Tumor derived exosomal ENTPD2 impair CD8+ T cell function in colon cancer through ATP-adenosine metabolism reprogramming.

BACKGROUND: Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer. METHODS: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. RESULTS: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. CONCLUSION: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

Enhancement of de novo lipogenesis by the IDH1 and IDH2-dependent reverse TCA cycle maintains the growth and angiogenic capacity of bone marrow-derived endothelial progenitor cells under hypoxia.

BACKGROUND: Bone marrow-derived endothelial progenitor cells (EPCs) play a dynamic role in maintaining the structure and function of blood vessels. But how these cells maintain their growth and angiogenic capacity under bone marrow hypoxic niche is still unclear. This study aims to explore the mechanisms from a perspective of cellular metabolism. METHODS: XFe96 Extracellular Flux Analyzer was used to analyze the metabolic status of EPCs. Gas Chromatography-Mass Spectrometry (GC-MS) was used to trace the carbon movement of 13C-labeled glucose and glutamine under 1 % O2 (hypoxia) and ∼20 % O2 (normoxia). Moreover, RNA interference, targeting isocitrate dehydrogenase-1 (IDH1) and IDH2, was used to inhibit the reverse tricarboxylic acid (TCA) cycle and analyze metabolic changes via isotope tracing as well as changes in cell growth and angiogenic potential under hypoxia. The therapeutic potential of EPCs under hypoxia was investigated in the ischemic hindlimb model. RESULTS: Compared with normoxic cells, hypoxic cells showed increased glycolysis and decreased mitochondrial respiration. Isotope metabolic tracing revealed that under hypoxia, the forward TCA cycle was decreased and the reverse TCA cycle was enhanced, mediating the conversion of α-ketoglutarate (α-KG) into isocitrate/citrate, and de novo lipid synthesis was promoted. Downregulation of IDH1 or IDH2 under hypoxia suppressed the reverse TCA cycle, attenuated de novo lipid synthesis (DNL), elevated α-KG levels, and decreased the expression of hypoxia inducible factor-1α (HIF-1α) and vascular endothelial growth factor A (VEGFA), eventually inhibiting the growth and angiogenic capacity of EPCs. Importantly, the transplantation of hypoxia-cultured EPCs in a mouse model of limb ischemia promoted new blood vessel regeneration and blood supply recovery in the ischemic area better than the transplantation of normoxia-cultured EPCs. CONCLUSIONS: Under hypoxia, the IDH1- and IDH2-mediated reverse TCA cycle promotes glutamine-derived de novo lipogenesis and stabilizes the expression of α-KG and HIF-1α, thereby enhancing the growth and angiogenic capacity of EPCs.

Targeting RPA promotes autophagic flux and the antitumor response to radiation in nasopharyngeal carcinoma.

BACKGROUND: Autophagy is involved in nasopharyngeal carcinoma (NPC) radioresistance. Replication protein A 1 (RPA1) and RPA3, substrates of the RPA complex, are potential therapeutic targets for reversing NPC radioresistance. Nevertheless, the role of RPA in autophagy is not adequately understood. This investigation was performed to reveal the cytotoxic mechanism of a pharmacologic RPA inhibitor (RPAi) in NPC cells and the underlying mechanism by which RPAi-mediated autophagy regulates NPC radiosensitivity. METHODS AND RESULTS: We characterized a potent RPAi (HAMNO) that was substantially correlated with radiosensitivity enhancement and proliferative inhibition of in vivo and in NPC cell lines in vitro. We show that the RPAi induced autophagy at multiple levels by inducing autophagic flux, AMPK/mTOR pathway activation, and autophagy-related gene transcription by decreasing glycolytic function. We hypothesized that RPA inhibition impaired glycolysis and increased NPC dependence on autophagy. We further demonstrated that combining autophagy inhibition with chloroquine (CQ) treatment or genetic inhibition of the autophagy regulator ATG5 and RPAi treatment was more effective than either approach alone in enhancing the antitumor response of NPC to radiation. CONCLUSIONS: Our study suggests that HAMNO is a potent RPAi that enhances radiosensitivity and induces autophagy in NPC cell lines by decreasing glycolytic function and activating autophagy-related genes. We suggest a novel treatment strategy in which pharmacological inhibitors that simultaneously disrupt RPA and autophagic processes improve NPC responsiveness to radiation.

Knockdown LIMP2 inhibits colorectal cancer cells migration, invasion, and metastasis.

Colorectal cancer (CRC) is a common malignancy worldwide nowadays and liver metastasis is the primary cause of death in patients with CRC. Although lysosomal integral membrane protein 2 (LIMP2) has been reported to play important roles in gastric cancer and prostate cancer, its role in CRC remains unclear. The aim of this study was to investigate the function of LIMP2 in CRC invasion and migration, along with the potential underlying molecular mechanisms. We found that LIMP2 levels were higher in CRC tissues compared to adjacent normal tissues. Kaplan-Meier survival analysis showed that high expression of LIMP2 was associated with worse prognosis in CRC patients. Knockdown of LIMP2 significantly inhibited invasion, migration, and wound healing abilities of CRC cells in vitro, and inhibited CRC liver metastasis in vivo. Additionally, LIMP2 knockdown inhibited autophagy in CRC. Therefore, LIMP2 plays an important role in CRC progression. High expression of LIMP2 was associated with worse prognosis in CRC patients. Knockdown LIMP2 can effectively inhibit CRC cell migration and invasion in vitro and prevent liver metastasis in vivo. These findings suggest that LIMP2 may serve as an independent prognostic factor and potential therapeutic target for CRC.

Promotion of NAD+ recycling by the hypoxia-induced shift in the lactate dehydrogenase isozyme profile reduces the senescence of human bone marrow-derived endothelial progenitor cells.

Expansion of bone marrow-derived endothelial progenitor cells (EPCs) in vitro to obtain required cell numbers for therapeutic applications faces the challenge of growing cell senescence under the traditional normoxic culture condition. We previously found that 1% O2 hypoxic culture condition is favorable for reducing senescence of EPCs, but the mechanisms underlying the favorability are still unclear. Here, we found that, compared with normoxia, hypoxia induced a shift in lactate dehydrogenase (LDH) isozyme profile, which manifested as decreased LDH2 and LDH1 and increased LDH5, LDH4 and total LDHs. Moreover, under hypoxia, EPCs presented higher LDH activity, which could promote the conversion of pyruvate to lactate, as well as a higher level of NAD+, Bcl2 interacting protein 3 (BNIP3) expression and mitophagy. Additionally, under hypoxia, knock-down of the LDHA subunit increased the LDH2 and LDH1 levels and knock-down of the LDHB subunit increased the LDH5 level, while the simultaneous knock-down of LDHA and LDHB reduced total LDHs and NAD+ level. Inhibition of NAD+ recycling reduced BNIP3 expression and mitophagy and promoted cell senescence. Taken together, these data demonstrated that 1% O2 hypoxia induces a shift in the LDH isozyme profile, promotes NAD+ recycling, increases BNIP3 expression and mitophagy, and reduces EPC senescence. Our findings contribute to a better understanding of the connection between hypoxic culture conditions and the senescence of bone marrow-derived EPCs and provide a novel strategy to improve in vitro expansion of EPCs.

Hypoxia-induced reprogramming of glucose-dependent metabolic pathways maintains the stemness of human bone marrow-derived endothelial progenitor cells.

The benefits of hypoxia for maintaining the stemness of cultured human bone marrow-derived endothelial progenitor cells (BM EPCs) have previously been demonstrated but the mechanisms responsible remain unclear. Growing evidences suggest that cellular metabolism plays an important role in regulating stem cell fate and self-renewal. Here we aimed to detect the changes of glucose metabolism and to explore its role on maintaining the stemness of BM EPCs under hypoxia. We identified the metabolic status of BM EPCs by using extracellular flux analysis, LC-MS/MS, and 13C tracing HPLC-QE-MS, and found that hypoxia induced glucose metabolic reprogramming, which manifested as increased glycolysis and pentose phosphate pathway (PPP), decreased tricarboxylic acid (TCA) and mitochondrial respiration. We further pharmacologically altered the metabolic status of cells by employing various of inhibitors of key enzymes of glycolysis, PPP, TCA cycle and mitochondria electron transport chain (ETC). We found that inhibiting glycolysis or PPP impaired cell proliferation either under normoxia or hypoxia. On the contrary, inhibiting pyruvate oxidation, TCA or ETC promoted cell proliferation under normoxia mimicking hypoxic conditions. Moreover, promoting pyruvate oxidation reverses the maintenance effect of hypoxia on cell stemness. Taken together, our data suggest that hypoxia induced glucose metabolic reprogramming maintains the stemness of BM EPCs, and artificial manipulation of cell metabolism can be an effective way for regulating the stemness of BM EPCs, thereby improving the efficiency of cell expansion in vitro.

Altered B cell immunoglobulin signature exhibits potential diagnostic values in human colorectal cancer.

Antibody-secreting B cells have long been considered the central element of gut homeostasis; however, tumor-associated B cells in human colorectal cancer (CRC) have not been well characterized. Here, we show that the clonotype, phenotype, and immunoglobulin subclasses of tumor-infiltrating B cells have changed compared to adjacent normal tissue B cells. Remarkably, the tumor-associated B cell immunoglobulin signature alteration can also be detected in the plasma of patients with CRC, suggesting that a distinct B cell response was also evoked in CRC. We compared the altered plasma immunoglobulin signature with the existing method of CRC diagnosis. Our diagnostic model exhibits improved sensitivity compared to the traditional biomarkers, CEA and CA19-9. These findings disclose the altered B cell immunoglobulin signature in human CRC and highlight the potential of using the plasma immunoglobulin signature as a non-invasive method for the assessment of CRC.

EWI2 and its relatives in Tetraspanin-enriched membrane domains regulate malignancy.

Experimental studies on immunoglobulin superfamily (IgSF) member EWI2 reveal that it suppresses a variety of solid malignant tumors including brain, lung, skin, and prostate cancers in animal models and inhibits tumor cell movement and growth in vitro. While EWI2 appears to support myeloid leukemia in mouse models and maintain leukemia stem cells. Bioinformatics analyses suggest that EWI2 gene expression is downregulated in glioblastoma but upregulated in melanoma, pancreatic cancer, and liver cancer. The mechanism of action for EWI2 is linked to its inhibition of growth factor receptors and cell adhesion proteins through its associated tetraspanin-enriched membrane domains (TEMDs), by altering the cell surface clustering and endolysosome trafficking/turnover of these transmembrane proteins. Recent studies also show that EWI2 modulates the nuclear translocation of ERK and TFEB to change the activities of these gene expression regulators. For EWI2 relatives including FPRP, IgSF3, and CD101, although their roles in malignant diseases are not fully clear and remain to be determined experimentally, FPRP and IgSF3 likely promote the progression of solid malignant tumors while CD101 seems to modulate immune cells of tumor microenvironment. Distinctive from other tumor regulators, the impacts of EWI subfamily members on solid malignant tumors are likely to be context dependent. In other words, the effect of a given EWI subfamily member on a tumor probably depends on the molecular network and composition of TEMDs in that tumor. Collectively, EWI2 and its relatives are emerged as important regulators of malignant diseases with promising potentials to become anti-cancer therapeutics and cancer therapy targets.

METTL3 promotes colorectal cancer metastasis by promoting the maturation of pri-microRNA-196b.

PURPOSE: Methyltransferase-like 3 (METTL3), a key member of the m6A methyltransferase complex, is upregulated in multiple human malignancies and plays a role in regulating tumor migration. This study aimed to reveal the underlying mechanism by which METTL3 in regulates the metastasis of colorectal cancer (CRC). METHODS: We compared METTL3 expression levels in CRC tumor tissues and adjacent nontumor tissues by immunohistochemistry (IHC). The functional roles of METTL3 in CRC were assessed by real-time cell migration assays, wound-healing assays and Transwell assays. miRNA sequencing (miRNA-seq), RNA-binding protein immunoprecipitation (RIP) assays and N6-methyladenosine immunoprecipitation (MeRIP) assays were performed to confirm the molecular mechanism underlying the involvement of METTL3 in CRC cell metastasis. RESULTS: We found that METTL3 was overexpressed in CRC tissues. METTL3 knockdown significantly inhibited CRC cell migration and invasion, while METTL3 overexpression had the opposite effects. Furthermore, we demonstrated that METTL3 regulates miR-196b expression via an N6-methyladenosine (m6A)-pri-miR-196b-dependent mechanism and thereby promotes CRC metastasis. CONCLUSION: This study shows the important role of METTL3 in CRC metastasis and provides novel insight into m6A modification in CRC metastasis.

Wild-Type Isocitrate Dehydrogenase-Dependent Oxidative Decarboxylation and Reductive Carboxylation in Cancer and Their Clinical Significance.

The human isocitrate dehydrogenase (IDH) gene encodes for the isoenzymes IDH1, 2, and 3, which catalyze the conversion of isocitrate and α-ketoglutarate (α-KG) and are required for normal mammalian metabolism. Isocitrate dehydrogenase 1 and 2 catalyze the reversible conversion of isocitrate to α-KG. Isocitrate dehydrogenase 3 is the key enzyme that mediates the production of α-KG from isocitrate in the tricarboxylic acid (TCA) cycle. In the TCA cycle, the decarboxylation reaction catalyzed by isocitrate dehydrogenase mediates the conversion of isocitrate to α-KG accompanied by dehydrogenation, a process commonly known as oxidative decarboxylation. The formation of 6-C isocitrate from α-KG and CO2 catalyzed by IDH is termed reductive carboxylation. This IDH-mediated reversible reaction is of great importance in tumor cells. We outline the role of the various isocitrate dehydrogenase isoforms in cancer, discuss the metabolic implications of interference with IDH, summarize therapeutic interventions targeting changes in IDH expression, and highlight areas for future research.

ICAT promotes colorectal cancer metastasis via binding to JUP and activating the NF-κB signaling pathway.

BACKGROUND: The inhibitor of ß-catenin and T-cell factor (ICAT) is a direct negative regulator of the canonical Wnt signaling pathway, which is an attractive therapeutic target for colorectal cancer (CRC). Accumulating evidence suggests that ICAT interacts with other proteins to exert additional functions, which are not yet fully elucidated. METHODS: The overexpression of ICAT of CRC cells was conducted by lentivirus infection and plasmids transfection and verified by quantitative real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) and Western blotting. The effect of ICAT on the mobility of CRC cells was assessed by wound healing assay and transwell assay in vitro and lung metastasis in vivo. New candidate ICAT-interacting proteins were explored and verified using the STRING database, silver staining, co-immunoprecipitation mass spectrometry analysis (Co-IP/MS), and immunofluorescence (IF) staining analysis. RESULT: Inhibitor of ß-catenin and T-cell factor overexpression promoted in vitro cell migration and invasion and tumor metastasis in vivo. Co-IP/MS analysis and STRING database analyses revealed that junction plakoglobin (JUP), a homolog of ß-catenin, was involved in a novel protein interaction with ICAT. Furthermore, JUP downregulation impaired ICAT-induced migration and invasion of CRC cells. In addition, ICAT overexpression activated the NF-κB signaling pathway, which led to enhanced CRC cell migration and invasion. CONCLUSION: Inhibitor of ß-catenin and T-cell factor promoted CRC cell migration and invasion by interacting with JUP and the NF-κB signaling pathway. Thus, ICAT could be considered a protein diagnostic biomarker for predicting the metastatic ability of CRC.

EWI2 prevents EGFR from clustering and endocytosis to reduce tumor cell movement and proliferation.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling. These changes in signaling make cancer cells (i) undergo partial epithelial-to-mesenchymal (EMT) for more tumor progression and (ii) proliferate faster for better tumor formation. Inhibition of EGFR or Erk kinase can abrogate the cancer cell phenotypes resulting from EWI2 removal. Thus, to inhibit cancer cells, EWI2 prevents EGFR from clustering and endocytosis to restrain its activation and signaling.

EWI2 promotes endolysosome-mediated turnover of growth factor receptors and integrins to suppress lung cancer.

As a partner of tetraspanins, EWI2 suppresses glioblastoma, melanoma, and prostate cancer; but its role in lung cancer has not been investigated. Bioinformatics analysis reveals that EWI2 gene expression is up regulated in lung adenocarcinoma and higher expression of EWI2 mRNA may predict poorer overall survival. However, experimental analysis shows that EWI2 protein is actually downregulated constantly in the tissues of lung adenocarcinoma and lung squamous cell carcinoma. Forced expression of EWI2 in human lung adenocarcinoma cells reduces total cellular and cell surface levels of various integrins and growth factor receptors, which initiates the outside-in motogenic and mitogenic signaling. These reductions result in the decreases in 1) cell-matrix adhesion, cell movement, and cell transformation in vitro and 2) tumor growth, burden, and metastasis in vivo, and result from the increases in lysosomal trafficking and proteolytic degradation of theses membrane receptors. EWI2 elevates lysosome formation by promoting nuclear retention of TFEB, the master transcription factor driving lysosomogenesis. In conclusion, EWI2 as a lung cancer suppressor attenuates lung cancer cells in a comprehensive fashion by inhibiting both tumor growth and tumor metastasis; EWI2 as an endolysosome regulator promotes lysosome activity to enhance lysosomal degradation of growth factor receptors and integrins and then reduce their levels and functions; and EWI2 can become a promising therapeutic candidate given its accessibility at the cell surface, dual inhibition on growth factor receptors and integrins, and broad-spectrum anti-cancer activity. More importantly, our observations also provide a novel therapeutic strategy to bypass the resistance to EGFR inhibitors.

ROS/JNK/C-Jun Pathway is Involved in Chaetocin Induced Colorectal Cancer Cells Apoptosis and Macrophage Phagocytosis Enhancement.

There is an urgent need for novel agents for colorectal cancer (CRC) due to the increasing number of cases and drug-resistance related to current treatments. In this study, we aim to uncover the potential of chaetocin, a natural product, as a chemotherapeutic for CRC treatment. We showed that, regardless of 5-FU-resistance, chaetocin induced proliferation inhibition by causing G2/M phase arrest and caspase-dependent apoptosis in CRC cells. Mechanically, our results indicated that chaetocin could induce reactive oxygen species (ROS) accumulation and activate c-Jun N-terminal kinase (JNK)/c-Jun pathway in CRC cells. This was confirmed by which the JNK inhibitor SP600125 partially rescued CRC cells from chaetocin induced apoptosis and the ROS scavenger N-acetyl-L-cysteine (NAC) reversed both the chaetocin induced apoptosis and the JNK/c-Jun pathway activation. Additionally, this study indicated that chaetocin could down-regulate the expression of CD47 at both mRNA and protein levels, and enhance macrophages phagocytosis of CRC cells. Chaetocin also inhibited tumor growth in CRC xenograft models. In all, our study reveals that chaetocin induces CRC cell apoptosis, irrelevant to 5-FU sensitivity, by causing ROS accumulation and activating JNK/c-Jun, and enhances macrophages phagocytosis, which suggests chaetocin as a candidate for CRC chemotherapy.

Overexpression of ICAT Inhibits the Progression of Colorectal Cancer by Binding with ß-Catenin in the Cytoplasm.

Inhibitor of ß-catenin and T-cell factor (ICAT) was first found as a polypeptide that blocks ß-catenin-TCF interaction. Abundant evidence has shown that ICAT has different functions in diverse cancers' progression. Nevertheless, the roles it plays in colorectal cancer (CRC) have not been described. Here, we documented that ICAT expression was higher in CRC tissue than in the adjacent normal tissue and that prognosis was better in high-ICAT expression patients. The overexpression of ICAT inhibited CRC cell proliferation both in vitro and in vivo. Wnt pathway transcriptional activity was suppressed in the CRC cells with ICAT overexpression, where the CCND1 and MYC expression, which occurs downstream of the Wnt signaling pathway, was inhibited. Co-immunoprecipitation experiments showed that ICAT bound with ß-catenin in stable overexpression cell lines; immunofluorescence showed the co-localization of ICAT and ß-catenin in the cytoplasm. Overall, our study reveals that ICAT inhibits CRC cell proliferation by binding to cytoplasm-located ß-catenin, and prevents its translocation, which results in Wnt signaling pathway inactivation. It may provide a scientific foundation for focusing on ICAT in treatments for CRC.

Hsa-miR-335 enhances cell migration and invasion in lung adenocarcinoma through targeting Copine-1.

Lung adenocarcinoma (LAC) is one of the most common pulmonary adenocarcinomas with a high peak of mortality, and metastasis is the main culprit of LAC deaths. microRNAs play important role in cancer metastasis, and thus are regarded as potential diagnostic and prognostic markers for human cancers. However, many miRNAs exhibit dual roles in diverse cellular contexts. Here, we revealed that hsa-miR-335, a previously reported tumor suppressor, exhibited an oncogenic role in LAC. Overexpression of miR-335 enhanced the abilities of A549 and H1299 cells to invade and migrate by regulating epithelial-mesenchymal transition, while inhibition of miR-335 exhibited an opposite effect in vitro and in vivo. Mechanically, miR-335 inhibited the expression of Copine-1 (CPNE1), an NF-κB suppressor, through interacting with its mRNA 3'UTR, while mutating the binding sites abolished this inhibitory effect. This finding not only highlights the suppressive effect of CPNE1 on cell motility, but also provides new insight into miR-335 in promoting LAC metastasis.

Improved diagnostic value by combining plasma PON1 level with tumor biomarkers in Colorectal Cancer patients.

The incidence of colorectal cancer (CRC) ranks third among all cancers in China and improvements in screening for CRC have an important impact on prevention and control of the disease. Paraoxonase 1 (PON1) is a calcium ion-dependent hydrolase that is widely distributed in tissue. Its diagnostic value in colorectal cancer has been reported, but the diagnostic value of combining PON1 with carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen 12-5 (CA12-5) in colorectal cancer has not been evaluated. Experiments were carried out in a total of 284 CRC patients and 90 healthy controls. The primary cohort was randomly divided into training and validation sets. The levels of PON1 in plasma of CRC patients were significantly lower than that in the healthy controls (P < 0.001). It showed excellent diagnostic value with the AUC reaching 0.750 for the training set and 0.742 for the validation set. Furthermore, combining PON1 with CEA, CA12-5, CA19-9 could better classify CRC patients (AUC rising from 0.821, 0.716, 0.712 to 0.875, 0.817 and 0.814, respectively, in the training set, from 0.818, 0.581, 0.593 to 0.854, 0.770, and 0.772 in the validation set). In conclusion, PON1 can serve as a diagnostic biomarker for CRC and raise the sensitivity and specificity when incorporated with traditional tumor biomarkers.

Circular RNA GLIS2 promotes colorectal cancer cell motility via activation of the NF-κB pathway.

Circular RNAs (circRNAs) are a newly discovered type of biological molecule that belongs to the noncoding RNA family. Abundant evidence has shown that circRNAs are involved in the progression of various cancers. However, the particular functions of circRNAs in colorectal cancer (CRC) remain elusive. In this study, we investigated the differentially expressed circRNAs in three pairs of cancer tissue and adjacent normal tissue of CRC. We revealed that circGLIS2 expression was higher in CRC tissue and cell lines. Gain-and-loss function assays showed that circGLIS2 was involved in the regulation of cell migration. Moreover, overexpressing circGLIS2 in CRC cells activated the NF-κB pathway and induced pro-inflammatory chemokine production, which evoked tumor-associated inflammation through recruiting leukocytes. In turn, when the cancer cells were exposed to the supernatant of circGLIS2 overexpressed cancer cells, they were endowed with the ability of migration and chemokines production. Furthermore, the rescue assay confirmed that circGLIS2 activated NF-κB signaling and promoted cell migration by sponging miR-671. Overall, our study reveals that circGLIS2, acting as a potential oncogene, maintains the abnormal activation state of the NF-κB signaling pathway via the miR-671 sponge mechanism in CRC cells. This study provides a scientific basis for targeting circGLIS2 in colorectal cancer interventions.

Toosendanin-induced apoptosis in colorectal cancer cells is associated with the κ-opioid receptor/ß-catenin signaling axis.

Developing new drugs for killing colorectal cancer (CRC) cells is urgently needed. Here, we explored the antitumor effects of toosendanin (TSN) in CRC, as well as explored its antitumor mechanisms and direct targets. Cell proliferation and apoptosis were analyzed by CCK8, colony formation, real-time cell impedance and flow cytometry. The signaling pathway and Wnt activity were analyzed by Wnt luciferase activity assay, quantitative real-time PCR and western blot. The interaction between TSN and the κ-opioid receptor was analyzed by a molecular docking simulation. BALB/c nude mice were used to detect the effects of TSN on tumor growth in vivo. We found that TSN inhibited proliferation, induced G1 phase arrest and caused caspase-dependent apoptosis in both 5-FU-sensitive and 5-FU-resistant CRC cells. Moreover, TSN effectively inhibited CRC growth in vivo. In terms of the mechanism, TSN inhibited Wnt/ß-catenin signaling in CRC cells, and the molecular docking results showed that TSN could bind to κ-opioid receptors directly. Additionally, TSN-induced apoptosis and ß-catenin decline were both reversed by the selective κ-opioid receptor agonist U50,488H. Our data demonstrate that TSN-induced apoptosis in CRC cells is associated with the κ-opioid receptor/ß-catenin signaling axis, and TSN has promising potential as an antitumor agent for CRC treatment.

MiR-27b-3p promotes migration and invasion in colorectal cancer cells by targeting HOXA10.

PURPOSE: Dysregulation of microRNAs (miRNAs) contributes to tumor progression via the regulation of the expression of specific oncogenes and tumor suppressor genes. One such example, miR-27b-3p, has reportedly been involved in tumor progression in many types of cancer. The aim of the present study was to delve into the role and the underlying mechanism of miR-27b-3p in colorectal cancer (CRC) cells. METHODS: In the present study, we detected the expression level of miR-27b-3p by RT-PCR. The effect of miR-27b-3p overexpression on cell proliferation in CRC cells was evaluated by cell counting and Edu assays. Transwell migration and invasion assays were used to examine the effects of cell migration and invasion. Bioinformatics, luciferase reporter assay and western blot assay were performed to identify the target of miR-27b-3p. RESULTS: Here, we have demonstrated that although miR-27b-3p can affect cell morphology, it has no observable effect on the proliferation of CRC cells. However, it significantly promotes the migration and invasion of CRC cells. We discovered that HOXA10 was a newly identified target of miR-27b-3p in CRC cells, as confirmed by bioinformatics, western blots and dual luciferase reporter assay. Furthermore, the overexpression of miR-27b-3p or the suppression of HOXA10 can activate the integrin ß1 signaling pathway. In conclusion, our results reveal a new function of miR-27b-3p that demonstrates its ability to promote CRC cell migration and invasion by targeting the HOXA10/integrin ß1 cell signal axis. CONCLUSION: This may provide a mechanism to explain why miR-27b-3p promotes CRC cell migration and invasion.