Phase separation of DDX21 promotes colorectal cancer metastasis via MCM5-dependent EMT pathway.

RNA binding proteins (RBPs) contributes to cancer progression, but the underlying mechanism reminds unclear. Here, we find that DDX21, a representative RBP, is highly expressed in colorectal cancer (CRC), which leads to CRC cell migration and invasion in vitro, and CRC to liver metastasis and lung metastasis in vivo. This effect of DDX21 on CRC metastasis is correlated to the activation of Epithelial-mesenchymal transition (EMT) pathway. Moreover, we reveal that DDX21 protein is phase separated in vitro and in CRC cells, which controls CRC metastasis. Phase-separated DDX21 highly binds on MCM5 gene locus, which is markedly reduced when phase separation is disrupted by mutations on its intrinsically disordered region (IDR). The impaired metastatic ability of CRC upon DDX21 loss is restored by ectopic expression of MCM5, indicating MCM5 is a key downstream target of DDX21 for CRC metastasis. Furthermore, co-higher expressions of DDX21 and MCM5 is significantly correlated with poor survival outcomes of stage III and IV CRC patients, indicating the importance of this mechanism in CRC late and metastatic stage. Altogether, our results elucidate a new model of DDX21 in regulating CRC metastasis via phase separation.

MELK predicts poor prognosis and promotes metastasis in esophageal squamous cell carcinoma via activating the NF­κB pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide with a low 5­year survival rate due to the lack of effective therapeutic strategies. Accumulating evidence has indicated that maternal embryonic leucine zipper kinase (MELK) is highly expressed in several tumors and associated with tumor development. However, the biological effects of MELK in ESCC remain unknown. In the present study, cell phenotypical experiments and animal metastasis assays were performed to detect the influence of MELK knockdown in vitro and in vivo. The potential molecular mechanism of MELK­mediated ESCC metastasis was further investigated by western blotting and immunofluorescence staining. The results revealed that the expression of MELK in human ESCC tissues was higher than that in adjacent normal tissues and was positively associated with the poor prognosis of patients. Reducing MELK expression resulted in growth inhibition and suppression of the invasive ability of ESCC cells in vitro and in vivo. MELK inhibition induced alterations of epithelial­mesenchymal transition­associated proteins. Mechanistically, MELK interacted with IκB kinase (IKK) and promoted the phosphorylation of IKK, by which MELK regulated activation of the NF­κB pathway. Collectively, the present study revealed the function and mechanism of MELK in the cell metastasis of ESCC, which may be a potential therapeutic target for ESCC.

TYMS-TM4SF4 axis promotes the progression of colorectal cancer by EMT and upregulating stem cell marker.

BACKGROUND: The expression of thymidylate synthase (TYMS) is significantly up-regulated in various cancers and associated with the poor prognosis of patients. However, the role of TYMS in the progression of colorectal cancer (CRC) is unclear. METHODS: Cell function assay, biology information analysis, and RNA sequencing were used to investigate the role of TYMS in the progression of CRC and underlining molecular mechanism. SPSS22.0 statistical software and GraphPad Prism 5 (Graphpad software) were used for statistical analysis. RESULTS: Our results showed that TYMS expression was higher in CRC tissues than that in non-tumor colorectal mucosa tissues. TYMS knockdown inhibited the proliferation, migration and invasion of HCT116 and HT29 cells, and the spheroid formation of HCT116 cells. The underling mechanism demonstrated that TYMS promoted the progression of CRC by regulating EMT-related proteins including E-cadherin, Vimentin, MMP-9 and stem cell biomarkers including CD133 and CD44. Furthermore, DEG sequencing showed that TYMS knockdown enriched the pathways of metastasis and metabolism by GO and KEGG analysis. We identified TM4SF4 was the downstream target of TYMS in CRC cells. TM4SF4 overexpression increased migration and invasion of CRC cells by regulating EMT and CD133 expression. CONCLUSIONS: Our findings suggest that TYMS-TM4SF4 axis may promote the progression of CRC by EMT and upregulating stem cell markers.

Development and Validation of a Ferroptosis-Related Gene Signature and Nomogram for Predicting the Prognosis of Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a common malignant tumor with high mortality and poor prognosis. Ferroptosis is a newly discovered form of cell death induced by iron-catalyzed excessive peroxidation of polyunsaturated fatty acids (PUFAs). However, the prognostic value of ferroptosis-related genes (FRGs) for ESCC remains unclear. Based on the ESCC dataset from the Gene Expression Omnibus (GEO) database, we identified 39 prognostic FRGs through univariate Cox regression analysis. After LASSO regression and multivariate Cox regression analyses, a multigene signature based on 10 prognostic FRGs was constructed and successfully divided ESCC patients into two risk groups. Patients in the low-risk group showed a significantly better prognosis than patients in the high-risk group. In addition, we combined the risk score with clinical predictors to construct a nomogram for ESCC. The predictive ability of the nomogram was further verified by ROC curves and calibration plots in both the training and validation sets. The predictive power of the nomogram was demonstrated to be better than that of either the risk score or clinical variable alone. Furthermore, functional analysis revealed that the 10-FRG signature was mainly associated with ferroptosis, differentiation and immune response. Connectivity map analysis identified potential compounds capable of targeting FRGs in ESCC. Finally, we demonstrated the prognostic value of SRC gene in ESCC using the clinical samples and found that SRC inhibition sensitized ESCC cells to ferroptosis inducers by in vitro experiments. In conclusion, we identified and verified a 10-FRG prognostic signature and a nomogram, which provide individualized prognosis prediction and provide insight into potential therapeutic targets for ESCC.

Vasorin stimulates malignant progression and angiogenesis in glioma.

Glioma, the most common human primary brain tumor, is characterized by invasive capabilities and angiogenesis. Vasorin (VASN), a transmembrane protein, is reported to be associated with vascular injury repair and is overexpressed in some human tumors. However, its role in tumor progression and angiogenesis in glioma is unknown. In this study, VASN was shown to be overexpressed in high-grade gliomas, and the expression level correlated with tumor grade and microvessel density in glioma specimens. Glioma patients with high VASN expression had a shorter overall survival time. Knockdown of VASN in glioma cells by shRNA significantly inhibited the malignancy of glioma, including cell proliferation, colony formation, invasion, and sphere formation. Ectopic expression of VASN increased glioma progression in vitro. The expression of VASN correlated with the mesenchymal type of glioblastoma multiforme (GBM) subtyped by gene set enrichment analysis (GSEA). Our results showed that the concentration of VASN was increased in the conditioned medium (CM) from glioma cells with VASN overexpression, and the CM from glioma cells with knockdown or overexpressed VASN inhibited or promoted HUVEC migration and tubulogenesis in vitro, respectively. Glioma growth and angiogenesis were stimulated upon ectopic expression of VASN in vivo. The STAT3 and NOTCH pathways were found to be activated and inhibited by VASN overexpression. Our findings suggest that VASN stimulates tumor progression and angiogenesis in glioma, and, as such, represents a novel therapeutic target for glioma.

A novel iron(II) phenanthroline complex exhibits anticancer activity against TFR1-overexpressing esophageal squamous cell carcinoma cells through ROS accumulation and DNA damage.

Esophageal squamous cell carcinoma (ESCC) is one of the most common and aggressive cancers worldwide, especially in China, with poor prognosis due to the lack of effective therapeutic strategies. Here, the anticancer effect and pharmacological mechanism of a newly synthesized Fe(II) phenanthroline complex was studied in ESCC. Our data showed that transferrin receptor 1 (TFR1) was specifically overexpressed in ESCC tissues compared to its expression in normal esophageal tissues, a finding further supported by public datasets. The newly synthesized Fe(II) complex was selectively transported into ESCC cells overexpressing TFR1 through TFR1-mediated endocytosis and exhibited anticancer activity in a dose-dependent manner. The mechanistic study elucidated that the Fe(II) complex caused cell cycle arrest at the G0/G1 phase by blocking the CDK4/6-cyclin D1 complex and induced mitochondria-mediated apoptosis. Furthermore, exposure to the Fe(II) complex led to excessive reactive oxygen species (ROS) accumulation by thioredoxin reductase (TrxR) inhibition and DNA double-strand breaks (DSBs), which in turn sequentially activated ATM, CHK1/2 and p53. Moreover, combination treatment with cisplatin and the Fe(II) complex exhibited a synergistic effect in ESCC cells. Taken together, our results initially suggest the potential application of the Fe(II) complex in ESCC chemotherapy, especially for patients with TFR1 overexpression.

UHRF1 is an Independent Prognostic Factor and a Potential Therapeutic Target of Esophageal Squamous Cell Carcinoma.

Purpose: Ubiquitin-like with plant homeodomain and ring-finger domains 1 (UHRF1) plays an essential role in DNA methylation, and the overexpression of UHRF1 is associated with poor prognosis in various cancers. Esophageal squamous cell carcinoma (ESCC) accounts for approximately 90% of esophageal cancer cases in China, but the five-year survival rate for patients is less than 10% due to limited clinical approaches for early diagnosis and treatment. The present research aimed to investigate the expression of UHRF1 in ESCC and its biological role in ESCC development. Methods: UHRF1 expression in ESCC and normal esophageal tissues was examined using immunohistochemical staining, followed by analysis of the correlation between UHRF1 expression and clinical features. In addition, the effects of lentivirus-mediated RNA interference of UHRF1 on global DNA methylation, cell proliferation, cell cycle progression and apoptosis and were investigated in ESCC cells. Results: UHRF1 was overexpressed in ESCC tissues and was an independent prognostic factor for ESCC patients. In ESCC cells, knockdown of UHRF1 caused global DNA hypomethylation, inhibited cell proliferation and induced apoptosis. Furthermore, UHRF1 depletion induced cell cycle arrest at the G2/M phase, accompanied by activation of Wee1 and DNA damage response pathway. Conclusions: Our findings identify UHRF1 as a promising prognostic marker for ESCC and suggest that UHRF1 may be a potential therapy target for ESCC patients with elevated UHRF1 expression.

Tetrandrine inhibits glioma stem-like cells by repressing ß-catenin expression.

Cancer stem cells (CSCs) in glioma are often responsible for relapse and resistance to therapy. The purpose of the present study was to confirm the self-renewal and migration inhibitory effects of tetrandrine (Tet), which is a compound extracted from the dried root of Stephania tetrandra S. Moore, toward glioma stem-like cells (GSLCs) and to examine the associated molecular mechanisms. Using a neurosphere culture technique, we enriched the GSLC population from the human glioblastoma cell lines U87 and U251. Cells were analyzed using cell counting kit-8 (CCK-8), western blotting, flow cytometry, transwell assay and immunofluorescence staining. GSLCs displayed properties of neural stem cells, including elevated expression of the cancer stem cell marker ALDH1 and ß-catenin. We found that Tet treatment decreased sphere formation in GSLCs in a dose-dependent manner using tumor spheroid formation assay. The GSK3ß inhibitor BIO maintained sphere formation and migration capacity in GSLCs, whereas the ß-catenin/TCF transcription inhibitor ICG-001 decreased sphere formation and the migration capacity of GSLCs. The proportion of apoptotic GSLCs also increased in response to ICG-001 treatment. These results indicate that ß-catenin activity is vital in maintaining neural stem cell traits of GSLCs. Tet inhibits cell viability, neurosphere formation and migration of GSLCs in vitro. Importantly, Tet treatment significantly repressed the nuclear translocation and expression of ß-catenin and induced apoptosis in GSLCs, as indicated in part by the upregulation of Bax, the cleavage of PARP and the downregulation of Bcl-2. The present study demonstrates that the inhibition of ß-catenin in CSCs by Tet could be an effective strategy for the treatment of glioma.

Tetrandrine suppresses human glioma growth by inhibiting cell survival, proliferation and tumour angiogenesis through attenuating STAT3 phosphorylation.

Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid, has been reported to possess anti-tumour activity. However, its effects on human glioma remain unknown. In this study, we demonstrated that Tet inhibited human glioma cell growth in vitro and in vivo. It has been hypothesised that Tet inhibits glioma growth by affecting glioma cell survival, proliferation and vasculature in and around the xenograft tumour in the chick CAM model and signal transducer and activator of transcription 3 (STAT3) mediated these activities. Therefore, we conducted a detailed analysis of the inhibitory effects of Tet on cell survival using a TUNEL assay and flow cytometric analysis; on cell proliferation based on the expression of proliferating cell nuclear antigen; and on angiogenesis using a CAM anti-angiogenesis assay. We used western blotting to investigate the role of STAT3 on the anti-glioma activities of Tet. The results revealed that Tet inhibited survival and proliferation in human glioma cells, impaired tumour angiogenesis and decreased the expression of phosphorylated STAT3 and its downstream proteins. In sum, our data indicate that STAT3 is involved in Tet-induced the regression of glioma growth by activating tumour cell apoptosis, inhibiting glioma cell proliferation and inhibiting angiogenesis.

EGCG inhibits properties of glioma stem-like cells and synergizes with temozolomide through downregulation of P-glycoprotein inhibition.

RATIONAL: Combination therapy to inhibit cancer stem cells may have important clinical implications. Here, we examine the molecular mechanisms by which epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, inhibits the stem cell characteristics of glioma stem-like cells (GSLCs) and synergizes with temozolomide (TMZ), a DNA-methylating agent commonly used as first-line chemotherapy in gliomas. GSLCs were enriched from the human glioblastoma cell line U87 using neurosphere culture. Cells were analyzed using flow cytometry, quantitative PCR, and western blotting. Compared to U87 cells, a higher percentage of U87 GSLCs remained in the G0/G1 phase, with downregulation of the cell-cycle protein CylinD1 and overexpression of stem cell markers CD133 and ALDH1. The drug-resistance gene ABCB1 (but not ABCG2 or MGMT) also showed high mRNA and protein expression. The resistance index of U87 GSLCs against TMZ and carmustine (BCNU) was 3.0 and 16.8, respectively. These results indicate that U87 GSLCs possess neural stem cell and drug-resistance properties. Interestingly, EGCG treatment inhibited cell viability, neurosphere formation, and migration in this cell model. EGCG also induced apoptosis, downregulation of p-Akt and Bcl-2, and cleaving PARP in a dose-dependent manner. Importantly, EGCG treatment significantly downregulated P-glycoprotein expression but not that of ABCG2 or MGMT and simultaneously enhanced sensitivity to TMZ. Our study demonstrates that the use of EGCG alone or in combination with TMZ may be an effective therapeutic strategy for glioma.

Dual high expression of STAT3 and cyclinD1 is associated with poor prognosis after curative resection of esophageal squamous cell carcinoma.

BACKGROUND: Signal transducer of activator of transcription 3 (STAT3) and cyclinD1 are overexpressed in various human cancers, and their overexpression positively correlates to tumor progression and poor prognosis. However, the clinical significance of dual high expression of these two proteins in esophageal squamous cell carcinoma (ESCC) has yet to be determined. METHODS: The expression of STAT3 and cyclinD1 was analyzed in tissue microarrays containing tumor and adjacent tissue samples from 82 patients who had undergone curative resection for histologically proven ESCC. Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the prognostic value of STAT3 and cyclinD1 expression. RESULTS: We discovered that expressions of STAT3 and cyclinD1 in cancer tissues were significantly higher than that in adjacent tissues. High expression of STAT3 and cyclinD1 was associated with malignant behaviors. Moreover, the expression of STAT3 was positively associated with the expression of cyclinD1. High STAT3 or cyclinD1 expression alone was associated with lower overall survival (OS) rates. Furthermore, dual high expression of STAT3 and cyclinD1 expression predict even worse survival outcome in both univariate and multivariate analysis. CONCLUSION: STAT3 and cyclinD1 correlate with more aggressive tumor behavior in ESCC. When STAT3 and cyclinD1 are considered together, they serve as effective prognostic markers in patients with surgically resected ESCC.