Squalene epoxidase promotes the chemoresistance of colorectal cancer via (S)-2,3-epoxysqualene-activated NF-κB.

BACKGROUND: While de novo cholesterol biosynthesis plays a crucial role in chemotherapy resistance of colorectal cancer (CRC), the underlying molecular mechanism remains poorly understood. METHODS: We conducted cell proliferation assays on CRC cells with or without depletion of squalene epoxidase (SQLE), with or without 5-fluorouracil (5-FU) treatment. Additionally, a xenograft mouse model was utilized to explore the impact of SQLE on the chemosensitivity of CRC to 5-FU. RNA-sequencing analysis and immunoblotting analysis were performed to clarify the mechanism. We further explore the effect of SQLE depletion on the ubiquitin of NF-κB inhibitor alpha (IκBα) and (S)-2,3-epoxysqualene on the binding of IκBα to beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) by using immunoprecipitation assay. In addition, a cohort of 272 CRC patients were selected for our clinical analyses. RESULTS: Mechanistically, (S)-2,3-epoxysqualene promotes IκBα degradation and subsequent NF-κB activation by enhancing the interaction between BTRC and IκBα. Activated NF-κB upregulates the expression of baculoviral IAP repeat containing 3 (BIRC3), sustains tumor cell survival after 5-FU treatment and promotes 5-FU resistance of CRC in vivo. Notably, the treatment of terbinafine, an inhibitor of SQLE commonly used as antifungal drug in clinic, enhances the sensitivity of CRC to 5-FU in vivo. Additionally, the expression of SQLE is associated with the prognosis of human CRC patients with 5-FU-based chemotherapy. CONCLUSIONS: Thus, our finding not only demonstrates a new role of SQLE in chemoresistance of CRC, but also reveals a novel mechanism of (S)-2,3-epoxysqualene-dependent NF-κB activation, implicating the combined potential of terbinafine for 5-FU-based CRC treatment.

TCL1A acts as a tumour suppressor by modulating gastric cancer autophagy via miR-181a-5p-TCL1A-Akt/mTOR-c-MYC loop.

Gastric cancer is the third most commonly cause of tumour-related death worldwide and one of the most prevalent malignancies in China. TCL1A, TCL1 family Akt coactivator A, can active Akt/mTOR pathway and regulate the autophagy. However, the action of TCL1A in gastric cancer is not well understood. The present study is investigating the mechanism of action of TCL1A in gastric cancer. TCL1A was lowly expressed in gastric cancer tissues. Subsequent experiments demonstrated that miR-181a-5p can regulate c-MYC through the TCL1A-Akt/mTOR pathway and c-MYC can in turn affect the expression of miR-181a-5p, thus confirming the existence of the miR-181a-5p-TCL1A-Akt/mTOR-c-MYC loop. Flow cytometric apoptosis assay and mRFP-eGFP-LC3 autophagy assay demonstrated that both miR-181a-5p and TCL1A can affect autophagy and apoptosis of gastric cancer cells through the loop. In vivo experiments confirmed that TCL1A can affect the proliferation of gastric cancer. These results illustrate that TCL1A can exert tumour suppressive effects and affect gastric cancer autophagy and progression via the miR-181a-5p-TCL1A-Akt/mTOR-c-MYC loop, which could be a potential therapeutic target for gastric cancer.

5-Lipoxygenase promotes epithelial-mesenchymal transition through the ERK signaling pathway in gastric cancer.

BACKGROUND AND AIM: 5-Lipoxygenase has been reported to enhance cell proliferation, migration, and invasion. Epithelial-mesenchymal transition is considered an important process for tumor metastasis and invasion. METHODS: The 5-lipoxygenase expression levels and the prognoses in patients with gastric cancer were evaluated by immunohistochemistry and by the log-rank test on Kaplan-Meier curves. We established 5-lipoxygenase-overexpressed and 5-lipoxygenase-silenced gastric cancer cells and measured migration, invasion, and epithelial-mesenchymal transition makers to examine the role of 5-lipoxygenase in gastric cancer in vitro. In vivo, 5-lipoxygenase-overexpressed gastric cancer cells were administered into mice by subcutaneous injection, intraperitoneal injection or splenic intravenous injection to study the proliferation or metastasis of 5-lipoxygenase in mice. Using the extracellular signal-regulated kinase pathway inhibitor U0126 and activator tumor growth factor-ß, we investigated the mechanism of epithelial-mesenchymal transition induced by 5-lipoxygenase in gastric cancer cells. RESULTS: 5-Lipoxygenase was upregulated in gastric cancer tissues and was related to poor overall survival in gastric cancer patients. 5-Lipoxygenase promoted gastric cancer cell proliferation, migration, and invasion and induced the process of epithelial-mesenchymal transition in gastric cancer cells. In the nude mouse model, mice with gastric cancer tumors overexpressing 5-LOX had a faster tumor growth rate and more severe abdominal and liver metastases than the control group. Inhibition of extracellular signal-regulated kinase signaling by U0126 or activation by tumor growth factor-ß neutralized the effect of 5-LOX overexpression or silencing on epithelial-mesenchymal transition. CONCLUSION: 5-Lipoxygenase promotes epithelial-mesenchymal transition in gastric cancer by activating the extracellular signal-regulated kinase signaling pathway.

Down-regulation of Barx2 predicts poor survival in colorectal cancer.

Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, has an important role in controlling the expression of cell adhesion molecules and has been reported in an increasing array of tumor types except colorectal cancer (CRC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in CRC. First, we analyzed the expression of Barx2 in two independent public datasets from Oncomine. Subsequently, we evaluated Barx2 mRNA and protein expression by quantitative real-time PCR and western blotting, respectively. It was determined that Barx2 expression was lower in tumor tissues than in adjacent non-tumorous colorectal tissues of CRC patients, consistent with results from the public datasets. Subsequently, a tissue microarray containing 196 CRC specimens was evaluated for Barx2 expression by immunohistochemical staining. It was found that low expression of Barx2 significantly correlated with TNM stage, AJCC stage, differentiation, and relapse in patients with CRC. Patients with lower levels of Barx2 expression showed reduced disease-free survival and overall survival. Furthermore, a trend toward shorter overall survival in the patient group with Barx2-negative tumors independent of advanced AJCC stage and poor differentiation was determined by Kaplan-Meier survival analysis. Based on univariate and multivariate analyses, Barx2 expression was an independent prognostic factor for determining CRC prognosis. Taken together, low Barx2 expression was associated with the progression of CRC and could serve as a potential independent prognostic biomarker for patients with CRC.

Development of an anoikis-related gene signature and prognostic model for predicting the tumor microenvironment and response to immunotherapy in colorectal cancer.

The effect of anoikis-related genes (ARGs) on clinicopathological characteristics and tumor microenvironment remains unclear. We comprehensively analyzed anoikis-associated gene signatures of 1057 colorectal cancer (CRC) samples based on 18 ARGs. Anoikis-related molecular subtypes and gene features were identified through consensus clustering analysis. The biological functions and immune cell infiltration were assessed using the GSVA and ssGSEA algorithms. Prognostic risk score was constructed using multivariate Cox regression analysis. The immunological features of high-risk and low-risk groups were compared. Finally, DAPK2-overexpressing plasmid was transfected to measure its effect on tumor proliferation and metastasis in vitro and in vivo. We identified 18 prognostic ARGs. Three different subtypes of anoikis were identified and demonstrated to be linked to distinct biological processes and prognosis. Then, a risk score model was constructed and identified as an independent prognostic factor. Compared to the high-risk group, patients in the low-risk group exhibited longer survival, higher enrichment of checkpoint function, increased expression of CTLA4 and PD-L1, higher IPS scores, and a higher proportion of MSI-H. The results of RT-PCR indicated that the expression of DAPK2 mRNA was significantly downregulated in CRC tissues compared to normal tissues. Increased DAPK2 expression significantly suppressed cell proliferation, promoted apoptosis, and inhibited migration and invasion. The nude mice xenograft tumor model confirmed that high expression of DAPK2 inhibited tumor growth. Collectively, we discovered an innovative anoikis-related gene signature associated with prognosis and TME. Besides, our study indicated that DAPK2 can serve as a promising therapeutic target for inhibiting the growth and metastasis of CRC.

LncRNA GAS6-AS1 contributes to 5-fluorouracil resistance in colorectal cancer by facilitating the binding of PCBP1 with MCM3.

5-Fluorouracil (5-FU) resistance has always been a formidable obstacle in the adjuvant treatment of advanced colorectal cancer (CRC). In recent years, long non-coding RNAs have emerged as key regulators in various pathophysiological processes including 5-FU resistance. TRG is a postoperative pathological score of the chemotherapy effectiveness for CRC, of which TRG 0-1 is classified as chemotherapy sensitivity and TRG 3 as chemotherapy resistance. Here, RNA-seq combined with weighted gene correlation network analysis confirmed the close association of GAS6-AS1 with TRG. GAS6-AS1 expression was positively correlated with advanced clinicopathological features and poor prognosis in CRC. GAS6-AS1 increased the 50% inhibiting concentration of 5-FU, enhanced cell proliferation and accelerated G1/S transition, both with and without 5-FU, both in vitro and in vivo. Mechanistically, GAS6-AS1 enhanced the stability of MCM3 mRNA by recruiting PCBP1, consequently increasing MCM3 expression. Furthermore, PCBP1 and MCM3 counteracted the effects of GAS6-AS1 on 5-FU resistance. Notably, the PDX model indicated that combining chemotherapeutic drugs with GAS6-AS1 knockdown yielded superior outcomes in vivo. Together, our findings elucidate that GAS6-AS1 directly binds to PCBP1, enhancing MCM3 expression and thereby promoting 5-FU resistance. GAS6-AS1 may serve as a robust biomarker and potential therapeutic target for combination therapy in CRC.

Cuproptosis-related miRNAs signature and immune infiltration characteristics in colorectal cancer.

BACKGROUND: A novel form of cell death termed cuproptosis was proposed recently. miRNAs play important roles in colorectal cancer (CRC). However, their relationships have not been reported. METHODS: miRNAs that negatively regulate 16 cuproptosis regulators were predicted using Targetscan database. The univariate Cox, LASSO, and multivariate Cox regression analyses were performed to select cuproptosis-related miRNAs. GSEA and ssGSEA analysis was carried out for functional enrichment analysis. The immune cell proportion score (IPS) and the efficiencies of multiple chemotherapy drugs were compared between different risk groups. The CCK8, cell colony, edu, and flow cytometry assays were performed to validate the roles of miRNA. Luciferase reporter assay confirmed the regulatory mechanism of miRNA on cuproptosis. RESULTS: Six cuproptosis-related miRNAs (hsa-miR-653, hsa-miR-216a, hsa-miR-3684, hsa-miR-4437, hsa-miR-641, and hsa-miR-552) were screened out for model construction. The risk score could act as an independent prognostic indicator in CRC (p < 0.001, 95% HR = 1.243 (1.129-1.369)). The nomogram could efficiently predict the overall survival rate (AUC = 0.836). Then, the level of immunosuppressive pathways, immunosuppressive cells, stromal-activated genes, and stromal score was higher in the high-risk group. The IPS analysis showed a better response to immunotherapy in the low-risk group. Also, the risk score was closely correlated with efficiencies of multiple chemotherapy drugs. Furthermore, miR-653 was highly expressed in CRC tissues (p < 0.001), closely correlated with T stage (p < 0.001), metastasis (p < 0.001), and tumor stage (p < 0.001). High expression of miR-653 predicted a shorter overall survival (p = 0.0282) and disease-free survival (p = 0.0056). In addition, miR-653 promoted cell proliferation, inhibited apoptosis, and negatively regulated the expression of DLD through directly binding to the 3'-UTR of DLD mRNA. CONCLUSION: We constructed a cuproptosis-related miRNA signature for the prediction of CRC patient survival and immunotherapy sensitivity. miR-653 was highly expressed in CRC tissues, promoted cell proliferation, and inhibited apoptosis by negatively regulating the expression of DLD.

circCAPRIN1 interacts with STAT2 to promote tumor progression and lipid synthesis via upregulating ACC1 expression in colorectal cancer.

BACKGROUND: Circular RNAs (circRNAs) generated by back-splicing of precursor mRNAs (pre-mRNAs) are often aberrantly expressed in cancer cells. Accumulating evidence has revealed that circRNAs play a critical role in the progression of several cancers, including colorectal cancer (CRC). However, the current understandings of the emerging functions of circRNAs in CRC lipid metabolism and the underlying molecular mechanisms are still limited. Here, we aimed to explore the role of circCAPRIN1 in regulating CRC lipid metabolism and tumorigenesis. METHODS: circRNA microarray was performed with three pairs of tumor and non-tumor tissues from CRC patients. The expression of circRNAs were determined by quantitative PCR (qPCR) and in situ hybridization (ISH). The endogenous levels of circRNAs in CRC cells were manipulated by transfection with lentiviruses overexpressing or silencing circRNAs. The regulatory roles of circRNAs in the occurrence of CRC were investigated both in vitro and in vivo using gene expression array, RNA pull-down/mass spectrometry, RNA immunoprecipitation assay, luciferase reporter assay, chromatin immunoprecipitation analysis, and fluorescence in situ hybridization (FISH). RESULTS: Among circRNAs, circCAPRIN1 was most significantly upregulated in CRC tissue specimens. circCAPRIN1 expression was positively correlated with the clinical stage and unfavorable prognosis of CRC patients. Downregulation of circCAPRIN1 suppressed proliferation, migration, and epithelial-mesenchymal transition of CRC cells, while circCAPRIN1 overexpression had opposite effects. RNA sequencing and gene ontology analysis indicated that circCAPRIN1 upregulated the expressions of genes involved in CRC lipid metabolism. Moreover, circCAPRIN1 promoted lipid synthesis by enhancing Acetyl-CoA carboxylase 1 (ACC1) expression. Further mechanistic assays demonstrated that circCAPRIN1 directly bound signal transducer and activator of transcription 2 (STAT2) to activate ACC1 transcription, thus regulating lipid metabolism and facilitating CRC tumorigenesis. CONCLUSIONS: These findings revealed the oncogenic role and mechanism of circCAPRIN1 in CRC. circCAPRIN1 interacted with STAT2 to promote CRC tumor progression and lipid synthesis by enhancing the expression of ACC1. circCAPRIN1 may be considered as a novel potential diagnostic and therapeutic target for CRC patients.

Cuproptosis-related molecular patterns and gene (ATP7A) in hepatocellular carcinoma and their relationships with tumor immune microenvironment and clinical features.

BACKGROUND: Cuproptosis has been studied in various aspects as a new form of cell death. AIMS: We hope to explore the molecular patterns and genes related to cuproptosis in evaluating and predicting the prognosis of hepatocellular carcinoma (HCC), as well as the impact of tumor immune microenvironment. METHODS AND RESULTS: Sixteen cuproptosis related gene (CRGs) and cuproptosis related molecular and gene characteristics were comprehensively analyzed from 492 HCC samples. Cuproptosis related molecular patterns were generated by consensus clustering algorithm, including cuproptosis clusters, cuproptosis gene clusters (CGC) and cuproptosis score (CS). The characteristics of tumor microenvironment (TME) and tumor immune cells were described by the ssGSEA and ESTIMATE algorithms. Cuproptosis score was established to assess the clinical characteristics, prognostic and immunotherapy. The role and mechanism of CRG (ATP7A) in HCC, as well as its relationship with TME and immune checkpoints, have been further explored. The results of somatic mutation, copy number variations (CNV), and CRGs expression in HCC suggested the CRGs might participate in the HCC oncogenesis. The cuproptosis clusters were closely related to the clinical pathological characteristics, biological processes, and prognosis of HCC. The three CGC was revealed to be consistent with the three immune infiltration characterizations, including immune-high, immune-mid, and immune-low subtypes. Higher CS was characterized by decreased TMB, activated immunity, higher immune cell proportion score (IPS) and better overall survival (OS), which indicated higher CS was immune-high type and with better treatment effect and prognosis. The ATP7A had the highest hazard ratio (HR = 1.465, p < .001), was high expression in HCC tissues and with a shorter 5-year OS. Knocking down ATP7A could enhance intracellular copper concentration, cause a decrease in DLAT expression, and induce cuproptosis and inhibit cell proliferation and migration. ATP7A was also positively correlated with most cancer immune cells and immune checkpoints. CONCLUSION: Taken together, this research revealed the cuproptosis related molecular patterns and genes associated with the clinical pathological characteristics, TME phenotype and prognosis of HCC. The CS will further deepen our understanding of the TME characteristics of HCC, and the involvement of ATP7A in cuproptosis will provide new ideas for predicting HCC prognosis and immunotherapy.

Prediction of prognosis, immune infiltration, and personalized treatment of hepatocellular carcinoma by analysis of cuproptosis-related long noncoding RNAs and verification in vitro.

Background: The correlations between cuproptosis and long noncoding RNAs (lncRNAs) with the tumor microenvironment (TME), immunotherapy, and some other characteristics of hepatocellular carcinoma (HCC) remain unclear. Methods: Sixteen cuproptosis regulators and 356 cuproptosis-related lncRNAs (CRLnc) were identified from 374 HCC profiles in The Cancer Genome Atlas (TCGA) database. Six differentially expressed CRLnc were selected, and a prognostic risk model based on the CRLnc signature (CRLncSig) was constructed. The prognostic power of the model was verified. Moreover, a cuproptosis-related gene cluster (CRGC) was generated based on six lncRNAs and differentially expressed genes. The relationship between immune cell infiltration in the TME, immunotherapy, CRLncSig, and CRGC was demonstrated through various algorithms, Tumor Immune Dysfunction and Exclusion (TIDE), tumor mutational burden (TMB), etc. Potential drugs and sensitivity to those agents were evaluated for the risk model. LncRNA AL158166.1 was selected and verified in HCC tissues and cell lines, the impact of its knockdown and overexpression in HCC cells was examined, and the copper (Cu) concentration and the cuproptosis-related gene expression were detected. Results: A CRLncSig prognostic risk model with good predictive ability was constructed. The low-risk group had a longer overall survival (OS), lower tumor purity, more extensive immune cell infiltration, higher immune score, enrichment in immune-activated pathways, and more positive response to immunotherapy versus the high-risk group. CRGC-B exhibited the best OS and the lowest tumor stage; the immune cell infiltration analysis was similar to the low-risk group in CRLncSig. CRGC-B belonged to the "immune-high" group of the TME. The low-risk group had a higher TIDE score and susceptibility to antitumor drugs. The lncRNA AL158166.1 had the highest hazard ratio. The levels of AL158166.1 were higher in HCC tissues versus healthy tissues. Knockdown of AL158166.1 could lead to an increase in intracellular Cu concentration, induce DLAT low expression, and inhibit the proliferation and migration of HCC cells, whereas overexpression of AL158166.1 exerted the reverse effect. Conclusion: Overall, a new CRLncSig prognostic risk model and a cuproptosis-related molecular signature were constructed and evaluated. The model and signature were associated with the prognosis, immune infiltration, and immunotherapy of HCC. Inhibiting the lncRNA AL158166.1 may induce cuproptosis and showed potential for the inhibition of tumors. Evaluation of the CRLnc, CRLncSig, and CRGC may enhance our understanding of the TME, determine the effectiveness of immunotherapy, and act as a marker for the prognosis of HCC.

A novel cuproptosis-related molecular pattern and its tumor microenvironment characterization in colorectal cancer.

Cuproptosis, or copper-induced cell death, has been reported as a novel noncanonical form of cell death in recent times. However, the potential roles of cuproptosis in the alteration of tumor clinicopathological features and the formation of a tumor microenvironment (TME) remain unclear. In this study, we comprehensively analyzed the cuproptosis-related molecular patterns of 1,274 colorectal cancer samples based on 16 cuproptosis regulators. The consensus clustering algorithm was conducted to identify cuproptosis-related molecular patterns and gene signatures. The ssGSEA and ESTIMATE algorithms were used to evaluate the enrichment levels of the infiltrated immune cells and tumor immune scores, respectively. The cuproptosis score was established to assess the cuproptosis patterns of individuals with principal component analysis algorithms based on the expression of cuproptosis-related genes. Three distinct cuproptosis patterns were confirmed and demonstrated to be associated with distinguishable biological processes and clinical prognosis. Interestingly, the three cuproptosis patterns were revealed to be consistent with three immune infiltration characterizations: immune-desert, immune-inflamed, and immune-excluded. Enhanced survival, activation of immune cells, and high tumor purity were presented in patients with low cuproptosisScore, implicating the immune-inflamed phenotype. In addition, low scores were linked to high tumor mutation burden, MSI-H and high CTLA4 expression, showing a higher immune cell proportion score (IPS). Taken together, our study revealed a novel cuproptosis-related molecular pattern associated with the TME phenotype. The formation of cuproptosisScore will further strengthen our understanding of the TME feature and instruct a more personalized immunotherapy schedule in colorectal cancer.

Exploring immunotherapy in colorectal cancer.

Chemotherapy combined with or without targeted therapy is the fundamental treatment for metastatic colorectal cancer (mCRC). Due to the adverse effects of chemotherapeutic drugs and the biological characteristics of the tumor cells, it is difficult to make breakthroughs in traditional strategies. The immune checkpoint blockades (ICB) therapy has made significant progress in the treatment of advanced malignant tumors, and patients who benefit from this therapy may obtain a long-lasting response. Unfortunately, immunotherapy is only effective in a limited number of patients with microsatellite instability-high (MSI-H), and segment initial responders can subsequently develop acquired resistance. From September 4, 2014, the first anti-PD-1/PD-L1 drug Pembrolizumab was approved by the FDA for the second-line treatment of advanced malignant melanoma. Subsequently, it was approved for mCRC second-line treatment in 2017. Immunotherapy has rapidly developed in the past 7 years. The in-depth research of the ICB treatment indicated that the mechanism of colorectal cancer immune-resistance has become gradually clear, and new predictive biomarkers are constantly emerging. Clinical trials examining the effect of immune checkpoints are actively carried out, in order to produce long-lasting effects for mCRC patients. This review summarizes the treatment strategies for mCRC patients, discusses the mechanism and application of ICB in mCRC treatment, outlines the potential markers of the ICB efficacy, lists the key results of the clinical trials, and collects the recent basic research results, in order to provide a theoretical basis and practical direction for immunotherapy strategies.

Comprehensive analysis of cuproptosis-related lncRNAs to predict prognosis and immune infiltration characteristics in colorectal cancer.

Background: Cuproptosis is a novel form of cell death discovered in recent. A great quantity of researches has confirmed the close relationships and crucial roles between long non-coding RNAs (lncRNAs) with the progression of colorectal cancer (CRC). However, the relationship between cuproptosis and lncRNAs remains unclear in CRC. Methods: 1,111 co-expressed lncRNAs with 16 cuproptosis regulators were retrieved from CRC samples of The Cancer Genome Atlas (TCGA) database. Through univariate Cox and least absolute shrinkage and selection operator regression analysis, a prognosis model was constructed with 15 lncRNAs. The Kaplan-Meier, receiver operating characteristic curve, C-index and principal component analysis identified the prognostic power. Furthermore, a cuproptosis-related cluster was generated based on the 15 lncRNAs by unsupervised methods. The correlations between the cuproptosis-related signatures with immune cell infiltration and anti-tumor therapy were explored by multiple algorithms. Results: A risk score and nomogram with great prediction ability were constructed for CRC prognosis evaluation. The immune activate pathways, immune infiltration cells, immune functions, immune score and immune activation genes were remarkably enriched in the high risk group. The cuproptosis-related cluster was generated, of which the cluster 2 showed longer overall survival. The immune cell infiltration analysis indicated the similar results of cluster 2 with the high risk group, implying a significant marker for "hot tumor." The cluster 2 also presented high expression of immune checkpoint molecules, MSI-H status and higher susceptibility to multiple immunotherapy drugs. Conclusion: We appraised a novel cuproptosis-related prognosis model and molecular signature associated with prognosis, immune infiltration and immunotherapy. The identification of cuproptosis-related lncRNAs improved our understanding of immune infiltration and provided a significant marker for prognosis and immunotherapy in CRC.

Circular RNA circZNF566 promotes hepatocellular carcinoma progression by sponging miR-4738-3p and regulating TDO2 expression.

As a recently discovered noncoding RNA, circular RNAs (circRNAs) have been identified to play key roles in cancer biology; however, the detailed functions and mechanisms of circRNAs in hepatocellular carcinoma (HCC) remain largely unclarified. RNA-seq analysis was used to screen the expression profiles of circRNAs in HCC. CircZNF566 expression in HCC tissues and cell lines was detected by qRT-PCR. In vitro CCK-8, colony formation, wound healing, transwell migration, and invasion assays and in vivo tumorigenesis and metastasis assays were conducted to determine the functions of circZNF566. Luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays were also performed to confirm the relationship between circZNF566 and miR-4738-3p. Bioinformatics analysis and luciferase reporter assays were employed to determine whether miR-4738-3p regulates tryptophan 2,3-dioxygenase (TDO2) expression. Finally, immunohistochemistry (IHC) was used to detect the level of TDO2 and determine its prognostic value. CircZNF566 was significantly upregulated in HCC tissues and cell lines. High circZNF566 expression in HCC tissues was positively correlated with clinicopathological features and poor prognosis. Functionally, in vitro experiments showed that circZNF566 promoted HCC cell migration, invasion, and proliferation, whereas in vivo experiments showed that circZNF566 promoted tumorigenesis and metastasis. Mechanistically, circZNF566 acted as a miR-4738-3p sponge to relieve the repressive effect of miR-4738-3p on its target TDO2. In addition, miR-4738-3p suppressed HCC cell migration, invasion, and proliferation, while TDO2 was positively correlated with pathological features and poor prognosis and promoted cell migration, invasion, and proliferation in HCC. CircZNF566 is a novel tumor promoter in HCC and functions through the circZNF566/ miR-4738-3p /TDO2 axis; in addition, circZNF566 may serve as a novel diagnostic marker, prognostic indicator, and target for the treatment of HCC.

TDO Promotes Hepatocellular Carcinoma Progression.

PURPOSE: Tryptophan 2,3-dioxygenase (TDO), encoded by the gene TDO2, is an enzyme that catalyses the first and rate-limiting step of tryptophan (Try) degradation in the kynurenine (Kyn) pathway in the liver. Recently, TDO has been demonstrated to be expressed in various human tumours, especially hepatocellular carcinoma (HCC). However, the role of TDO in HCC is still not very clear. Here, we studied the role of TDO in HCC. METHODS: We demonstrated that TDO is overexpressed in human HCC tissues and is significantly correlated with malignant phenotype characteristics, including tumour size, tumour differentiation, vascular invasion, etc. Kaplan-Meier analysis showed a poor overall survival rate in patients with TDO-overexpressing tumours. In addition, the effects of TDO on HCC tumour growth and metastasis were detected both in vivo and in vitro. TDO overexpression facilitated HCC cell growth, invasion and migration. CONCLUSION: Our results suggest that TDO positively regulates HCC proliferation and invasion and acts as a new prognostic biomarker of HCC.

Feasibility and efficacy evaluation of metallic biliary stents eluting gemcitabine and cisplatin for extrahepatic cholangiocarcinoma.

BACKGROUND: Effective endoscopic management is fundamental for the treatment of extrahepatic cholangiocarcinoma (ECC). However, current biliary stents that are widely used in clinical practice showed no antitumor effect. Drug-eluting stents (DESs) may achieve a combination of local chemotherapy and biliary drainage to prolong stent patency and improve prognosis. AIM: To develop novel DESs coated with gemcitabine (GEM) and cisplatin (CIS)-coloaded nanofilms that can maintain the continuous and long-term release of antitumor agents in the bile duct to inhibit tumor growth and reduce systemic toxicity. METHODS: Stents coated with different drug-eluting components were prepared by the mixed electrospinning method, with poly-L-lactide-caprolactone (PLCL) as the drug-loaded nanofiber membrane and GEM and/or CIS as the antitumor agents. Four different DESs were manufactured with four drug-loading ratios (5%, 10%, 15%, and 20%), including bare-loaded (PLCL-0), single-drug-loaded (PLCL-GEM and PLCL-CIS), and dual-drug-loaded (PLCL-GC) stents. The drug release property, antitumor activity, and biocompatibility were evaluated in vitro and in vivo to confirm the feasibility and efficacy of this novel DES for ECC. RESULTS: The in vitro drug release study showed the stable, continuous release of both GEM and CIS, which was sustained for over 30 d without an obvious initial burst, and a higher drug-loaded content induced a lower release rate. The drug-loading ratio of 10% was used for further experiments due to its ideal inhibitory efficiency and relatively low toxicity. All drug-loaded nanofilms effectively inhibited the growth of EGI-1 cells in vitro and the tumor xenografts of nude mice in vivo; in addition, the dual-loaded nanofilm (PLCL-GC) had a significantly better effect than the single-drug-loaded nanofilms (P < 0.05). No significant differences in the serological analysis (P > 0.05) or histopathological changes were observed between the single-loaded and drug-loaded nanofilms after stent placement in the normal porcine biliary tract. CONCLUSION: This novel PLCL-GEM and CIS-eluting stent maintains continuous, stable drug release locally and inhibits tumor growth effectively in vitro and in vivo. It can also be used safely in normal porcine bile ducts. We anticipate that it might be considered an alternative strategy for the palliative therapy of ECC patients.

CTHRC1 promotes gastric cancer metastasis via HIF-1α/CXCR4 signaling pathway.

Metastasis is the main cause of gastric cancer (GC) related death and the underlying mechanisms still remain unclear. Collagen triple helix repeat containing 1 (CTHRC1) protein is known to be involved in tissue remodeling processes and closely associated with carcinogenesis and metastasis in solid tumors, but the functional role of CTHRC1 and its underlying mechanism with tumor metastasis in GC have not been fully illuminated. In the present study, CTHRC1 was highly expressed in tumor tissues and associated with poor prognosis of GC according to TCGA and GEO database. Functional studies revealed that CTHRC1 overexpression in GC significantly increased cell migration and invasion capacity. However, the promoting effects were abolished subsequent to silencing of CXCR4. In addition, CTHRC1 increased CXCR4 expression through upregulating HIF-1α expression, which eventually contributed to the promotion of cell migration and invasion. Inhibiting HIF-1α expression decreased CXCR4 expression and suppressed cell migration and invasion in GC. These results substantiated our hypothesis that HIF-1α/CXCR4 signaling pathway mediated the promoting effect of CTHRC1 on cell migration and invasion in GC.

PCDHGA9 represses epithelial-mesenchymal transition and metastatic potential in gastric cancer cells by reducing ß-catenin transcriptional activity.

Gastric cancer (GC) has a high mortality rate, and metastasis is the main reason for treatment failure. It is important to study the mechanism of tumour invasion and metastasis based on the regulation of key genes. In a previous study comparing the expression differences between GES-1 and SGC-7901 cells, PCDHGA9 was selected for further research. In vitro and in vivo experiments showed that PCDHGA9 inhibited invasion and metastasis. A cluster analysis suggested that PCDHGA9 inhibited epithelial-mesenchymal transition (EMT) through the Wnt/ß-catenin and TGF-ß pathways. Laser confocal techniques and western blotting revealed that PCDHGA9 inhibited the nuclear translocation of ß-catenin, regulated T cell factor (TCF)/ /lymphoid enhancer factor (LEF) transcriptional activity, directly impacted the signal transmission of the TGF-ß/Smad2/3 pathway, strengthened the adhesion complex, weakened the effects of TGF-ß, and blocked the activation of the Wnt pathway. In addition, PCDHGA9 expression was regulated by methylation, which was closely related to poor clinical prognosis. The aim of this study was to elucidate the molecular mechanism by which PCDHGA9 inhibits EMT and metastasis in GC to provide a new theoretical basis for identifying GC metastasis and a new target for improving the outcome of metastatic GC.

Elevated HOXA13 expression promotes the proliferation and metastasis of gastric cancer partly via activating Erk1/2.

PURPOSE: HOXA13 is a transcription factor of the Homeobox (HOX) gene family, which is highly evolutionarily conserved. HOXA13 is upregulated and associated with oncogenic properties in some cancers. Here, we studied the potential mechanism of HOXA13-mediated proliferation and metastasis in gastric cancer (GC). METHODS: Quantitative real-time PCR, Western blot, and immunohistochemistry were used to detect HOXA13 expression levels in GC. In vitro and in vivo assays were performed to investigate the function of HOXA13 in GC cell proliferation, migration, and invasion. RNA-Seq transcriptome analysis was performed to study the underlying mechanism of HOXA13-mediated aggressiveness in GC. RESULTS: HOXA13 mRNA and protein expression levels were upregulated in GC tissues. According to Cell Counting Kit-8 and colony formation assays, we found that HOXA13 over-expression promoted proliferation. Flow cytometry analysis showed that HOXA13 overexpression or knockdown led to G1-S phase transition or G1 phase arrest, respectively. Western blot analysis results showed that HOXA13 overexpression increased cyclin D1 expression, while knockdown decreased its expression. Wound healing and transwell assay results demonstrated that HOXA13 overexpression promoted the migration and invasion of GC cells. Western blot analysis results also showed that HOXA13 overexpression upregulated N-cadherin and vimentin and downregulated E-cadherin, while HOXA13 knockdown led to the opposite results, indicating that HOXA13 might participate in epithelial to mesenchymal transition. These results were verified in vivo by tumor xenograft and metastasis assays. Mechanistically, using RNA-Seq transcriptome analysis, we found that Erk1/2 activation played an important role in HOXA13-induced GC progression. CONCLUSION: Our results show that HOXA13 plays an important role in GC development. HOXA13 overexpression promotes proliferation and metastasis partly via activation of Erk1/2 in GC. Thus, HOXA13, together with Erk1/2, may be promising targets for novel anticancer strategies.

RBBP6, a RING finger-domain E3 ubiquitin ligase, induces epithelial-mesenchymal transition and promotes metastasis of colorectal cancer.

RBBP6 has been implicated in tumorigenesis but its role in tumor metastasis and progression has not been evaluated. Interestingly, here we show that RBBP6 is upregulated in colorectal cancer (CRC) where its expression level is positively correlated with distant metastasis. In this study, we identified RBBP6, a RING Finger-domain E3 ubiquitin ligase, served as an independent prognostic factor and predicted poor outcome for CRC patients. RBBP6 promoted cell proliferation, migration, and invasion in CRC cells and promoted tumor growth, lung metastasis, and liver metastasis in mouse models. Mechanistically, we revealed that RBBP6 bound and ubiquitylated IκBα, an inhibitor of the NF-κB-signaling pathway. RBBP6-mediated ubiquitination and degradation of IκBα significantly enhanced p65 nuclear translocation, which triggered the activation of NF-κB pathway and then induced the epithelial-mesenchymal transition (EMT) process and cell metastasis. Furthermore, by DNA methylation results and ChIP analysis, we demonstrated that the promoter of RBBP6 was hypomethylated, and was activated by multi-oncogenic transcription factors. In conclusion, our findings suggest that RBBP6 may be a potential prognostic biomarker and therapeutic target for CRC invasion and metastasis.