PADI1 and Its Co-Expressed Gene Signature Unveil Colorectal Cancer Prognosis and Immunotherapy Efficacy.

Peptidyl arginine deiminase 1 (PADI1) catalyzes protein citrullination and has a role in regulating immune responses. The tumor immune microenvironment has been reported to be important in colorectal cancer (CRC), which was correlated with the ability of CRC patients to benefit from immunotherapy. However, there is a lack of molecular markers for matching CRC immunotherapy. Previously, single-gene risk models have only considered the effect of individual genes on intrinsic tumor properties, ignoring the role of genes and their co-expressed genes as a whole. In this study, we analyzed the differential expression of PADI1 in colorectal cancer (CRC). We found that PADI1 was highly expressed in CRC. Subgroup survival analysis revealed a prognostic survival difference for PADI1 in CRC patients aged less than 65 years, male, T stage, N0, M0, and stage I-II (p < 0.05). In addition, we analyzed the functions and signaling pathways associated with PADI1 in CRC and found that it was highly enriched in several immune-related functions and pathways. Then, a set of PADI1 co-expressed genes (PCGs) risk-prognosis scores was developed with PADI1 as the core, which could accurately predict the prognosis of CRC (p < 0.05). PCGs risk score can be an independent prognostic factor for CRC. A new set of Norman plot models were developed for clinical characteristics with age, sex, and TNM stage, which can accurately predict CRC 1, 3, and 5 years survival, and calibration curves and decision curve analysis (DCA) validated the accuracy of the models. The risk score assessed the immune microenvironment of CRC and found that the immune score was higher in the low-risk group, and CD4+ T cells, helper T cells, and eosinophils were more infiltrated in the low-risk group (p < 0.05). Immunotherapy efficacy was better in the low-risk group (p < 0.05). The underlying mechanism may be that the high-risk group of PCGs was enriched in some pathways that promote immune escape and immune dysfunction. In conclusion, PCGs may better predict CRC prognosis and immunotherapeutic response.

Endostatin induces normalization of blood vessels in colorectal cancer and promotes infiltration of CD8+ T cells to improve anti-PD-L1 immunotherapy.

Introduction: The purpose of this study was to evaluate recombinant human endostatin (rHE)-induced normalization of the tumor vasculature in colorectal cancer (CRC) and to evaluate the therapeutic effects of combined treatment with rHE and a programmed death ligand-1 (PD-L1) inhibitor. Methods: A mouse subcutaneous tumorigenesis model was established to evaluate the antitumor effects of endostatin combined with a PD-L1 inhibitor on CRC. Intravoxel incoherent motion diffusion-weighted magnetic resonance imaging (IVIM-DW MRI) was used to evaluate changes in the intratumor microcirculation in response to combined treatment with endostatin and a PD-L1 inhibitor. The infiltration density and function of CD8+ T cells in tumors were evaluated using flow cytometry. Finally, clinical specimens were used to evaluate the expression area of tumor vascular pericytes and CD8+ T cells in tumor tissues. Results: The antitumor effects of endostatin combined with a PD-L1 inhibitor were significantly greater than those of endostatin or a PD-L1 inhibitor alone. On the ninth day of intervention, the endostatin group showed significantly higher pseudo diffusion parameter (D*) and microvascular volume fraction (F) values in tumors than those in the control group or PD-L1 group. After 27 days of intervention, the endostatin groups showed significantly lower levels of vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-ß than those in the control group. Treatment of CD8+ T cells with endostatin for 24 h did not alter the expression levels of markers of reduced T-cell activity. However, endostatin reversed the VEGF-mediated inhibition of the secretion of interferon (IFN)-γ from T cells. The results in CRC clinical samples showed that treatment with endostatin induced significantly higher infiltration of CD8+ T cells compared with treatment that did not include endostatin. Furthermore, the expression area of pericytes was significantly positively related to the infiltration density of CD8+ T cells and overall survival time. Conclusion: Endostatin improved the antitumor effects of PD-L1 inhibitors on CRC, significantly increased the activity of CD8+ T cells, and synergistically improved the tumor treatment effect of the two inhibitors.

Thrombospondin-2 holds prognostic value and is associated with metastasis and the mismatch repair process in gastric cancer.

BACKGROUND: This study aims to investigate thrombospondin 2 (TSP2) expression levels in gastric cancer (GC) and determine the relationship between TSP2 and clinical characteristics and prognosis. METHODS: The online database Gene Expression Profile Interactive Analysis (GEPIA) was used to analyse TSP2 mRNA expression levels in GC. The Kaplan-Meier plotter prognostic analysis tool was used to evaluate the influence of TSP2 expression on clinical prognosis in GC patients. TSP2 expression levels were analysed in paraffin-embedded GC samples and adjacent normal tissues by immunohistochemistry. The relationship between the clinicopathological characteristics and prognosis of GC patients was assessed. Transwell experiments were used to evaluate the effect of TSP2 on HGC27 and AGS cell invasion and migration. The EdU experiment was used to detect the effect of transfection of TSP2 on cell proliferation, and the flow cytometry experiment was used to detect the effect of TSP2 on cell apoptosis and the cell growth cycle. Western blotting (Wb) technology was used to detect MMP, E-cadherin, N-cadherin, Vimentin, Snail, AKT, PI3K, and VEGF protein expression in HGC27 cells. RESULTS: Compared with normal tissues, TSP2 mRNA expression in GC was significantly upregulated and was closely related to the clinical stage of GC. High TSP2 expression significantly affected the OS, FP and PPS of patients with GC. Among these patients, TSP2 expression levels did not affect the prognosis of patients with GC in the N0 subgroup but significantly affected the prognosis of patients with GC in the N (1 + 2 + 3) subgroup. TSP2 protein expression levels were significantly higher in GC tissue compared with normal tissues (P < 0.01). The overall survival (OS) and relapse-free survival (RFS) of patients with high TSP2 expression were lower than those of patients with low TSP2 expression. Cells transfected with the TSP2-silencing sequence exhibited increased apoptosis and inhibition of proliferation, migration and invasion. AKT and PI3K expression in cells was significantly downregulated (P < 0.01). AKT, PI3K and VEGF expression in cells transfected with the TSP2 silencing sequence was significantly reduced. Proliferation, migration, invasion ability, and TSP2 expression levels significantly correlated with mismatch repair genes, such as PMS2, MSH6, MSH2, and MLH1 (P < 0.05). CONCLUSION: TSP2 expression is significantly increased in GC. TSP2 expression is closely related to metastasis and the mismatch repair process in GC patients and affects GC patient prognosis. The mechanism may involve regulating gastric cancer cell proliferation and migration by modulating the VEGF/PI3K/AKT signalling pathway. TSP2 is a potential marker and therapeutic target for the prognosis of GC patients.

Comprehensive Analysis of N6-Methyladenosine-Related lncRNA Signature for Predicting Prognosis and Immune Cell Infiltration in Patients with Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most common tumor worldwide. Aberrant N6-methyladenosine (m6A) modification can influence the progress of the CRC. Additionally, long noncoding RNA (lncRNA) plays a critical role in CRC and has a close relationship with m6A modification. However, the prognostic potential of m6A-related lncRNAs in CRC patients still remains to be clarified. METHODS: We use "limma" R package, "glmnet" R package, and "survival" R package to screen m6A-related-lncRNAs with prognostic potential. Then, we comprehensively analysed and integrated the related lncRNAs in different TNM stages from TCGA database using the LASSO Cox regression. Meanwhile, the relationship between functional enrichment of m6A-related lncRNAs and immune microenvironment in CRC was also investigated using the TCGA database. A prognostic model was constructed and validated to determine the association between m6A-related lncRNAs in different TNM stages and the prognosis of CRC. RESULT: We demonstrated that three related m6A lncRNAs in different TNM stages were associated with the prognosis of CRC patients. Patients from the TCGA database were classified into the low-risk and the high-risk groups based on the expression of these lncRNAs. The patients in the low-risk group had longer overall survival than the patients in the high-risk group (P < 0.001). We further constructed and validated a prognostic nomogram based on these genes with a C-index of 0.80. The receiver operating characteristic curves confirmed the predictive capacity of the model. Meanwhile, we also found that the low-risk group has the correlation with the dendritic cell (DC). Finally, we discovered the relationship between the m6A regulators and the three lncRNAs. CONCLUSION: The prognostic model based on three m6A-related lncRNAs exhibits superior predictive performance, providing a novel prognostic model for the clinical evaluation of CRC patients.

The Application of and Strategy for Gold Nanoparticles in Cancer Immunotherapy.

Immunotherapy of malignant tumor is a verified and crucial anti-tumor strategy to help patients with cancer for prolonging prognostic survival. It is a novel anticancer tactics that activates the immune system to discern and damage cancer cells, thereby prevent them from proliferating. However, immunotherapy still faces many challenges in view of clinical efficacy and safety issues. Various nanomaterials, especially gold nanoparticles (AuNPs), have been developed not only for anticancer treatment but also for delivering antitumor drugs or combining other treatment strategies. Recently, some studies have focused on AuNPs for enhancing cancer immunotherapy. In this review, we summarized how AuNPs applicated as immune agents, drug carriers or combinations with other immunotherapies for anticancer treatment. AuNPs can not only act as immune regulators but also deliver immune drugs for cancer. Therefore, AuNPs are candidates for enhancing the efficiency and safety of cancer immunotherapy.

Relationship between Tertiary Lymphoid Structure and the Prognosis and Clinicopathologic Characteristics in Solid Tumors.

Background: An increasing number of studies had shown that tertiary lymphoid structure (TLS) plays an important role in tumor progression. However, the prognostic role of TLS in various tumors remains controversial. This meta-analysis aims to investigate the clinicopathological and prognostic values of TLS in solid tumors. Methods: A systematic search was conducted in PubMed, EMBASE and Cochrane Library undated to November 2, 2020. Odds ratios of clinical parameters, hazard ratio (HR) of overall survival (OS), relapse-free survival (RFS), disease-free survival (DFS) and relapse rate were calculated in order to evaluate the relationship between TLS expression and clinicopathological or prognostic values in different tumors. Result: 27 eligible studies including 6647 patients with different types of tumors were analyzed. High TLS expression was associated with a longer OS (HR = 0.66, 95% CI: 0.50 - 0.86, P = 0.002) and RFS (HR = 0.61, 95% CI: 0.47 - 0.79, P = 0.0001). Moreover, high TLS levels in tumor were associated with a low risk of recurrence (HR = 0.43, 95% CI: 0.32 - 0.57, P < 0.0001). However, there was no relationship between TLS expression and DFS. Meanwhile, high TLS expression was associated with smaller tumor size (P < 0.00001) and higher tumor infiltrating lymphocytes (TILs). Furthermore, the subgroup analysis showed high TLS expression that may be associated with a lower clinical grading and N stage in breast cancer and colorectal cancer. Conclusion: High TLS expression is associated with the longer OS and RFS in solid tumors, and a lower risk of cancer relapse. Meanwhile, high TLS expression is also associated with a smaller tumor size, higher infiltration of TILs, lower clinical grading and N stage in the tumor. Therefore, high TLS expression in the tumor is a favorable prognostic biomarker for solid tumor patients.

Long Noncoding RNA KCNQ1OT1 is a Prognostic Biomarker and mediates CD8+ T cell exhaustion by regulating CD155 Expression in Colorectal Cancer.

Background: Long noncoding RNA KCNQ1 opposite strand/antisense transcript 1 (lncRNA KCNQ1OT1) is abnormally expressed in various solid tumors. The purpose of this study was to explore the prognostic value and potential functional role of lncRNA KCNQ1OT1 across cancers. Methods: We performed a meta-analysis of published literature to evaluate the prognostic value of lncRNA KCNQ1OT1 across cancers. Verification, functional analysis, and genomic variation analysis were performed using the GEPIA, TIMER, and LnCeVar databases. According to the immune cell infiltration level, we established a prognostic model of lncRNA KCNQ1OT1 expression using public datasets of TIMER. We used quantitative real-time polymerase chain reaction (RT-qPCR) and western blot to detect the expression levels of lncRNA KCNQ1OT1 and the CD155 protein in colorectal cancer (CRC) tissues and cell lines. Then, a lncRNA KCNQ1OT1-knockdown cell line was cocultured to explore the role of lncRNA KCNQ1OT1 and CD155 in the T cell response by flow cytometric analysis. Results: Our results showed that the high expression of lncRNA KCNQ1OT1 was significantly related to poor overall survival across cancers, especially CRC. Interestingly, we found that COAD patients with high lncRNA KCNQ1OT1 expression and high CD8+ T cell infiltration levels had a worse prognosis than those with low lncRNA KCNQ1OT1 expression and high CD8+ T cell infiltration levels. Moreover, lncRNA KCNQ1OT1 and CD155 showed significantly higher expression in CRC tissue than in normal tissue, and lncRNA KCNQ1OT1 expression was positively correlated with CD155 expression in CRC. Finally, knockdown of lncRNA KCNQ1OT1 reduced CD155 expression in HCT116 and SW620 cells and enhanced the immune response in coculture with CD8+ T cells. Conclusions: High lncRNA KCNQ1OT1 expression is significantly correlated with poor prognosis of CRC patients and mediates the CD8+ T cell response in CRC. These findings indicate that lncRNA KCNQ1OT1 is a prognostic biomarker and potential immune therapeutic target for enhancing the CD8+ T cell response in CRC.

Clinicopathological and prognostic significance of Fusobacterium nucleatum infection in colorectal cancer: a meta-analysis.

Background: This study aimed to clarify the relationship between F. nucleatum levels and the prognosis of CRC, which is still controversial. Methods: Relevant articles were searched on PubMed, Web of Science, PMC and Embase up to April 7, 2020. Outcomes of interest included clinical characteristics, molecular characteristic and survival analysis. HR (OR), odds ratios (OR) and 95% confidence interval (CI) were calculated to explore the prognostic value and relationship of clinical characteristics of Fusobacterium nucleatum in CRC. Results: A total of 3626 CRC patients from 13 eligible studies were included. High levels of F. nucleatum were associated with worse prognosis, as such parameters as overall survival (OS) (hazard ratio [HR] = 1.40, 95% confidence interval [CI]: 1.40 - 1.63, P < 0.0001), disease-free survival (DFS) (HR = 1.71, 95% CI: 1.29-2.26, P = 0.0002), and cancer-specific survival (OR= 1.93, 95% CI: 1.42-2.62, P <0.0001). F. nucleatum levels were related with T3-T4 stage (OR = 2.20, 95% CI: 1.66-2.91, P < 0.00001), M1 stage (OR = 2.11, 95% CI: 1.25-3.56, P = 0.005), poor tumor differentiation (OR = 1.83, 95% CI: 1.11-3.03, P =0.02), microsatellite instability-high (OR = 2.53, 95% CI: 1.53-4.20, P = 0.0003), and KRAS mutation (OR =1.27, 95% CI: 1.00-1.61, P=0.05) showed. Conclusions: High levels of F. nucleatum suggest a poor prognosis and are associated with tumor growth, distant metastasis, poor differentiation, MSI-high, and KRAS mutation in CRC patients.

A network pharmacology approach for investigating the multi-target mechanisms of Huangqi in the treatment of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most prevalent cancer globally. In the treatment of CRC, surgical resection is commonly adopted, and neoadjuvant chemotherapy or immunotherapy is mainly administered for patients with advanced disease. However, despite the developments in the field of cancer treatment, the mortality rate of CRC has remained high. Therefore, novel treatments for CRC need to be explored. Astragalus membranaceus, commonly known in China as Huangqi (HQ), a traditional Chinese medicine, has been reported to be a potential antitumorigenic agent. This study aimed to investigate the mechanisms of action of HQ. METHODS: Active ingredients and putative targets of HQ were obtained through a comprehensive search of the Traditional Chinese Medicine Systems Pharmacology database. CRC-related targets were retrieved from the GeneCards database and then overlapping targets were acquired. After visualization of the compound-disease network and protein-protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the overlapping genes were performed. Additionally, HCT116 cells were treated with the active components of HQ at a 20-µM concentration. Cell Counting Kit-8 was used to detect cell activity, and real-time quantitative polymerase chain reaction was carried out to detect the expression of genes downstream of the interleukin (IL)-17 signaling pathway. RESULTS: A PPI network comprising 177 nodes and 318 edges was obtained. The GO analysis of the overlapping genes showed enrichment in response to lipopolysaccharide and oxidative process. For the KEGG analysis, the AGE-RAGE signaling pathway and inflammation-related pathways, such as the IL-17 and tumor necrosis factor (TNF) signaling pathways, were enriched. The in vitro experiments showed that HQ promoted the apoptosis of CRC cells by inhibiting the expression of the CCL2, CXCL8, CXCL10, and PTGS2 genes. CONCLUSIONS: This study systematically revealed the multitarget mechanism of HQ in CRC through a network pharmacology approach. We verified that HQ promotes CRC cell death via the IL-17 signaling pathway. This finding provides indications for further mechanistic studies and the development of HQ as a potential treatment for CRC patients.

[Apoptosis induced by transferring cytosine deaminase suicide gene into kinds of cancer cells with novel polyamidoamine dendrimers].

This study demonstrates efficacy of a novel polyamidoamine dendrimers(PAMAM dendrimers) with pentaerythritol derivatives as the core(G5 PD dendrimer) in deliver of the cytosine deaminase(CD) gene and EGFP gene fusion plasmid into different tumor cell lines to induce apoptosis. The physical and chemical properties of G5 PD dendrimers in terms of DNA complexation, particulate properties and transfection efficiencies were investigated and compared with commercial gene vectors PEI 25 k Da. The optimum ratio of G5 PD dendrimer complexed with plasmid DNA was 0.2∶1, and the particle size of the complex was(100 ± 5) nm. Compared with the commercial gene carriers PEI, G5 PD dendrimer exhibited a higher transfection efficiency at the weight ratio of 1∶1 in three different cell lines, given the fact that PEI are different from PAMAM dendrimers in terms of molecular structure. Furthermore, the cytotoxicity assays of the cell lines transfected with G5 PD dendrimer/p CD-EGFP-N1 followed by exposure to various concentrations of 5-fluorocytosine(5-FC) also showed that the transfected cell lines could generate a very low amount of 5-FC to 5-fluorouracil(5-FU) in a short period of time, which indicating the high expression level of CD gene in the cell line. These results indicate that the CD/5FC system of G5 PD dendrimer has an excellent efficacy in gene delivery.

Inhibitory effects of osteoprotegerin on osteoclast formation and function under serum-free conditions.

The purpose of this study was to determine whether osteoprotegerin (OPG) could affect osteoclat differentiation and activation under serum-free conditions. Both duck embryo bone marrow cells and RAW264.7 cells were incubated with macrophage colony stimulatory factor (M-CSF) and receptor activator for nuclear factor kB ligand (RANKL) in serum-free medium to promote osteoclastogenesis. During cultivation, 0, 10, 20, 50, and 100 ng/mL OPG were added to various groups of cells. Osteoclast differentiation and activation were monitored via tartrate-resistant acid phosphatase (TRAP) staining, filamentous-actin rings analysis, and a bone resorption assay. Furthermore, the expression osteoclast-related genes, such as TRAP and receptor activator for nuclear factor κB (RANK), that was influenced by OPG in RAW264.7 cells was examined using real-time polymerase chain reaction. In summary, findings from the present study suggested that M-CSF with RANKL can promote osteoclast differentiation and activation, and enhance the expression of TRAP and RANK mRNA in osteoclasts. In contrast, OPG inhibited these activities under serum-free conditions.

Influence of osteoprotegerin on differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts in vitro.

ABSTRACT The aim of this study was to determine the influence of osteoprotegerin (OPG) on the differentiation, activation, and apoptosis of Gaoyou duck embryo osteoclasts cultured in vitro. Bone marrow cells were harvested from 23-d-old Gaoyou duck embryos and cultured in the presence of different concentrations of OPG (group A: no added factors, group B: 30 ng/mL of OPG, and group C: 100 ng/mL of OPG). Tartrate-resistant acid phosphatase (TRAP) staining, pit formation assay, and co-staining with tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin and Hoechst 33258 were all performed to determine the number of TRAP-positive cells, bone resorption activity, and the level of apoptosis, respectively. The number of TRAP-positive cells and the net expansion of pit formations area peaked on d 7 of culture in all 3 groups. The number of osteoclasts and the total volume of pit formations in OPG-treated groups were significantly lower compared with group A (P < 0.05). At each time point, the net expansion of pit formations area correlated with the number of TRAP-positive cells. The OPG inhibited the de novo formation of filamentous (F)-actin rings and promoted the disruption of existing F-actin rings in mature osteoclasts. In addition, OPG induced apoptosis in mature osteoclasts, as demonstrated by morphological changes in the nuclei. In osteoclast precursors, OPG inhibited differentiation and downregulated the formation of F-actin rings. In mature osteoclasts, OPG suppressed activation and enhanced the development of apoptosis, observed as a decrease in the number of TRAP-positive cells, the disruption of F-actin rings and morphological changes of the nuclei.

Osteoprotegerin influences the bone resorption activity of osteoclasts.

The aim of the present study was to determine whether osteoprotegerin (OPG) influences the bone resorption activity of osteoclasts. RAW264.7 cells were induced by macrophage colony-stimulating factor (M-CSF) + receptor activator of nuclear factor-κB ligand (RANKL) and 0, 10, 20, 50 and 100 ng/ml OPG were added into various groups in the presence of the two cytokines. The OPG treatment was continued for 24 h. Osteoclast differentiation and activation were estimated via TRAP staining assay, TRITC-conjugated phalloidin staining, resorption activity analysis. Furthermore, the expression levels of the osteoclastic bone resorption-related genes MMP-9, cathepsin K and carbonic anhydrase II (CA II) were examined using real-time polymerase chain reaction (PCR). The data demonstrated that high concentrations of OPG could inhibit the differentiation and activation of osteoclasts. Furthermore, real-time PCR analysis illustrated that OPG decreased the expression of MMP-9 and cathepsin K in different concentrations of OPG and it decreased the expression of CA II genes at 10 and 20 ng/ml concentrations of OPG. For the time gradient study, OPG decreased the expression of MMP-9 and CA II genes but not that of the cathepsin K gene. In summary, the resorption activity of osteoclasts was suppressed by high concentrations of OPG and, at the molecular level, OPG decreased the expression of osteoclastic bone resorption-related genes.

Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108.

AIM: YCP, a novel (1,4)-alpha-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation. METHODS: Fluorescence-labeled YCP (fl-YCP) was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting (FACS) were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 6 (TLR6) and complement receptor 3 (CR3). We also examined the role of the p38 kinase pathway in mediating nitric oxide (NO) production. RESULTS: YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fl-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b (CR3) antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation. CONCLUSION: We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.Acta Pharmacologica Sinica (2009) 30: 1008-1014; doi: 10.1038/aps.2009.93.