HIC-5 in cancer-associated fibroblasts contributes to esophageal squamous cell carcinoma progression.

Esophageal squamous cell carcinoma (ESCC) remains one of the most common malignancies in China and has a high metastasis rate and poor prognosis. Cancer-associated fibroblasts (CAFs), a prominent component of the tumor microenvironment, can affect tumor progression and metastasis, but the underlying mechanism remains unclear. There are no studies that explore the role of hydrogen peroxide-inducible clone 5 (HIC-5) in ESCC or compare the role of HIC-5 in CAFs and adjacent noncancerous normal fibroblasts (NFs). In this study, we isolated primary CAFs and NFs from ESCC patients. HIC-5 was highly expressed in CAFs from the tumor stroma of human ESCC patients. HIC-5 knockdown in CAFs inhibited the migration and invasion of ESCC cells in vitro. Supernatant CCL2 levels of CAFs were significantly higher after TGF-ß stimulation and lower after knocking down HIC-5 expression, independent of TGF-ß treatment. HIC-5 knockdown in CAFs led xenograft tumors derived from ESCC cells mixed with CAFs to present more regular morphology, express higher CDH1, and lower CCL2. Further RNA-seq data showed that HIC-5 has distinct biological functions in CAFs vs. NFs, especially in cell movement and the Rho GTPase signaling kinase pathway, which was verified by wound-healing assays and western blotting. An ESCC tissue microarray revealed that increased HIC-5 expression in the tumor stroma was associated with positive lymph node metastasis and a higher TNM stage. In summary, we identified that stromal HIC-5 was a predictive risk factor for lymph node metastasis in human ESCC and that CAF-derived HIC-5 regulated ESCC cell migration and invasion by regulating cytokines and modifying the ECM.

Transcriptional activation of ANO1 promotes gastric cancer progression.

The prognosis of gastric cancer (GC) remains poor due to local invasion and distal metastasis. The GC-related molecular mechanisms underlying invasion and metastasis are not well understood. In this study, we investigated the functional role of ANO1 in GC progression. We found that ANO1 is overexpressed in GC tissues and correlated with GC tumor-node-metastasis stage. Knockdown of ANO1 significantly inhibited GC cell migration and invasion in vitro, and loss of ANO1 resulted in inhibition of tumor metastasis in vivo. Mechanistically, SP1 increased ANO1 transcription, recruited MLL1 to the ANO1 promoter region, facilitated H3K4 trimethylation, and subsequently promoted ANO1 expression. Together, our findings provide a mechanistic assessment of ANO1 overexpression, which represents a GC progression-related molecule and a potentially valuable target for future research.

Asparaginyl endopeptidase may promote liver sinusoidal endothelial cell angiogenesis via PI3K/Akt pathway

Background and aims: pathological angiogenesis plays an important role in the progression of chronic liver diseases. Asparaginyl endopeptidase (AEP) participates in tumor angiogenesis and was recently shown to be associated with liver fibrosis. This study aimed to explore the effect of AEP on liver sinusoidal endothelial cell (LSECs) angiogenesis and determine the underlying mechanism. Methods: cultured LSECs were infected with lentiviruses in order to suppress AEP expression (AEP-KD1, AEP-KD2). The effect of AEP on LSECs proliferation, apoptosis and migration were subsequently determined by a CCK8 assay, flow cytometry and wound-healing and Transwell assays, respectively, in AEP knocked-down and control LSECs. The expression of the endothelial cell surface markers CD31, CD34 and von Willebrand factor (vWF) were detected by immunofluorescence assay and western blot. The angiogenic factors, vascular endothelial growth factor receptor 2 (VEGFR2) and interleukin 8 (IL 8) were detected by real-time PCR and western blot. The effect of AEP on vessel tube formation by LSECs was examined by Matrigel(TM) tube-formation assay. Phosphoinositide 3-kinase (PI3K)/Akt expression and phosphorylation were detected by western blot. Results: AEP was effectively knocked down by lentivirus infection in LSECs. Down-regulation of AEP expression significantly decreased proliferation and migration and increased apoptosis of LSECs. Moreover, expression levels of the endothelial cell surface markers CD31, CD34 and vWF, as well as angiogenic factors VEGFR2 and IL 8, were also reduced after AEP was knocked-down. The vessel tube formation abilities of AEP-KD1 and AEP-KD2 LSECs were significantly inhibited compared with LSECs without AEP knocked-down. Down-regulation of AEP also inhibited the phosphorylation of PI3K and Akt. Conclusion: AEP promotes LSECs angiogenesis in vitro, possibly via the PI3K/Akt pathway. AEP may therefore be a potential therapeutic target for preventing the progression of liver fibrosis

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Asparaginyl endopeptidase may promote liver sinusoidal endothelial cell angiogenesis via PI3K/Akt pathway.

BACKGROUND AND AIMS: pathological angiogenesis plays an important role in the progression of chronic liver diseases. Asparaginyl endopeptidase (AEP) participates in tumor angiogenesis and was recently shown to be associated with liver fibrosis. This study aimed to explore the effect of AEP on liver sinusoidal endothelial cell (LSECs) angiogenesis and determine the underlying mechanism. METHODS: cultured LSECs were infected with lentiviruses in order to suppress AEP expression (AEP-KD1, AEP-KD2). The effect of AEP on LSECs proliferation, apoptosis and migration were subsequently determined by a CCK8 assay, flow cytometry and wound-healing and Transwell assays, respectively, in AEP knocked-down and control LSECs. The expression of the endothelial cell surface markers CD31, CD34 and von Willebrand factor (vWF) were detected by immunofluorescence assay and western blot. The angiogenic factors, vascular endothelial growth factor receptor 2 (VEGFR2) and interleukin 8 (IL 8) were detected by real-time PCR and western blot. The effect of AEP on vessel tube formation by LSECs was examined by Matrigel™ tube-formation assay. Phosphoinositide 3-kinase (PI3K)/Akt expression and phosphorylation were detected by western blot. RESULTS: AEP was effectively knocked down by lentivirus infection in LSECs. Down-regulation of AEP expression significantly decreased proliferation and migration and increased apoptosis of LSECs. Moreover, expression levels of the endothelial cell surface markers CD31, CD34 and vWF, as well as angiogenic factors VEGFR2 and IL 8, were also reduced after AEP was knocked-down. The vessel tube formation abilities of AEP-KD1 and AEP-KD2 LSECs were significantly inhibited compared with LSECs without AEP knocked-down. Down-regulation of AEP also inhibited the phosphorylation of PI3K and Akt. CONCLUSION: AEP promotes LSECs angiogenesis in vitro, possibly via the PI3K/Akt pathway. AEP may therefore be a potential therapeutic target for preventing the progression of liver fibrosis.

Erratum: Label-free quantitative proteomic analysis identifies CTNNB1 as a direct target of FOXP3 in gastric cancer cells.

[This corrects the article DOI: 10.3892/ol.2018.8277.].

Label-free quantitative proteomic analysis identifies CTNNB1 as a direct target of FOXP3 in gastric cancer cells.

Forkhead box protein 3 (FOXP3) is expressed in numerous types of tumor cell and is associated with tumor progression and prognosis. A previous study reported that FOXP3 inhibited cellular proliferation and induced apoptosis of gastric cancer (GC) cells by activating the apoptosis signaling pathway. In the present study, label-free quantitative proteomic analysis and chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) was performed to investigate the mechanism by which the anticancer role of FOXP3 was mediated and the proteins that with which it may interact. Label-free quantitative proteomic analysis was used to screen for proteins differentially expressed between FOXP3-overexpressing GC (AF) and vector (ANC) cells. Catenin ß1 (CTNNB1) was one of the proteins that exhibited the greatest difference between AF and ANC among 3,313 proteins identified by liquid chromatography with tandem mass spectrometry analysis. The expression of CTNNB1 was evaluated by reverse transcription-quantitative PCR and western blotting. The association between FOXP3 and CTNNB1 was confirmed by ChIP-PCR in AGS cells. The changes in expression of epithelial-mesenchymal transition-associated proteins were analyzed by western blotting. The level of FOXP3 expression was positively associated with CTNNB1 and E-cadherin expression, but not with vimentin and N-cadherin expression. FOXP3 positively regulates CTNNB1 and binds to it directly. Along with the upregulation of glycogen synthase kinase 3ß (GSK3ß), which was also a protein whose expression was found to change significantly in proteomic analysis and has a key role in the Wnt pathway. This association is an attractive and novel hypothesis for the mechanism by which FOXP3 inhibits the invasion and metastasis of GC cells.

Quantitative proteomic Analysis Reveals up-regulation of caveolin-1 in FOXP3-overexpressed human gastric cancer cells.

Forkhead box protein 3 (FOXP3) is implicated in tumor progression and prognosis in various types of tumor cells. We have recently reported that FOXP3 inhibited proliferation of gastric cancer (GC) cells through activating the apoptotic signaling pathway. In this study, we found that over-expression of FOXP3 inhibited GC cell migration, invasion and proliferation. Then, the label-free quantitative proteomic approach was employed to further investigating the down-stream proteins regulated by FOXP3, resulting in a total of 3,978 proteins quantified, including 186 significantly changed proteins. Caveolin-1 (CAV1), as a main constituent protein of caveolae, was one of those changed proteins up-regulated in FOXP3-overexpressed GC cells, moreover, it was assigned as one of the node proteins in the protein-protein interaction network and the key protein involved in focal adhesion pathway by bioinformatics analysis. Further biological experiments confirmed that FOXP3 directly bound to the promoter regions of CAV1 to positively regulate CAV1 transcription in GC cells. In summary, our study suggested that FOXP3 can be considered as a tumor suppressor in GC via positively regulating CAV1 through transcriptional activation, and this FOXP3-CAV1 transcriptional regulation axis may play an important role in inhibiting invasion and metastasis of GC cells. Data are available via ProteomeXchange under identifier PXD007725.

Kruppel-like factor 2 inhibit the angiogenesis of cultured human liver sinusoidal endothelial cells through the ERK1/2 signaling pathway.

Kruppel-like factor 2 (KLF2) is a crucial anti-angiogenic factor. However, its precise role in hepatic angiogenesis induced by liver sinusoidal endothelial cells (LSECs) remain unclear. This study was aimed to evaluate the effect of KLF2 on angiogenesis of LSECs and to explore the corresponding mechanism. Cultured human LSECs were infected with different lentiviruses to overexpress or suppress KLF2 expression. The CCK-8 assay, transwell migration assay and tube formation test, were used to investigate the roles of KLF2 in the proliferation, migration and vessel tube formation of LSECs, respectively. The expression and phosphorylation of ERK1/2 were detected by western blot. We discovered that the up-regulation of KLF2 expression dramatically inhibited proliferation, migration and tube formation in treated LSECs. Correspondingly, down-regulation of KLF2 expression significantly promoted proliferation, migration and tube formation in treated LSECs. Additionally, KLF2 inhibited the phosphorylation of ERK1/2 pathway, followed by the function of KLF2 in the angiogenesis of LSECs disrupted. In conclusion, KLF2 suppressed the angiogenesis of LSECs through inhibition of cell proliferation, migration, and vessel tube formation. These functions of KLF2 may be mediated through the ERK1/2 signaling pathway.

[Clinical significance of circulating hypoxia inducible factor-1α and hemeoxygenase-1 in patients with liver cirrhosis].

OBJECTIVE: To explore the levels of circulating hypoxia inducible factor-1α (HIF-1α) and hemeoxygenase-1 (HO-1) in liver cirrhosis and to explore their diagnostic values as noninvasive methods. METHODS: The levels of circulating HIF-1α and HO-1 were quantitatively detected in 34 patients with liver cirrhosis and 10 healthy controls by ELISA from May 2012 to May 2013. The diagnostic values were analyzed by ROC curve and their correlation with clinicopathological characters were also compared. RESULTS: The serum levels of HIF-1α and HO-1 were significantly higher than those in healthy controls ((10.99 ± 0.24) vs (5.79 ± 0.84) µg/L, (63.04 ± 1.87) vs (16.35 ± 2.07) µg/L, both P < 0.01). No significant difference existed among Child-Pugh classification. The expressions of HIF-1α and HO-1 had a linear correlation between each other, and closely related with rebleeding and portal thrombosis during a one-year follow-up (all P < 0.05). The area under ROC curve of HIF-1α and HO-1 was 0.894 and 0.994 respectively. With 9.45 µg/L as a critical point for HIF-1α, the diagnosis of liver cirrhosis might be predicted with a positive predictive value of 91.2%. And HO-1 >31.86 µg/L predicted the diagnosis of liver cirrhosis with a sensitivity of 97.1% and a specificity of 100%. CONCLUSION: The serum levels of HIF-1α and HO-1 may serve as useful molecular markers for a noninvasive diagnosis of liver cirrhosis.