Transforming growth factor­ß/miR­143­3p/cystatin B axis is a therapeutic target in human ovarian cancer.

We previously reported that cystatin B (CSTB) is a progression marker of human ovarian cancer (OC); however, the regulatory mechanism of CSTB and its function in OC remain unclear. The present study aimed to explore the mechanism underlying transforming growth factor-ß (TGF­ß) 1­mediated CSTB regulation, and to examine the function of CSTB on OC cell proliferation and apoptosis. Using the online program, miRWalk, a microRNA (miR)­143­3p was detected, which contains a homologous sequence of the potential binding site to the 3'­untranslated region (3'­UTR) of CSTB. A dual­luciferase reporter assay confirmed the interaction between miR­143­3p and CSTB 3'­UTR. Treating OC cells with miR­143­3p mimics or inhibitors resulted in a decrease or an increase of CSTB expression at mRNA and protein levels, respectively. Additionally, CSTB was significantly overexpressed, whereas miR­143­3p was downregulated in human OC tissues compared with normal ovarian tissues. A negative correlation between miR­143­3p and CSTB mRNA expression was observed in ovarian malignant tumors. The levels of primary and mature miR­143­3p expression were upregulated in OC cells after TGF­ß1 treatment; the action of TGF­ß1 was abolished in the presence of an inhibitor of TGF­ß type I receptor. These results indicated an axis between TGF­ß, miR­143­3p and CSTB in OC cells. Furthermore, high levels of CSTB expression were associated with the poor overall survival of patients with OC. Knockdown of CSTB resulted in a decrease in OC cell proliferation and arrested cells in G2/M phase. In addition, suppression of CSTB induced cell apoptosis. In conclusion, CSTB was overexpressed and miR­143­3p was downregulated in ovarian malignant tumors. Mature miR­143­3p directly bound CSTB 3'­UTR, leading to a decrease in CSTB expression in OC cells, which was regulated by TGF­ß1. Our findings suggest the potential therapeutic application of targeting the TGF­ß/miR­143­3p/CSTB axis for treating patients with OC.

Physical interaction of STAT1 isoforms with TGF-ß receptors leads to functional crosstalk between two signaling pathways in epithelial ovarian cancer.

BACKGROUND: The signal transducer and activator of transcription (STAT) and transforming growth factor-ß (TGF-ß) signaling pathways play important roles in epithelial ovarian cancer (EOC). However, the mechanism of crosstalk between two pathways is not completely understood. METHODS: The expression of STAT1 protein was detected by tissue microarray and immunoblotting (IB). The interaction of STAT1 isoforms with TGF-ß receptors was confirmed by immunoprecipitation and IB. The effect of TGF-ß signaling on STAT1 activation was examined in EOC and non-tumorous HOSEpiC cells treated with TGF-ß1 in the presence or absence of the inhibitor of TGF-ß type I receptor. The gain-of-function and loss-of-function approaches were applied for detecting the role of STAT1 on EOC cell behaviours. RESULTS: The high level of STAT1 was observed in patients with high-grade serous EOC. STAT1 expression was higher in ovarian cancer cells than noncancerous cells. TGF-ß1 activated the STAT1 pathway by inducing the phosphorylation of STAT1α on S727 residue. The full-length STAT1α and the truncated STAT1ß directly interacted with TGF-ß receptors (ALK1/ALK5 and TßRII), which was mediated by TGF-ß1. STAT1α and STAT1ß blocked the activation of the TGF-ß1 signaling pathway in EOC cells by reducing Smad2 phosphorylation. STAT1 overexpression induced EOC cell proliferation, migration, and invasion; whereas its inhibition enhanced TGF-ß1-induced phospho-Smad2 and suppressed EOC cell proliferation, migration, and invasion. CONCLUSIONS: Our data unveil a novel insight into the molecular mechanism of crosstalk between the STAT1 and TGF-ß signaling pathways, which affected the cancer cell behavior. Suppression of STAT1 may be a potential therapeutic strategy for targeting ovarian cancer.

MALAT1 affects ovarian cancer cell behavior and patient survival.

Epithelial ovarian cancer (EOC) is one of the most lethal malignancies of the female reproductive organs. Increasing evidence has revealed that long non­coding RNAs (lncRNAs) participate in tumorigenesis. Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is an lncRNA and plays a role in various types of tumors. However, the function of MALAT1 on cellular behavior in EOC remains unclear. The current study explored the expression of MALAT1 in ovarian cancer tissues and in EOC cell lines. Quantitative RT­PCR analysis revealed that the expression of MALAT1 was higher in human ovarian malignant tumor tissues and EOC cells than in normal ovarian tissues and non­tumorous human ovarian surface epithelial cells, respectively. By analyzing the online database Kaplan­Meier Plotter, MALAT1 was identified to be correlated with the overall survival (OS) and progression­free survival (PFS) of patients with ovarian cancer. Furthermore, knockdown of MALAT1 by small interfering RNA (siRNA) significantly decreased EOC cell viability, migration, and invasion. Finally, dual­luciferase reporter assays demonstrated that MALAT1 interacted with miR­143­3p, a miRNA that plays a role in EOC as demonstrated in our previous study. Inhibition of MALAT1 resulted in an increase of miR­143­3p expression, leading to a decrease of CMPK protein expression. In conclusion, our results indicated that MALAT1 was overexpressed in EOC. Silencing of MALAT1 decreased EOC cell viability and inhibited EOC cell migration and invasion. These data revealed that MALAT1 may serve as a new therapeutic target of human EOC.

Biological Function of Ribosomal Protein L10 on Cell Behavior in Human Epithelial Ovarian Cancer.

Ribosomal protein L10 (RPL10) is one of large ribosomal proteins and plays a role in Wilms' tumor and premature ovarian failure. However, the function of RPL10 in human epithelial ovarian cancer (EOC) remains unknown. The purpose of this study was to examine the expression level and function of RPL10 in EOC. RPL10 protein expression was detected by immunohistochemistry and Western blot. The association RPL10 expression with clinical features was analyzed. Loss-of-function and gain-of-function approaches were applied in cellular assays, including cell viability, migration, invasion, and apoptosis. Our study demonstrated for the first time that RPL10 was upregulated in human EOC compared with normal ovarian tissues. Knockdown of RPL10 inhibited cell viability, migration, and invasion, and increased cell apoptosis. On the contrary, upregulation of RPL10 increased cell viability, migration, invasion, and decreased cell apoptosis. Furthermore, miR-143-3p regulated RPL10 expression. Our data indicate that RPL10 is a potential tissue biomarker of patients with EOC and may be a therapeutic target of ovarian cancer.

Overexpression of collagen type V α1 chain in human breast invasive ductal carcinoma is mediated by TGF-ß1.

Collagen type V α1 chain (COL5A1) is a minor fibrillar collagen in mammals that co-polymerizes with type I collagen to adjust the diameter of collagen molecules. However, the function of COL5A1 in invasive ductal carcinoma (IDC) of the human breast remains unknown. In the present study, our group examined the expression of COL5A1 in IDC compared with its adjacent normal tissue and fibroadenoma of the breast. COL5A1 was revealed to be overexpressed in IDC compared with benign tumor and adjacent normal control tissues, and was associated with the expression of estrogen receptor and progesterone receptor. No association between COL5A1 expression and tumor size, lymph node metastasis, clinical stage, age, or Her2 expression was identified. High expression of COL5A1 mRNA was associated with distant metastasis free survival in patients with breast cancer. Knockdown of COL5A1 led to a decrease of cell viability, as detected by the WST-1 assay, and an inhibition of migration and invasion, as detected by wound healing and Transwell assays, respectively, in the breast cancer cell line MCF-7. The expression of COL5A1 in MCF-7 cells was downregulated by transforming growth factor (TGF)­ß1, which was abolished in the presence of SB-431542, an inhibitor of TGF-ß type I receptor. In conclusion, these data indicated that COL5A1 is overexpressed in IDC and regulated by TGF-ß1, suggesting that an increase of COL5A1 reflects tumor progression and may serve as a novel biomarker and therapeutic target for the treatment of breast IDC.

Prognostic value of endoglin-assessed microvessel density in cancer patients: a systematic review and meta-analysis.

BACKGROUND: Endoglin (ENG, CD105), an auxiliary receptor for several TGF-ß superfamily ligands, is constitutively expressed in tumor microvessels. The prognostic value of ENG-assessed microvessel density (MVD) has not been systemically analyzed. This meta-analysis reviews and evaluates the association between ENG expression and prognosis in cancer patients. MATERIALS AND METHODS: Thirty published studies involving in 3613 patients were included after searching of PubMed, Web of Science, and EMBASE. The pooled hazard ratios (HRs) and 95% confidence intervals (CIs) for overall survival (OS), disease-free survival (DFS), and cancer-specific survival (CSS) were calculated using random-effects models. The publication bias was detected by a Begg's test and Egger's test. The outcome stability was verified by sensitivity analysis. RESULTS: The high ENG-assessed MVD was significantly associated with poor OS (HR = 2.14, 95% CI 1.62-2.81; P < 0.001), DFS (HR = 3.23, 95% CI 2.10-4.95; P < 0.001), CSS (HR = 3.33, 95% CI 1.32-8.37; P < 0.001). Furthermore, subgroup analysis revealed that the association between the overexpression of ENG in tumor microvessels and the outcome endpoints (OS or DFS) were also significant in the Asians and Caucasians patients with different cancer types. CONCLUSIONS: ENG of tumor microvessels is a predictor of poor OS, DFS and CSS and may be a prognostic marker of patients with cancer.

Dual Effects of N,N-dimethylformamide on Cell Proliferation and Apoptosis in Breast Cancer.

N,N-dimethylformamide (DMF) has been widely used as an organic solvent in industries. DMF is a potential medication. However, the antitumorigenic role of DMF in breast cancer remains unclear. Here, we examined dose-dependent effects of DMF on proliferation and apoptosis in breast cancer MCF-7 and nontumorous MCF-12A cells. We found that DMF had a growth inhibitory effect in MCF-12A cells in a dose-dependent manner. By contrast, however, DMF had dual effects on cell proliferation and apoptosis in MCF-7 cells. DMF at a high dose (100 mM) significantly inhibited MCF-7 cell growth while at a low dose (1 mM) significantly stimulated MCF-7 cell growth (both P < .05). The inhibitory effect of DMF on cell proliferation was accompanied by the decrease of cyclin D1 and cyclin E1 protein expression, leading to the cell cycle arrest at the G0/G1 phase. Furthermore, a high-dose DMF significantly increased the number of early apoptotic cells by increasing cleaved caspase-9 and proapoptotic protein Bax expression and decreased the ratio of Bcl-xL/Bax (P < .01). Thus, our data demonstrated for the first time that DMF has dual effects on breast cancer cell behaviors depending upon its dose. Caution must be warranted in determining its effective dose for targeting breast cancer.

Pyridoxine 5'-phosphate oxidase is a novel therapeutic target and regulated by the TGF-ß signalling pathway in epithelial ovarian cancer.

Pyridoxine 5'-phosphate oxidase (PNPO) is an enzyme that converts pyridoxine 5'-phosphate into pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 implicated in several types of cancer. However, the role of PNPO and its regulatory mechanism in epithelial ovarian cancer (EOC) are unknown. In the present study, PNPO expression in human ovarian tumour tissue and its association with the clinicopathological features of patients with EOC were examined. Further, the biological function of PNPO in EOC cells and in xenograft was evaluated. We demonstrated for the first time that PNPO was overexpressed in human EOC. Knockdown of PNPO induced EOC cell apoptosis, arrested cell cycle at G2/M phase, decreased cell proliferation, migration and invasion. Xenografts of PNPO-shRNA-expressing cells into the nude mouse attenuated tumour growth. PNPO at mRNA and protein levels in EOC cells was decreased after transforming growth factor-ß1 (TGF-ß1) treatment. The inhibitory effect of TGF-ß1 on PNPO expression was abolished in the presence of SB-431542, a TGF-ß type I receptor kinase inhibitor. Moreover, we found that TGF-ß1-mediated PNPO expression was at least in part through the upregulation of miR-143-3p. These data indicate a mechanism underlying PNPO regulation by the TGF-ß signalling pathway. Furthermore, PLP administration reduced PNPO expression and decreased EOC cell proliferation, suggesting a feedback loop between PLP and PNPO. Thus, our findings reveal that PNPO can serve as a novel tissue biomarker of EOC and may be a potential target for therapeutic intervention.

Data on the association of CMPK1 with clinicopathological features and biological effect in human epithelial ovarian cancer.

Human epithelial ovarian cancer (EOC) is the most lethal gynecological disease. However, the molecular mechanisms by which transforming growth factor-ß (TGF-ß) regulates ovarian tumor progression markers remain unclear. The present data show cytidine monophosphate kinase (CMPK) as an EOC biomarker and are related to the article entitled "Cytidine monophosphate kinase is inhibited by the TGF-ß signalling pathway through the upregulation of miR-130b-3p in human epithelial ovarian cancer" [1]. CMPK, as well as cystatin B [2] and ß-2-microglobulin [3], is overexpressed in human epithelial-type ovarian tumors. CMPK is an enzyme required for nucleic acid biosynthesis [4] and is regulated by the TGF-ß signaling pathway in EOC cells [1]. Furthermore, the data show the effect of CMPK-shRNA on EOC cell apoptosis and TGF-ß-induced Smad2 phosphorylation. CMPK expression in two EOC cell lines OVCAR-3 and SK-OV-3 is regulated by multiple miRNAs and some of these miRNAs may affect EOC chemoresistance [5].

Cytidine monophosphate kinase is inhibited by the TGF-ß signalling pathway through the upregulation of miR-130b-3p in human epithelial ovarian cancer.

Cytidine monophosphate kinase (CMPK), a member of the nucleoside monophosphate kinase family, plays an important role in the biosynthesis of nucleoside metabolism, DNA repair and tumour development. In this study, we demonstrated for the first time that CMPK was overexpressed in human ovarian epithelial borderline and malignant tumours using tissue microarray. Knockdown of CMPK significantly inhibited epithelial ovarian cancer (EOC) cell proliferation, migration and invasion. Furthermore, CMPK-shRNA inhibited PCNA, MMP-2, MMP-9 and vimentin expression, increased E-cadherin expression and arrested cell cycle at the G2/M phase. Suppression of CMPK resulted in a decrease of EOC cell microtissue formation and colony formation in vitro. Overexpression of miR-130b-3p decreased CMPK expression, whereas anti-miR-130b-3p increased CMPK expression. Moreover, TGF-ß1 inhibited the expression of CMPK, which was blocked in the presence of a TGF-ß type I receptor, SB431542, and was abolished by the inhibitor of miR-130b-3p, indicating that CMPK is regulated by the TGF-ß signalling pathway through the upregulation of miR-130b-3p. Thus, our data identify that overexpression of CMPK occurs in EOC and reveal a mechanism underlying the regulation of CMPK by the TGF-ß signalling pathway. We could consider CMPK as an EOC biomarker and targeting CMPK by decreasing its expression may be beneficial in patients with EOC.