of tumor aggressiveness, and significantly affects exosome release by tumor cells. However, the effects of exosomes derived from hypoxic lung adenocarcinoma (LUAD) cells are poorly understood. METHODS: Samples of miRNA isolated from hypoxic LUAD cell-derived exosomes (HExo) and normoxic LUAD cell-derived exosomes (NExo) were sequenced to identify miRNAs that might mediate tumor progression. Exosomal miRNA was co-cultured with LUAD cells to assess its biological effects on cell migration and metastasis both in vitro and in vivo. The cellular target of exosomal miRNA was confirmed by dual-luciferase assays. Western blot studies showed that exosomal miRNA regulated the related pathway. The availability of circulating exosomal miRNA derived from plasma was also evaluated. RESULTS: We found that HExo could significantly enhance the migration and invasion of normoxic LUAD cells. MiRNA sequencing results suggested that miR-31-5p was largely internalized within HExo and could be taken up by normoxic LUAD cells. Exosomal miR-31-5p was found to directly target Special AT-Rich Sequence-Binding Protein 2 (SATB2)-revered epithelial mesenchymal transition and significantly increase activation of MEK/ERK signaling, thereby contributing to tumor progression both in vitro and in vivo. Furthermore, higher levels of circulating exosomal miR-31-5p were detected in LUAD patients, especially in patients with metastatic disease. CONCLUSIONS: Our findings demonstrate that exosomal miR-31-5p exerts a crucial role in LUAD progression, and could serve as a diagnostic biomarker for LUAD.

Prognostic Significance of Rab27A and Rab27B Expression in Esophageal Squamous Cell Cancer.

PURPOSE: Rab27A and Rab27B, members of the Rab family of small GTPases, have aberrant expression and exert different roles in various cancers. However, their expression and potential prognostic values in esophageal squamous cell cancer (ESCC) still remain elusive. In the present study, we explored the association of Rab27A and Rab27B expression with clinical significance and prognosis in ESCC. PATIENTS AND METHODS: A total of 100 surgically resected ESCC tissues were examined to evaluate Rab27A and Rab27B expression levels using the immunohistochemistry method. The relationship of Rab27A and Rab27B with clinicopathological features and prognosis was analyzed. We also investigated the correlation between Rab27A and Rab27B through external and internal validation. RESULTS: High-expression Rab27A was found to be significantly correlated with N (p=0.045) and TNM (p=0.005) stage, while up-regulated Rab27B was remarkably associated with N stage (p=0.033), TNM stage (p=0.009), and differentiation (p=0.013). High expression of both Rab27A and Rab27B had a worse overall survival (OS) rate. In addition, multivariate Cox regression analyses were utilized to validate that Rab27B expression is an independent prognostic factor for unfavorable OS. Further combined analyses showed that the Rab27Alow/Blow group had a superior OS rate than the Rab27Ahigh/Blow group, Rab27Alow/Bhigh group, and Rab27Ahigh/Bhigh group. Nevertheless, the latter three groups displayed rare significance between each two comparisons. Furthermore, our data demonstrated that Rab27A expression was positively correlated with Rab27B expression, which were also verified in TCGA datasets. CONCLUSION: Rab27A and Rab27B expression levels could be potentially novel prognostic biomarkers in ESCC.

C-Myc regulates PD-L1 expression in esophageal squamous cell carcinoma.

Immunotherapy using antibodies blocking the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway has achieved great success in preclinical models and the clinical treatment of esophageal squamous cell carcinoma (ESCC). The c-Myc proto-oncogene helps prevent immune cells from attacking tumor cells by inducing PD-L1 expression. However, the underlying mechanisms of c-Myc and PD-L1 in ESCC remain unclear, and a thorough understanding of this regulation would allow the development of new approaches to enhance antitumor immunity. In the present study, the positive relationship between c-Myc and PD-L1 was explored in the Cancer Genome Atlas dataset using the bioinformatics tool GEPIA, and was confirmed in 105 ESCC tissues by immunostaining (r=0.516, P<0.001). The patients positive for both proteins had a poorer overall survival (P=0.032). Furthermore, in ESCC cell lines, c-Myc overexpression, depletion, and inhibition was able to regulate the expression of PD-L1. Also, the ChIP assays showed that the increase in PD-L1 expression was likely due to the binding of c-Myc to the PD-L1 promoter. Taken together, c-Myc and PD-L1 levels were significantly correlated, and c-Myc expression regulated the expression of PD-L1 in ESCC cells. In addition, a small molecule inhibitor of c-Myc effectively regulated PD-L1 expression. This indicates that synergistic therapy combining a c-Myc inhibitor with PD-L1 immunotherapy might be a promising new treatment strategy for ESCC.

HER4 promotes the growth and metastasis of osteosarcoma via the PI3K/AKT pathway.

Osteosarcoma is the most common primary malignant bone tumor, which occurs in adolescents. As reported by our previous studies, HER4 indicates a poor prognosis of primary osteosarcoma. However, its mechanisms in the pathogenesis of osteosarcoma have not yet been studied. The purpose of this study was to investigate the role of HER4 in osteosarcoma and whether the PI3K/AKT pathway is involved. In this study, western blot analysis was used to investigate the expression of HER4 protein in osteosarcoma tissues and cell lines. CCK8 and transwell assays were used to detect the effects of HER4 on the proliferation, migration, and invasion of osteosarcoma cells in vitro. The effects of HER4 on the growth and metastasis of osteosarcoma in vivo were detected by tumor formation and immunofluorescence in nude mice. The role of the PI3K/AKT pathway in HER4 regulation of the growth and metastasis of osteosarcoma was examined by western blot analysis and immunofluorescence assay. We found that HER4 protein was highly expressed in clinical osteosarcoma specimens and osteosarcoma cells. HER4 markedly promoted the proliferation, migration, and invasion of osteosarcoma cells in vitro as well as the growth and metastasis of osteosarcoma in vivo. HER4 overexpression upregulated the expression of phosphorylated protein kinase B (pAKT), proliferation marker antigen Ki67, and metastasis cell marker matrix metalloproteinase 9 (MMP9). Notably, PI3K/AKT inhibitor LY294002 significantly inhibited the effects of HER4 via the downregulation of pAKT, Ki67, and MMP9. Moreover, LY294002 markedly blocked the effects of HER4-induced upregulation of tumor malignancy. The present study suggests that HER4 may promote the growth and metastasis of osteosarcoma via the PI3K/AKT pathway. The HER4/PI3K/AKT pathway could serve as a potential target for the treatment of osteosarcoma.

High programmed death-ligand 1 expression is a poor prognostic indicator for esophageal squamous cell carcinoma and is correlated with two-field lymph node metastasis.

BACKGROUND: This study detected programmed death-ligand 1 (PD-L1) expression in esophageal squamous cell carcinoma (ESCC) samples and investigated the association between the clinicopathological features of ESCC and PD-L1 expression. Furthermore, this study evaluated whether lymph node metastasis (LNM) status affected the prognosis of ESCC patients with diverse PD-L1 expression. METHODS: A total of 108 ESCC paraffin-embedded specimens were collected and further used to detect the expression of PD-L1 by immunohistochemistry. The correlation between PD-L1 expression and clinicopathological characteristics and its prognostic significance were evaluated. Finally, the clinical outcomes were investigated in the subgroup of patients with simultaneous high PD-L1 expression and LNM. RESULTS: PD-L1 expression was observed in 85.2% of ESCC cases, 43.5% of which showed high expression. Only N stage (P=0.038) was associated with high PD-L1 expression. Univariate and multivariate Cox regression analyses showed that T stage (P=0.012), N stage (P=0.029), and PD-L1 expression (P=0.044) were independent prognostic factors in ESCC patients. Moreover, further analysis of the N stage subgroup showed that skip LNM (P=0.286) or laryngeal nerve LNM (P=0.387) showed no prognostic significance, while two-field LNM was highly correlated with a worser 5-year overall survival (P=0.037). CONCLUSIONS: High PD-L1 expression was significantly correlated with N stage and can be used as an independent prognostic biomarker in ESCC. Its expression level can also be used to guide appropriate lymph node dissection in ESCC.

Upregulation of Long Non-Coding RNA GCC2-AS1 Facilitates Malignant Phenotypes and Correlated With Unfavorable Prognosis for Lung Adenocarcinoma.

BACKGROUND: The role played by long noncoding RNA GCC2-AS1 in primary malignant tumors remains poorly understood. This study aimed to determine the expression levels and evaluate the clinical significance and biological effects of GCC2-AS1 in lung adenocarcinoma (LUAD). METHODS: We used data obtained from tissue samples and the TCGA database to determine the levels of GCC2-AS1 expression LUAD patients, and the prognostic value of those levels. Functional experiments were performed to investigate the effect of GCC2-AS1 on LUAD cells. Genes that were differentially expressed in GCC2-AS1-low and -high groups were analyzed by an enrichment analysis. Additionally, a nomogram model was created and subgroup analyses were performed to further determine the prognostic value of GCC2-AS1 in LUAD. RESULTS: GCC2-AS1 expression was significantly upregulated in lung adenocarcinoma tissues as compared with normal tissues. Depletion of GCC2-AS1 inhibited the proliferation and invasion of LUAD cells in vitro. An elevated level of GCC2-AS1 was strongly correlated with shorter overall survival time and was identified as an independent prognostic marker for LUAD patients. Enrichment analyses conducted using GO, KEGG, and GSEA databases were performed to identify biological pathways that might involve GCC2-AS1. Several subgroups were found to have a significant prognostic value for patients in the GCC2-AS1-low and -high groups. CONCLUSIONS: Our findings suggest that GCC2-AS1 can serve as a diagnostic and prognostic biomarker for LUAD patients.

ACTN4 Promotes the Proliferation, Migration, Metastasis of Osteosarcoma and Enhances its Invasive Ability through the NF-κB Pathway.

Alpha-actinin-4 (ACTN4) is associated with different types of tumors, but its role in osteosarcoma (OS) is not known. We aimed to investigate the effect of ACTN4 on the growth, migration, invasion and metastasis of OS. We further explored the possible mechanism of how ACTN4 affects the development of OS. First, the expression of ACTN4 in OS tissues and OS cell lines was analyzed by PCR. Second, the role of ACTN4 in the development of OS was explored by the proliferation, scratch, and invasion assays. We further explored the effect of ACTN4 on OS growth in an orthotopic xenograft model of nude mice. In addition, we used hematoxylin and eosin (HE) staining of lung tissues in nude mice to observe the effect of ACTN4 on lung metastasis of OS. Finally, rescue experiments further investigated the role of NF-κB on ACTN4 in the development of OS. ACTN4 was highly expressed in OS tissues and OS cell lines. In vitro experiments demonstrated that reducing ACTN4 expression inhibited the proliferation, migration, and invasion of OS. In contrast, overexpression of ACTN4 promotes these effects. In vivo experiments further validated that ACTN4 promoted the growth of OS. The HE staining of lungs in nude mice revealed that ACTN4 promoted lung metastasis of OS. In addition, we found that ACTN4 enhanced the ability of OS to invade, through the NF-κB pathway. ACTN4 promotes the proliferation, migration, metastasis of OS and enhances its invasion ability through the NF-κB pathway.

Clinicopathological and prognostic values of fibronectin and integrin αvß3 expression in primary osteosarcoma.

BACKGROUND: Osteosarcoma is a malignant bone tumor with a high potential for lung metastasis, and the prognosis for patients with metastatic disease is very poor. The interaction between fibronectin (FN) and integrin αvß3 in soft-tissue sarcoma promotes cell migration, invasion, and lung metastasis. This study aimed to investigate the prognostic significance of FN and αvß3 in osteosarcoma. METHODS: Immunohistochemistry and western blotting were used to detect the expression of FN and αvß3 in 60 osteosarcoma specimens and in 30 osteochondroma specimens. Furthermore, correlations of FN and αvß3 with the clinicopathological features of osteosarcoma patients were analyzed using the χ2 test and Fisher's exact test. Disease-free survival and overall survival of osteosarcoma patients were assessed using the Kaplan-Meier method and Cox proportional hazards model. The predictive accuracy of the model was determined by the Harrell concordance index. RESULTS: FN (P < 0.05) and αvß3 (P < 0.05) were overexpressed in osteosarcoma specimens compared with osteochondroma specimens. High FN expression was associated with a poor response to chemotherapy (P = 0.001) and poor disease-free (P < 0.001) and overall (P < 0.001) survival. High expression of αvß3 was linked to an advanced surgical stage (P = 0.028), a poor response to chemotherapy (P = 0.002), and both poor disease-free survival (P < 0.001) and overall survival (P < 0.001). FN and αvß3 co-expression were associated with sex (P = 0.011), an advanced surgical stage (P = 0.013), and a poor response to chemotherapy (P = 0.002). Moreover, high expression of both proteins can serve as an independent prognostic value for reduced survival time in osteosarcoma patients. CONCLUSIONS: The results of this study suggest that FN and αvß3 expression is associated with an unfavorable clinical outcome of osteosarcoma, and these molecules may constitute attractive therapeutic targets for osteosarcoma treatment. To improve the survival of osteosarcoma patients, further investigations are required to clarify their prognostic values in a larger population.

MicroRNA-885-5p promotes osteosarcoma proliferation and migration by downregulation of cell division cycle protein 73 homolog expression.

Osteosarcoma (OS) is the most common primary malignant bone tumor. Numerous studies have strongly implicated the ectopic expression of microRNAs (miRNAs/miRs), including miR-885-5p, which is aberrantly expressed in several cancer types, in multiple cancer-related processes. However, the role of miR-885-5p in OS remains unknown. In the present study, it was found that the expression of miR-885-5p was markedly upregulated in OS cell lines and clinical tissues. Moreover, high expression of miR-885-5p was significantly associated with the development of OS. The human OS MG-63 cell line was transfected with recombinant lentivirus to regulate miR-885-5p expression. Overexpressed miR-885-5p significantly promoted the proliferation and migration of MG-63 cells in vitro, while downregulating miR-885-5p expression reversed these effects. Furthermore, bioinformatic analysis was used to predict the potential target genes of miR-885-5p, and cell division cycle protein 73 homolog (CDC73) was identified as a novel and direct target of miR-885-5p. This interaction was further confirmed using reverse transcription-quantitative polymerase chain reaction, western blotting and luciferase activity assays. These findings suggest that miR-885-5p serves a critical role in facilitating OS proliferation and migration, and can regulate CDC73 expression in OS cells and tissues. Thus, miR-885-5p could be a promising novel therapeutic biomarker for OS.

Prognostic significance of the expression of HER family members in primary osteosarcoma.

The prognosis of patients with metastatic osteosarcoma is poor and has shown no significant improvement in nearly 20 years. The human epidermal growth factor (EGF) receptor (HER) family is frequently overexpressed in the majority of human carcinomas, and is involved in promoting the proliferation and survival of cancer cells. However, the role of EGFR and HER-2 expression in osteosarcoma survival remains controversial and no previous study has simultaneously investigated the association of the expression of all the four HER family members with the prognostic significance of osteosarcoma. Therefore, the present study investigated the expression levels of the complete members of the HER family in osteosarcoma specimens, as well as their associations with the clinicopathological parameters, progression-free survival (PFS) and overall survival (OS) time of patients with osteosarcoma. The expression of HER family members was detected in osteosarcoma tumor specimens from 60 patients using immunohistochemistry. The association of the expression of HER receptors in osteosarcoma with clinicopathological parameters was analyzed using χ2 test and Fishers exact test. Survival analyses were evaluated by Kaplan-Meier method and Cox proportional hazards regression model. Overall, 18 (30%), 13 (22%), 23 (38%) and 19 (32%) patients presented with high expression of EGFR, HER-2, HER-3 and HER-4, respectively, and the co-expression of 2, 3 and all 4 members of the HER family was observed. High expression of EGFR and HER-4 was associated with distant metastasis. High HER-3 expression was significantly associated with an advanced Enneking stage and distant metastasis. Multivariate analysis demonstrated that the expression of EGFR, HER-3, HER-4, EGFR/HER-3, EGFR/HER-4 and HER-3/HER-4 was an independent predictor of poor PFS and OS time in osteosarcoma patients with stage I-IIB disease. In patients with stage IIB osteosarcoma, the expression of HER-4 and EGFR/HER-4 demonstrated a more significant effect on PFS and OS time. In conclusion, therapies targeting EGFR, HER-3 and HER-4 may provide promising strategies for primary osteosarcoma.