PITX1 suppresses osteosarcoma metastasis through exosomal LINC00662-mediated M2 macrophage polarization.

Paired-like homeodomain transcription factor 1 (PITX1) is frequently downregulated in cancers, including osteosarcoma (OS). However, its role in OS remains unknown. Therefore, we aimed to explore the functions and potential mechanisms of PITX1 in OS malignant progression. Elevated PITX1 suppressed OS cell proliferation and migration, based on transwell, proliferation, and colony formation assays. Pathway enrichment analysis of differentially-expressed genes between PITX1-overexpressing and control OS cells indicated that PITX1 expression was associated with the FAK/Src and PI3k/Akt signaling pathways. Mechanistically, ubiquitination assays and rescue experiments showed that PITX1 interacted with transcription factor STAT3, leading to decreased STAT3 transcriptional activity, which repressed the expression of LINC00662. Specific knockdown of LINC00662 reduced the tumor growth and invasion of OS cells induced by downregulated PITX1. Moreover, exosomal LINC00662, derived from PITX1 knockdown OS cell lines activated M2 macrophages in cell co-culture assays. M2 macrophage secreted several cytokines, among which CCL22 was found to cause OS cell EMT. Collectively, our data indicate that PITX1 suppresses OS cell proliferation and metastasis by downregulating LINC00662. Moreover, LINC00662 can be packaged into OS cell-derived exosomes to mediate M2 macrophage polarization to promote OS metastasis via CCL22.

Erratum: H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts: Erratum.

[This corrects the article DOI: 10.7150/thno.51245.].

The miR-19b-3p-MAP2K3-STAT3 feedback loop regulates cell proliferation and invasion in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most refractory malignancies worldwide. Mitogen-activated protein kinase 3 (MAP2K3) has a contradictory role in tumor progression, and the function and expression patterns of MAP2K3 in ESCC remain to be determined. We found that MAP2K3 expression to be downregulated in ESCC, and MAP2K3 downregulation correlated with clinically poor survival. MAP2K3 inhibited ESCC cell proliferation and invasion in vitro and in vivo. MAP2K3 suppressed STAT3 expression and activation. Mechanistically, MAPSK3 interacted with MDM2 to promote STAT3 degradation via the ubiquitin-proteasome pathway. Furthermore, exosomal miR-19b-3p derived from the plasma of patients with ESCC could suppress MAP2K3 expression to promote ESCC tumorigenesis. STAT3 was found to bind to the MIR19B promoter and increased the expression of miR-19b-3p in ESCC cells. In summary, our results demonstrated that the miR-19b-3p-MAP2K3-STAT3 feedback loop regulates ESCC tumorigenesis and elucidates the potential of therapeutically targeting this pathway in ESCC.

H3K27 acetylation activated-COL6A1 promotes osteosarcoma lung metastasis by repressing STAT1 and activating pulmonary cancer-associated fibroblasts.

Background: Collagen type VI alpha 1 (COL6A1) has been found to be dysregulated in several human malignancies. However, the role of COL6A1 in osteosarcoma (OS) progression remains largely unclear. Here, we aimed to explore the clinical significance and biological involvement of COL6A1 in the OS cell migration and invasion. Material and Methods: We used immunohistochemistry, qRT-PCR and western blot to detect the expression of COL6A1 in 181 OS patient samples. Chromatin immunoprecipitation (ChIP) and PCR were carried out to verify the regulatory interaction of p300, c-Jun and COL6A1 promoter. The invasion and migration function of COL6A1 in OS was detected in vitro and in vivo. RNA sequence was performed to detect the downstream pathway of COL6A1, and then co-immunoprecipitation (co-IP), ubiquitination assays and rescue experiments were performed to determine the regulatory effect of COL6A1 and signal transducers and activators of transcription (STAT1). Exosomes derived from OS cell lines were assessed for the ability to promote cancer progression by co-cultured assay and exosomes tracing. Results: COL6A1 was commonly upregulated in OS tissues, especially in lung metastasis tissues, which was associated with a poor prognosis. c-Jun bound p300 increased the enrichment of H3K27ac at the promoter region of the COL6A1 gene, which resulted in the upregulation of COL6A1 in OS. Overexpression of COL6A1 promoted OS cell migration and invasion via interacting with SOCS5 to suppress STAT1 expression and activation in an ubiquitination and proteasomal degradation manner. Most interestingly, we found that exosomal COL6A1 derived from OS cells convert normal fibroblasts to cancer-associated fibroblasts (CAFs) by secreting pro-inflammatory cytokines, including IL-6 and IL-8. The activated CAFs could promote OS cell invasion and migration by mediating TGF-ß/COL6A1 signaling pathway. Conclusion: Our data demonstrated that upregulation of COL6A1 activated by H3K27 acetylation promoted the cell migration and invasion by suppressing STAT1 pathway in OS cells. Moreover, COL6A1 can be packaged into OS cell-derived exosomes and activate CAFs to promote OS metastasis.

Room Temperature Versus Warm Irrigation Fluid Used for Patients Undergoing Arthroscopic Shoulder Surgery: A Systematic Review and Meta Analysis.

PURPOSE: The aim of this study was to analyze whether warm irrigation fluid could reduce postoperative adverse effects in patients undergoing arthroscopic shoulder surgery compared with room temperature irrigation fluid. DESIGN: A systematic review and meta-analysis of clinical trials was performed. METHODS: A computerized search of electronic databases was performed. The inclusion criteria were studies comparing the clinical effects of room temperature and warm irrigation fluid on patients undergoing arthroscopic shoulder surgery. FINDINGS: Warm irrigation fluid reduced the degree of core body temperature drop and the incidence of hypothermia. A statistically lower incidence of shivering also occurred in the warm irrigation fluid group. CONCLUSIONS: The use of warm irrigation fluid better maintains core body temperature and reduces incidence of shivering than room temperature irrigation fluid. Therefore, warm irrigation fluid is a better choice for arthroscopic shoulder surgery.

Promoter methylation and clinical significance of GPX3 in esophageal squamous cell carcinoma.

BACKGROUND: Glutathione peroxidase 3 (GPX3) provides critical protection against reactive oxygen species (ROS) in cells. Downregulation of GPX3 may contribute to carcinogenesis of esophageal squamous cell carcinoma (ESCC), but the mechanisms are not clear. MATERIALS AND METHODS: We examined the differences in gene expression between ESCC and normal esophageal epithelial, by using GEO datasets, and collected 136 ESCC tumors and adjacent normal tissues to confirm our findings. GPX3 expression was measured, at the mRNA and protein levels, by qRT-PCR, Western Blot and immunohistochemistry (IHC). RESULTS: GPX3 mRNA and protein levels were 3.3-fold higher in normal epithelium compared with case-matched ESCC tissues. There was no significant correlation between clinical parameters and expression levels of GPX3 in ESCC. Promoter methylation of the GPX3 gene correlated with decreased expression. CONCLUSION: Downregulation of GPX3 might be a key factor in the process of ESCC carcinogenesis.

STAT1 inhibits STAT3 activation in esophageal squamous cell carcinoma.

BACKGROUND: Signal transducer and activator of transcription (STAT) 1 is an important transcription factor and has been reported to be a tumor suppressor in many types of cancer. However, another STAT family member, STAT3, is considered to be an oncogene. The cross-talk between STAT1 and STAT3 in cancer has not been fully demonstrated. MATERIALS AND METHODS: Esophageal squamous cell carcinoma (ESCC) was used as a model to examine STAT1-STAT3 cross-regulation in cancer. We detected STAT1-STAT3 binding by co-immunoprecipitation (co-IP) and measured the transcription activity by using a luciferase reporter gene. DNA binding was detected by a DNA probe. Expression of STAT1 and STAT3 in ESCC was detected by immunohistochemistry. RESULTS: We found that STAT1 attenuated STAT3 activity upon oncostatin M treatment by decreasing STAT3 transcription activity and DNA binding ability of STAT3. Furthermore STAT3 downregulation increased the phosphorylation and transcriptional activation of STAT1. Finally, STAT1 expression and STAT3 expression were negatively correlated in ESCC cases. CONCLUSION: Altogether, this paper demonstrated STAT1 and STAT3 cross-regulation in ESCC and proposed that STAT3 downregulation and/or STAT1 accumulation may be a therapeutic approach to treat ESCC.

ERK is a negative feedback regulator for IFN-γ/STAT1 signaling by promoting STAT1 ubiquitination.

BACKGROUND: We recently reported that STAT1 plays a tumor suppressor role, and ERK was inversely correlation with STAT1 expression in esophageal squamous cell carcinoma (ESCC). Here, we investigated the mechanism(s) that are responsible for the ERK regulates STAT1 in ESCC. METHODS: We performed the immunoprecipitation (IP) to detect the ubiquitin of STAT1 upon MEK transfection or U0126 treatment and co-IP to confirm the binding of STAT1 and ERK in ESCC cell lines. RESULTS: We found evidence that the ubiquitin-proteasome pathway can efficiently degrade STAT1 in ESCC cells, as MG132 treatment rapidly and dramatically increased STAT1 expression in these cells. This process is not dependent on the phosphorylation of the two important STAT1 residues, Y701 and S727, as site-directed mutagenesis of these two sites did not affect STAT1 degradation. We also found that ERK promotes proteasome degradation of STAT1, supported by the observations that pharmacologic inhibition of ERK resulted in a substantial increase of STAT1 whereas expression of constitutively active ERK further reduced the STAT1 protein level. In addition to suppressing STAT1 expression, ERK limited STAT1 signaling by decreasing the production of IFNγ. CONCLUSION: To conclude, ERK is an effective negative regulator of STAT1 signaling in ESCC, by promoting its proteasome degradation and decreasing IFNγ production. Our data further supports that targeting ERK and/or STAT1 may be useful for treating ESCC.

Association of interleukin-18 gene polymorphism and its protein expression with the lower extremity deep venous thrombosis in the chinese han population: A case-control study.

OBJECTIVE: We aim to explain the correlation among IL-18 gene polymorphism, its protein expression and LEDVT in the Chinese Han population. METHODS: A total of 138 LEDVT patients and 150 healthy people volunteered as LEDVT and control groups. All the data, including the gender, age, BMI, levels of TG, LDL/HDL, TC, GLU, APTT, BUN, Cr, ALT, AST, ApoA1, ApoB, and Fg was detected. IL-18 level, IL-18 -137G/C and -607C/A polymorphism, and risk factors of LEDVT were detected using ELISA, PCR-RFLP and multivariate logistic regression analysis, respectively. RESULTS: Increased BMI, GLU, Fg, BUN, ApoB and IL-18 and decreased APTT were found in the LEDVT group. The GC + CC genotype and C allele in -137G/C polymorphism was elevated in the control group when compared to that in the LEDVT group. The IL-18 level was elevated in the case group when compared to the control group with respect to the same genotype in -607C/A and -137G/C polymorphisms, and in the LEDVT group, IL-18 level was higher in the GG genotype than that in the GC + CC genotype of -137G/C polymorphism. BUN, GG genotype and IL-18 level were independent risk factors, but APTT was a protective factor of LEDVT. CONCLUSION: On the basis of our results, we concluded that the GG genotype of -137G/C polymorphism and IL-18 level are independent risk factors of LEDVT, and IL-18 gene polymorphism affects the level of IL-18 in LEDVT patients.

STAT1ß enhances STAT1 function by protecting STAT1α from degradation in esophageal squamous cell carcinoma.

STAT1, which carries tumor suppressor functions in several models, consists of two isoforms, namely STAT1α and STAT1ß. The biological function and significance of STAT1ß has never been examined in human cancer. We examined STAT1ß function in esophageal squamous cell carcinoma (ESCC) by transfecting a STAT1ß gene into various ESCC cell lines. The interaction between STAT1α and STAT1ß was examined by using co-immunoprecipitation and confocal microscopy. The prognostic significance of STAT1ß expression, detectable by immunohistochemistry and western blot, was evaluated in a large cohort of ESCC patients. Enforced expression of STAT1ß induced and prolonged the expression and phosphorylation of STAT1α in ESCC cells, and these effects were amplified by gamma-interferon (IFN-γ). We also found that STAT1ß interacts with STAT1α and decreases STAT1α degradation by the proteasome. Moreover, STAT1ß substantially increased the DNA binding and transcription activity of STAT1. STAT1ß also sensitized ESCC cells to chemotherapeutic agents, including cisplatin and 5-flurouracil. Using western blot and immunohistochemistry, we found that STAT1ß was frequently decreased in esophageal cancer, as compared to their adjacent benign esophageal epithelial tissue. Loss of STAT1ß significantly correlated with lymph node metastasis, invasion and shorter overall survival in ESCC patients. Therefore, STAT1ß plays a key role in enhancing the tumor suppressor function of STAT1α, in ESCC, in a manner that can be amplified by IFN-γ.

Clinical significance of SPARC in esophageal squamous cell carcinoma.

BACKGROUND: Secreted protein acidic and rich in cysteine (SPARC) is matricellular protein that modulates interactions between cells and the extracellular matrix. The role of SPARC in carcinogenesis is controversialin that SPARC can be a tumor suppressor, but overexpression of SPARC is associated with poorer prognosis. METHODS: We collected 145 esophageal squamous cell carcinoma and adjacent normal tissues in Shantou, a high incidence region for esophageal cancer. The mRNA and protein expression levels of SPARC in cancer tissue and in adjacent normal mucosa were measured by qRT-PCR, western blot and immunohistochemistry (IHC). RESULTS: The mRNA and protein levels of SPARCwere5.78-fold higher in cancer tissues compared with the case-matched normal epithelium. High expression levels of SPARC in ESCC parenchyma, as detected by IHC, were related to lymph node metastasis and poor prognosis (p = 0.049 and p = 0.04). CONCLUSION: High expression of SPARC in the parenchyma may be a potential predictor of prognosis, suggesting SPARC could serve as a therapeutic target in ESCC.

ERK expression and its correlation with STAT1 in esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma is one of leading causes of cancer-related deaths in Chaoshan region a high-risk region for esophageal cancer. Extracellular regulated protein kinases (ERK) usually play an important role in cell proliferation and differentiation. However, accumulating evidence has shown that the ERK was aberrantly expressed in cancers and correlated with STAT1 depression. RESULTS: The activated ERK downregulates STAT1 expression in ESCC cell lines and U0126 increases expression of STAT1. Our immunohistochemistry result also confirms that the expression of ERK inversely correlated with that of STAT1 in ESCC tumors. In addition, a significantly higher expression of ERK/p-ERK was found in ESCC tissues in comparison with case-matched normal esophageal tissues (p < 0.05). Moreover, the immunohistochemical analysis demonstrated that ERK expression was paralleled with the differentiation and clinical stage. In 74 patients with follow-up data, those with ERKlow tumors survived significantly longer than those with ERKhigh tumors (p = 0.04); patients with ERKlow/STAT1high tumors had the longest survival (p = 0.001). MATERIALS AND METHODS: To investigate whether ERK can mediated STAT1 expression in ESCC, we used the MEK plasmid and U0126, a MEK inhibitor, to treat the cell. To further confirm our in-vitro study, we detected the ERK, p-ERK and STAT1 expression in 131 ESCC cases and 22 case-matched normal esophageal tissues adjacent to the tumors specimens. CONCLUSIONS: These findings provide pathological evidence that ERK/p-ERK is negatively correlated with STAT1 in ESCC. Our data suggests that inhibition of ERK and/or restoration of STAT1 expression maybe useful therapeutic strategies for ESCC.

Prognostic evaluation of high sensitivity-C reactive protein in peripheral T-cell lymphoma / 中国肿瘤临床

Objective:To investigate the prognostic significance of high sensitivity-C reactive protein (Hs-CRP) in patients with peripher-al T-cell lymphoma (PTCL). Methods:A total of 234 newly diagnosed PTCL patients with a median age of 48 years were analyzed retro-spectively. Serum Hs-CRP levels and other factors, including tumor stage and international prognostic index (IPI), were determined. Af-ter a median follow-up of 23 months, the relationship between Hs-CRP and overall survival (OS) was observed. Results:Serum Hs-CRP level positively correlated with IPI score (r=0.132, P<0.001), tumor stage (r=0.183, P=0.005), B symptoms (r=0.225, P=0.001), and lactic dehydrogenase (r=0.169, P=0.009), but negatively correlated with plasma albumin levels (r=?0.343, P<0.001), hemoglobin concentra-tion (r=?0.239, P<0.001), and platelet count (r=0.131, P=0.045), and is uncorrelated with age (P>0.05), gender (P>0.05), fitness score (P>0.05), and leukocyte count (P>0.05). Patients with serum Hs-CRP levels≤10 mg/L had better OS than patients with serum Hs-CRP levels>10 mg/L. Univariate and multivariate Cox regression models showed that platelet count, Hs-CRP, albumin levels, and IPI score were independent adverse prognostic factors. Conclusion:The baseline Hs-CRP level can serve as a major indicator of prognosis in PT-CL patients.

Prognostic evaluation of high sensitivity-C reactive protein in peripheral T-cell lymphoma / 中国肿瘤临床

Objective:To investigate the prognostic significance of high sensitivity-C reactive protein (Hs-CRP) in patients with peripher-al T-cell lymphoma (PTCL). Methods:A total of 234 newly diagnosed PTCL patients with a median age of 48 years were analyzed retro-spectively. Serum Hs-CRP levels and other factors, including tumor stage and international prognostic index (IPI), were determined. Af-ter a median follow-up of 23 months, the relationship between Hs-CRP and overall survival (OS) was observed. Results:Serum Hs-CRP level positively correlated with IPI score (r=0.132, P<0.001), tumor stage (r=0.183, P=0.005), B symptoms (r=0.225, P=0.001), and lactic dehydrogenase (r=0.169, P=0.009), but negatively correlated with plasma albumin levels (r=?0.343, P<0.001), hemoglobin concentra-tion (r=?0.239, P<0.001), and platelet count (r=0.131, P=0.045), and is uncorrelated with age (P>0.05), gender (P>0.05), fitness score (P>0.05), and leukocyte count (P>0.05). Patients with serum Hs-CRP levels≤10 mg/L had better OS than patients with serum Hs-CRP levels>10 mg/L. Univariate and multivariate Cox regression models showed that platelet count, Hs-CRP, albumin levels, and IPI score were independent adverse prognostic factors. Conclusion:The baseline Hs-CRP level can serve as a major indicator of prognosis in PT-CL patients.

Inhibition of lipopolysaccharide-stimulated NO production by a novel synthetic compound CYL-4d in RAW 264.7 macrophages involving the blockade of MEK4/JNK/AP-1 pathway.

In the present study, a novel synthetic compound 4-(2-(cyclohex-2-enylidene)hydrazinyl)quinolin-2(1H)-one (CYL-4d) was found to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production without affecting cell viability or enzyme activity of expressed inducible NO synthase (iNOS) in RAW 264.7 macrophages. CYL-4d exhibited parallel inhibition of LPS-induced expression of iNOS protein, iNOS mRNA and iNOS promoter activity in the same concentration range. LPS-induced activator protein-1 (AP-1) DNA binding, AP-1-dependent reporter gene activity and c-Jun nuclear translocation were all markedly inhibited by CYL-4d with similar efficacy, whereas CYL-4d produced a weak inhibition of nuclear factor-kappaB (NF-kappaB) DNA binding, NF-kappaB-dependent reporter gene activity and p65 nuclear translocation without affecting inhibitory factor-kappa B alpha (I kappa B alpha) degradation. CYL-4d had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and its upstream activator MAPK kinase (MEK) 3, whereas it significantly attenuated the phosphorylation of c-Jun, c-Jun NH(2)-terminal kinase (JNK) and its upstream activator MEK4 in a parallel concentration-dependent manner. Other Toll-like receptors (TLRs) ligands (peptidoglycans, double-stranded RNA, and oligonucleotide containing unmethylated CpG motifs)-induced iNOS protein expression were also inhibited by CYL-4d. Furthermore, the NO production from BV-2 microglial cells as well as rat alveolar macrophages in response to LPS was diminished by CYL-4d. These results indicate that the blockade of NO production by CYL-4d in LPS-stimulated RAW 264.7 cells is attributed mainly to interference in the MEK4-JNK-AP-1 signaling pathway. CYL-4d inhibition of NO production is not restricted to TLR4 activation and immortalized macrophage-like cells.