Role of CD61+ low-density neutrophils in promoting hepatocellular carcinoma metastasis through CCDC25 upregulation.

BACKGROUND: A subset of neutrophils isolated from the peripheral blood mononuclear cells (PBMC) layer has recently been described in cancer patients. METHODS: Double-gradient centrifugation was used to separate the neutrophil subsets. Western blotting and immunohistochemical assays were performed to assess CCDC25 expression levels. RESULTS: In this study, we found that low-density neutrophils (LDNs) were more highly enriched in metastatic hepatocellular carcinoma (HCC) patients than in non-metastatic HCC patients. We then showed a CD61+ LDNs subset, which displayed distinct functions and gene expression, when compared with high-density neutrophils (HDNs) and CD61- LDNs. Transcriptomic analysis revealed that the CD61+ LDNs were predominantly enhanced in the transcription of glycolysis and angiogenesis associated gene, HMGB1 associated gene and granulation protein gene. These CD61+ LDNs displayed a prominent ability to trigger metastasis, compared with HDNs and CD61- LDNs. Specifically, CD61+ LDN-derived HMGB1 protein increased the invasion of HCC cells by upregulating CCDC25. Mechanistically, the CD61+ LDN-derived HMGB1 protein enhanced the invasiveness of HCC cells and triggered their metastatic potential, which was mediated by TLR9-NF-κB-CCDC25 signaling. Blocking this signaling pathway reversed the invasion of the CD61+ LDN-induced HCC cells. In vivo, we consistently showed that CD61+ LDN-derived HMGB1 enhances HCC metastasis to the lungs. CONCLUSIONS: Overall, our findings showed that a subset of CD61+ LDNs has pro-metastatic effects on HCC, and may be used to target HCC in the clinical setting.

CCDC25 suppresses clear cell renal cell carcinoma progression by LATS1/YAP-mediated regulation of the hippo pathway.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is one of the most prevalent renal cancers, and the molecular mechanisms underlying its progression are still not fully understood. The expression of CCDC25, a notably underexpressed gene in many tumors, has been understudied in ccRCC. This research aims to explore the role of CCDC25 in ccRCC's clinical outcomes and uncover the molecular pathways influenced by it. METHODS: A multi-tiered approach was adopted involving bioinformatic analysis, tissue sample evaluation, in vitro and in vivo experiments. CCDC25 expression levels in tumor vs. normal tissues were quantified using Western blot and immunofluorescence studies. Cell proliferation and migration were analyzed using CCK8, EDU, Transwell assays, and wound healing assays. RNA sequencing was performed to elucidate the molecular pathways affected, followed by detailed protein-protein interaction studies and mouse xenograft models. RESULTS: CCDC25 was predominantly underexpressed in ccRCC tumors and associated with advanced clinical stages and poor prognosis. Overexpression of CCDC25 in renal cancer cell lines resulted in reduced proliferation and migration. RNA sequencing revealed significant alterations in the Hippo pathway. Overexpression of CCDC25 inhibited the expression of downstream Hippo pathway proteins ITGA3 and CCND1 and promoted YAP phosphorylation. Mechanistic studies showed that CCDC25 interacts with YAP and influences YAP phosphorylation through LATS1. In vivo, CCDC25 overexpression inhibited tumor growth and promoted apoptosis. CONCLUSION: CCDC25 acts as a potential tumor suppressor in ccRCC by inhibiting cell proliferation and migration, potentially through regulating the Hippo signaling pathway. These findings highlight the potential of CCDC25 as a therapeutic target in ccRCC treatment.

The oncolytic bacteria-mediated delivery system of CCDC25 nucleic acid drug inhibits neutrophil extracellular traps induced tumor metastasis.

BACKGROUND: Neutrophil extracellular traps (NETs), antibacterial weapons of neutrophils (NEs), have been found to play a crucial role in cancer metastasis in recent years. More and more cancer research is focusing on anti-NETs. However, almost all anti-NETs treatments have limitations such as large side effects and limited efficacy. Therefore, exploring new anti-NETs therapeutic strategies is a long-term goal. RESULTS: The transmembrane protein coiled-coil domain containing 25 (CCDC25) on tumor cell membranes can bind NETs-DNA with high specificity and affinity, enabling tumor cells to sense NETs and thus promote distant metastasis. We transformed shCCDC25 into VNP20009 (VNP), an oncolytic bacterium, to generate VNP-shCCDC25 and performed preclinical evaluation of the inhibitory effect of shCCDC25 on cancer metastasis in B16F10 lung metastasis and 4T1 orthotopic lung metastasis models. VNP-shCCDC25 effectively blocked the downstream prometastatic signaling pathway of CCDC25 at tumor sites and reduced the formation of NETs while recruiting more neutrophils and macrophages to the tumor core, ultimately leading to excellent metastasis inhibition in the two lung metastasis models. CONCLUSION: This study is a pioneer in focusing on the effect of anti-NET treatment on CCDC25. shCCDC25 is effectively delivered to tumor sites via the help of oncolytic bacteria and has broad application in the inhibition of cancer metastasis via anti-NETs.

Serum CCDC25 Levels as a Potential Marker for Metabolic Syndrome.

BACKGROUND/AIM: Metabolic syndrome (MetS) stands as a significant risk for developing various severe health problems. Therefore, the discovery of biomarkers capable of predicting the progression of metabolic conditions is crucial for improving overall health outcomes. Recently, we reported that coiled-coil domain containing 25 (CCDC25) might be associated with key proteins involved in metabolic pathways, by bioinformatics analysis. Thus, we assumed that serum CCDC25 levels might have an association with MetS status. PATIENTS AND METHODS: In this study, based on the modified National Cholesterol Education Program-Adult Treatment Panel III (modified NCEP-ATP III) criteria, the participants who had three or more of abnormal criteria were defined as MetS, and those who had 1 or 2 abnormal criteria as pre-MetS groups; those who had no abnormal criteria were classified as the healthy control (HC) group. Serum CCDC25 levels were measured using the dot blot assay. RESULTS: The results showed that serum CCDC25 levels of the MetS group (0.072±0.026 ng/µl) were significantly higher (p<0.001) than that of pre-MetS (0.031±0.011 ng/µl) or HC groups (0.018±0.007 ng/µl). We can discern a consistent trend indicating that serum CCDC25 level is well correlated with the number of abnormal criteria of MetS of each participant. Although serum CCDC25 levels correlated with the distribution of all 5 MetS criteria, the highest correlation was seen in serum CCDC25 levels and triglyceride (TG) levels, with r=0.563, followed by systolic blood pressure (SBP) levels (r=0.557) and high-density lipoprotein-cholesterol (HDL-C) levels (r=-0.545). CONCLUSION: CCDC25 showed correlations with all MetS parameters, particularly with TG, SBP, and HDL-C. This prompts speculation that heightened CCDC25 levels may indicate the development and/or progression of those MetS-associated diseases.

Coiled-coil domain containing 25 (CCDC25) regulates cell proliferation, migration, and invasion in clear cell renal cell carcinoma by targeting the ILK-NF-κB signaling pathway.

Increasing evidence has demonstrated that the expression of coil domains containing 25 (CCDC25) in various malignancies is abnormally high. However, the potential regulatory role and mechanism of CCDC25 in the development of clear cell renal cell carcinoma (ccRCC) are still unclear. In this experiment, we combined in vitro experiments such as wound healing, CCK8, and transwell assay with in vivo experiments on tumor formation in nude mice to evaluate the effect of CCDC25 on the proliferation, migration, and invasion of renal cancer cells. In addition, we also used Western blotting and qPCR to evaluate the role of CCDC25 in activating the integrin-linked kinase (ILK)-NF-κB signaling pathway. Here, we demonstrate that compared to normal tissues and cell lines, CCDC25 is overexpressed in both human ccRCC tissues and cell lines. After CCDC25 knockdown, it has obvious inhibitory effect on the proliferation, migration, and invasion of cancer cells in vitro and in vivo. In contrast, CCDC25 overexpression promotes these effects. Additionally, we also discovered that CCDC25 interacts with ILK and coordinates the activation of the NF-κB signaling pathway downstream. Generally, our study suggests that CCDC25 plays a vital role in the development of ccRCC, which also means that it may be a potential therapeutic target for ccRCC.

Cannabidiol alleviates perfluorooctane sulfonate-induced macrophage extracellular trap mediate inflammation and fibrosis in mice liver.

As a new type of persistent organic pollutant, perfluorooctane sulphonate (PFOS) has received extensive attention worldwide. Cannabidiol (CBD) is a non-psychoactive natural cannabinoid extract that has been proved to have antioxidation, regulation of inflammation and other functions. However, the effects of PFOS on liver injury and whether CBD can alleviate PFOS-induced liver injury are still unclear. Therefore, in this study, we used CBD (10 mg/kg) and/or PFOS (5 mg/kg) to intraperitoneally inject mice for 30 days. We found that PFOS exposure led to inflammatory infiltration in the liver of mice, increased the formation of macrophage extracellular trap (MET), and promoted fibrosis. In vitro, we established a coculture system of RAW264.7, AML12 and LX-2 cells, and treated them with CBD (10 µM) and/or PFOS (200 µM). The results showed that PFOS could also induce the expression of MET, inflammation and fibrosis marker genes in vitro. Coiled-coil domain containing protein 25 (CCD25), as a MET-DNA sensor, was used to investigate its ability to regulate inflammation and fibrosis, we knocked down CCDC25 and its downstream proteins (integrin-linked kinase, ILK) by siRNA technology, and used QNZ to inhibit NF-κB pathway. The results showed that the knockdown of CCDC25 and ILK and the inhibition of NF-κB pathway could inhibit MET-induced inflammation and fibrosis marker gene expression. In summary, we found that PFOS-induced MET can promote inflammation and fibrosis through the CCDC25-ILK-NF-κB signaling axis, while the treatment of CBD showed a protective effect, and it is proved by Macromolecular docking that this protective effect is achieved by combining CBD with peptidylarginine deiminase 4 (PAD4) to alleviate the release of MET. Therefore, regulating the formation of MET and the CCDC25-ILK-NF-κB signaling axis is an innovative treatment option that can effectively reduce hepatotoxicity. Our study reveals the mechanism of PFOS-induced hepatotoxicity and provides promising insights into the protective role of CBD in this process.

Neutrophil extracellular traps promote angiogenesis in gastric cancer.

Although antiangiogenic therapy has been used in gastric cancer, disease progression due to drug resistance remains common. Neutrophils play an important role in the occurrence and progression of cancer via neutrophil extracellular traps (NETs). However, few studies have investigated angiogenic regulation in gastric cancer. We aimed to determine the role of NETs in promoting angiogenesis in gastric cancer. Multiple immunohistochemical staining was used to analyze the spatial distribution of NETs and microvessels in patient tissue samples. A mouse subcutaneous tumor model was established to determine the effect of NETs on tumor growth, and changes in microvessel density were observed via immunohistochemical staining. We screened differentially expressed proteins in HUVECs stimulated by NETs via proteomics. Cell Counting Kit-8, EdU labeling, and tubule formation assays were used to verify the effect of NETs on HUVEC proliferation, migration, and tubule formation. Blocking NETs, which was related to decreased microvessel density, significantly inhibited tumor growth in the murine subcutaneous tumor model. Compared with those of the control group, tumor volume and mass among mice in the inhibition group decreased by 61.3% and 77.9%, respectively. The NET-DNA receptor CCDC25 was expressed in HUVECs, providing a platform for NETs to promote HUVEC proliferation, migration, and tubulation. In an in vitro rat aortic explant model, NETs induced HUVEC proliferation, survival, and chemotaxis, which were not significantly different from those observed in the VEGF stimulation group. Our results confirm that NETs promote angiogenesis in gastric cancer, providing a theoretical basis for identifying new anti-vascular therapeutic targets. Video Abstract.

Ccdc25 regulates neurogenesis during the brain development.

During brain development, the proliferation and differentiation of neural stem cells (NSCs) are precisely regulated. Defects in embryonic brain development can lead to serious developmental disorders. The cerebral cortex is the most evolved and complicated structure in the mammalian brain. The process of neuronal production, also known as neurogenesis, plays crucial roles in cerebral development and can affect the function of the neocortex. Ccdc25 is a newly discovered molecule. It has been proved that it can play an important role in tumor. However, its function in neural systems is unclear. In this study, we find that in early embryonic development, Ccdc25 can express in the brain. Suppression of the Ccdc25 mediated by shRNAs causes the increase of the Ki67- or BrdU-positive NSCs proliferation and inhibits the premature terminal mitosis and neuronal differentiation. Simultaneously, overexpression of Ccdc2525 inhibits the proliferation and promotes the differentiation of NSCs. Knockdown of Ccdc25 also affects neuronal maturation, the number of branches of neurons cultured in vitro decreased, and the number of axons became shorter. We also examined the expression profile of NSCs when Ccdc25 was knocked down by RNA sequencing technique. We found that Ccdc25 regulates the development of NSCs through Egr1. Egr1 knockdown can result in a phenotype similar to Ccdc25, while the overexpression of Egr1 can also rescue the phenotype of Ccdc25 knockdown. In conclusion, Ccdc25 can affect the proliferation and differentiation of NSCs and the maturation of neuron.

High Level of Serum Coiled-coil Domain Containing 25 (CCDC25) as a Diagnostic Marker for Cholangiocarcinoma But Not for Other Cancers.

BACKGROUND/AIM: Recently, we reported that coiled-coil domain containing 25 (CCDC25) protein is elevated in the sera of patients with cholangiocarcinoma (CCA) and is suggested to be a diagnostic biomarker for CCA. This study aimed to examine whether serum CCDC25 level can be a unique biomarker for CCA. Bioinformatic analyses using Human Protein Atlas (HPA) database and Gene Expression Profiling Interactive Analysis 2 (GEPIA2) indicated that CCDC25 protein and mRNA are expressed not only in CCA but also in other cancers, such as colorectal cancer (CRC), breast cancer (BC), and hepatocellular carcinoma (HCC), all of which are the top 5 cancers highly prevalent in Thailand. MATERIALS AND METHODS: Using a quantitative dot blot assay, serum CCDC25 levels were measured for 30 healthy controls (HC), 34 CRC, 42 BC, 43 HCC, and 83 CCA. RESULTS: The serum CCDC25 levels of CCA patients (0.193±0.039 ng/µl) were significantly higher than those of CRC (0.019±0.006 ng/µl), BC (0.036±0.015 ng/µl), HCC (0.035±0.016 ng/µl), and higher than those of HC (0.012±0.003 ng/µl). The serum CCDC25 level can discriminate CCA from the HC, CRC, BC, and HCC with a sensitivity of 100, 99, 94, and 94%, respectively, and specificity of 100, 100, 98, and 95%, respectively. CONCLUSION: CCDC25 is a candidate diagnostic biomarker for CCA.

Nanoparticulate Cationic Poly(amino acid)s Block Cancer Metastases by Destructing Neutrophil Extracellular Traps.

Cancer metastasis that is resistant to conventional therapies has become a major cause of patient death. Recent reports indicate that the neutrophil extracellular trap (NET) is closely associated with cancer distant metastases, and the cell-free DNA of NETs has been identified as the ligand of the transmembrane protein CCDC25 of cancer cells, acting as a chemokine to induce cancer cell migration to distant organs. In this work, we present the poly(aspartic acid) based-cationic materials to interfere with the interaction between NET-DNA and CCDC25, and furthermore to inhibit NET-DNA-mediated cancer cell chemotaxis and migration. Because of a stronger binding affinity to DNA and favorable retention in the liver, nanoparticulate poly(aspartic acid) derivatives (cANP) efficiently reduce the level of hepatic NET-DNA infiltration, leading to a significant suppression of cancer metastases in mice and several human metastatic models. Moreover, the cANP exhibits no toxicity to organs of animals during the entire treatment. Thus, this work suggests a strategy for controlling cancer metastases, which will benefit patients in clinics.

Kallikrein-11, in Association with Coiled-Coil Domain Containing 25, as a Potential Prognostic Marker for Cholangiocarcinoma with Lymph Node Metastasis.

Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes. Currently, the treatment and prognosis for CCA are mostly poor. Recently, we have reported that coiled-coil domain containing 25 (CCDC25) protein level in the sera may be a diagnostic marker for CCA. Subsequently, we identified three binding proteins of CCDC25 and found that kallikrein-11 (KLK11) expression was highest among those binding proteins. In this study, we investigated CCDC25 and KLK11 expression in CCA and adjacent normal tissues (n = 18) using immunohistochemistry. The results demonstrated that the expressions of CCDC25 and KLK11 in CCA tissues were both significantly higher than the adjacent tissues (p < 0.001 and p = 0.001, respectively). Then, using GEPIA bioinformatics analysis, KLK11 mRNA was significantly overexpressed in CCA tumor tissues compared with normal tissues (p < 0.05). Moreover, CCDC25 expression was positively correlated with KLK11 expression in CCA with lymph node metastasis (p = 0.028, r = 0.593). An analysis for the interaction of KLK11 with CCDC25 and other proteins, using STRING version 11.0, revealed that CCDC25 and KLK11 correlated with metastasis-related proteins. In addition, Kaplan-Meier survival curve analysis revealed that a high expression of KLK11 was associated with the poor prognosis of CCA. In conclusion, KLK11 is, as a binding protein for CCDC25, possibly involved in the metastatic process of CCA. KLK11 may be used as a prognostic marker for CCA.

Local strong acids: A driving force for metastasis.

Carcinogenesis was postulated as the result of the local buildup of strong acids such as hydrogenchloride which may trigger metastasis. A previous study revealed that bicarbonate raised tumor pH and suppressed metastases. The phosphate groups in DNA on neutrophil extracellular traps possess hydrogen bonding capacity and can accept protons. The proteins commonly found in neutrophil extracellular traps such as CCDC25, myeloperoxidase (MPO), histone H3, peptidylarginine deiminase 4 (PAD4) possess basic amino acid content at about 20.2%, 12.8%, 24.3% and 13.0% respectively, which attracts anions such as chloride. The striking 20.2% basic amino acid content in CCDC25 is similar to that of typical oncoproteins. Local hydrogenchloride may be the dominant impetus for metastasis, accounting for the anticancer effects of virtually all weak organic acids, acetic acid and lactic acid in particular.

High expression of CCDC25 in cholangiocarcinoma tissue samples.

Cholangiocarcinoma (CCA) is a malignant transformation of biliary epithelial cells. It is a slow growing tumor, but is also highly metastatic with a poor prognosis. Bile acids are known to transactivate the epidermal growth factor receptor (EGFR) in cholangiocytes and induce cyclooxygenase-2 expression. The protein expression profiles of bile acid-treated CCA cells were studied using a proteomic approach. To elucidate the possible mechanisms involved in the bile acid-mediated enhancement of CCA cell migration, the effects of six bile acids, including cholic, deoxycholic, taurocholic, taurodeoxycholic, glycocholic and glycodeoxycholic acid, on the migration of CCA cells were examined in vitro using wound healing assays. Subsequently, the possible proteins involved in enhanced CCA cell migration were investigated using a proteomic approach. Changes to the protein expression profiles of CCA cells following bile acid treatment was examined using two-dimensional electrophoresis and mass spectrometry. The results demonstrated that cholic and deoxycholic acid significantly enhanced the migration of CCA cells, compared with the treated MMNK-1 control cells. CCA cells had 77 overexpressed protein spots following cholic acid treatment, and 50 protein spots following deoxycholic acid treatment, compared with the treated MMNK-1 control cells. Liquid chromatography tandem-mass spectrometry analysis revealed that coiled-coil domain containing 25 (CCDC25) was significantly overexpressed in cholic acid-treated CCA cells compared with in cholic acid-treated control cells. When the expression levels of CCDC25 were investigated using western blot analysis, CCDC25 was demonstrated to be highly expressed in CCA tissues, but not in the adjacent non-cancerous tissue samples. The identified proteins were further analyzed for protein-chemical interactions using STITCH version 3.1 software. CCDC25 protein was identified to be associated with Son of sevenless homolog 1 and growth factor receptor-bound protein 2, which are involved in EGFR signaling. The results of the present study demonstrated that following cholic acid treatment, CCDC25 is overexpressed in CCA cells, which is associated with significantly enhanced cell migration. This suggests that CCDC25 is a potential therapeutic target for the treatment of patients with CCA.