Human epididymis protein 4 promotes P­glycoprotein­mediated chemoresistance in ovarian cancer cells through interactions with Annexin II.

The aim of the present study was to investigate the effects of human epididymis protein 4 (HE4) on drug resistance and its underlying mechanisms. The associations among proteins were detected by immunoprecipitation and immunofluorescence assays. Then, stably transfected cell lines CAOV3­HE4­L and CAOV3­A2­L expressing HE4 short hairpin (sh)RNAs and ANXA2 shRNAs, respectively, were constructed. MTT assay, immunocytochemistry, western blotting, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and flow cytometry were employed to examine drug sensitivity, as well as the expression and activity of P­glycoprotein (P­gp). HE4 and P­gp in epithelial ovarian cancer tissues were assessed via immunohistochemistry. MicroRNAs that targeted the P­gp gene, ABCB1, were predicted using bioinformatics methods, and their expression was evaluated by RT­qPCR. The common signaling pathways shared by HE4, ANXA2 and P­gp were selected by Gene Set Enrichment Analysis (GSEA). The interaction of HE4, ANXA2 and P­gp were confirmed. P­gp expression was positively associated with HE4 and ANXA2 expression, respectively. Moreover, it was observed that there was no significant rescue of P­gp expression in CAOV3­A2­L cells following the administration of active HE4 protein. In addition, the expression of HE4 and P­gp in ovarian cancer tissues of drug­resistant patients were higher compared with that of the drug­sensitive group (P<0.05). Furthermore, the results revealed that hsa­miR­129­5p was significantly increased accompanied by decreased HE4 or ANXA2 expression and P­gp expression in CAOV3­HE4­L and CAOV3­A2­L cells. GSEA analyses disclosed that HE4, ANXA2 and P­gp genes were commonly enriched in the signaling pathway involved in regulating the actin cytoskeleton. These results indicated that HE4 promotes P­gp­mediated drug resistance in ovarian cancer cells through the interactions with ANXA2, and the underlying mechanism may be associated with decreased expression of hsa­miR­129­5p and dysregulation of the actin cytoskeleton signaling pathway.

TMEFF1 overexpression and its mechanism for tumor promotion in ovarian cancer.

BACKGROUND: Transmembrane protein with epidermal growth factor-like and two follistatin-like domains 1 (TMEFF1) has an anticarcinogenic effect in brain tumors. However, little is known about the role of TMEFF1 in epithelial ovarian cancer (EOC). MATERIALS AND METHODS: TMEFF1 expression in EOC was detected by immunohistochemistry; its relationship with clinical pathological parameters and its influence on prognosis were analyzed. The MTT, scratch, Transwell assays, and flow cytometry were used to assess the malignant behavior of ovarian cancer cell. Changes in node proteins in MAPK and PI3K/AKT signaling pathways and the expression of epithelial-mesenchymal transformation markers were measured by Western blot. The regulatory effect of p53 on TMEFF1 was verified by chromatin immunoprecipitation (ChIP) assay and Western blot. RESULTS: TMEFF1 expression was higher in the EOC group than in the borderline and benign tumor groups and normal ovary group; its high expression was significantly related to International Federation of Gynecology and Obstetrics stage (P=0.024) and independently predicted shorter overall survival (P<0.01). TMEFF1 overexpression in ovarian cancer cells induced increased cellular proliferation, migration, and invasion but reduced apoptosis. In addition, the percentage of phosphorylated node proteins in MAPK and PI3K/AKT signaling pathways increased significantly. The expression of E-cadherin decreased but that of vimentin and N-cadherin increased. After the addition of MAPK (PD98059) and PI3K (GDC-0941) pathway inhibitors, ovarian cancer cells overexpressing TMEFF1 showed suppressed malignant behavior. TMEFF1 protein expression in an ovarian cancer cell lines (CAOV3 and ES-2) was downregulated after the inhibition of TP53. The transcription factor, p53, bound the promoter region of the TMEFF1 gene according to ChIP. CONCLUSION: TMEFF1 is a carcinogenic gene in ovarian cancer and can be regulated by p53 transcription. Through MAPK and PI3K/AKT signaling pathways, TMEFF1 promotes the malignant behavior in EOC. Therefore, TMEFF1 may be considered as a potential therapeutic target for ovarian cancer.

Lewis Y regulates signaling molecules of the transforming growth factor ß pathway in ovarian carcinoma-derived RMG-I cells.

LeY (Lewis Y) is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of LeY is frequently observed in epithelial-derived cancers and is correlated to pathological staging and prognosis. To study the role of LeY on cancer cells, a stably LeY-overexpressing cell line, RMG-I-H, was developed previously by transfection of the α1,2-fucosyltransferase gene, a key enzyme that catalyzes the synthesis of LeY, into ovarian carcinoma-derived RMG-I cells. Our studies have shown that LeY is involved in the changes in biological behavior of RMG-I-H cells. However, the mechanism is still largely unknown. In this study, we determined the structural relationship and co-localization between LeY and TßRI/TßRII, respectively, and the potential cellular signaling mechanism was also investigated. We found that both TßRI and TßRII contain the LeY structure, and the level of LeY in TßRI and TßRII in RMG-I-H cells was significantly increased. Overexpression of LeY up-regulates the phosphorylation of ERK, Akt and down-regulates the phosphorylation of Smad2/3. In addition, the phosphorylation intensity was attenuated significantly by LeY monoantibody. These findings suggest that LeY is involved in the changes in biological behavior through TGF­ß receptors via Smad, ERK/MAPK and PI3K/Akt signaling pathways. We suggest that LeY may be an important composition of growth factor receptors and could be an attractive candidate for cancer diagnosis and treatment.

Lewis(y) antigen stimulates the growth of ovarian cancer cells via regulation of the epidermal growth factor receptor pathway.

Lewis(y) antigen is an oligosaccharide containing two fucoses, and is expressed variously in 75% of ovarian tumors, where its high expression level predicts poor prognosis. The effect and the possible mechanism of Lewis(y) on the proliferation of human ovarian cancer cells are still largely unkown. We report here that transfecting alpha1,2-FT gene into RMG-I cells increased the expression of Lewis(y) and promoted cell proliferation. In alpha1,2-FT-transfected cells, the Lewis(y) content of EGFR was increased dramatically. Tyrosine phosphorylation of EGFR was elevated. Concomitantly, tyrosine phosphorylation of Akt, ERK1/2 was also upregulated. Moreover, the expression of HER2/neu mRNA and protein, the tyrosine phosphorylation of HER2/ neu were also elveated, while the expression of p27 was significantly reduced. However, the expression of EGFR and the relative content of Lewis(y) on HER2/neu were unchanged. The above-mentioned alterations were correlated with the Lewis(y) content of EGFR and alpha1,2-FT expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of alpha1,2-FT were attenuated significantly by the inhibitor of EGFR tyrosine kinase and by the mono-antibody to Lewis(y). Meanwhile, the reduction in p27 and the difference in its expression among the two cell lines were also blocked by the Lewis(y) antibody. The PI3K signaling pathway was more important than the MAPK pathway in the regulation of p27 expression. These findings provide strong evidence that increased expression of Lewis(y) promotes cell proliferation through regulating the phosphorylation and expression of some molecules involved in the EGFR/PI3K-signaling pathway.