HERPUD1 promotes ovarian cancer cell survival by sustaining autophagy and inhibit apoptosis via PI3K/AKT/mTOR and p38 MAPK signaling pathways.

HERPUD1 is an important early marker of endoplasmic reticulum stress (ERS) and is involved in the ubiquitination and degradation of several unfolded proteins. However, its role in tumorigenesis is seldom studied, and its role in ovarian cancer is unclear. Lewis y antigen is a tumor-associated sugar antigen that acts as an 'antenna' on the cell surface to receive signals from both inside and outside the cell. We previously reported that Lewis y can promote ovarian cancer by promoting autophagy and inhibiting apoptosis. In this study, we detect the expression of HERPUD1 and Lewis y antigens in 119 different ovarian cancer tissues, determine their relationship with clinicopathological parameters, analyze the correlation between these two proteins, and explore the related cancer-promoting mechanisms through MTT, flow cytometry, western blotting, and bioinformatics. HERPUD1 is highly expressed in ovarian cancer, especially in the early stage, and the expression of HERPUD1 and Lewis y antigen was positively correlated. After overexpression of Lewis y antigen, the expression level of HERPUD1 increased. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) analysis showed that HERPUD1 and its related genes are enriched in regulating immunity, endoplasmic reticulum stress, ubiquitin-dependent degradation, ERS-induced apoptosis, and other key signaling pathways. We also clarified the HERPUD1 network of kinases, microRNA and transcription factor targets, and the impact of HERPUD1 mutations on prognosis. In addition, HERPUD1 promotes the proliferation of ovarian cancer cells, inhibits apoptosis, affects the cell cycle, promotes the occurrence of autophagy, and inhibits EMT and PI3K/AKT/mTOR and p38MAPK pathways. Overall, HERPUD1, regulated by the expression of tumor-associated protein Lewis y, promotes cell survival in the early stages of tumors, suggesting that HERPUD1 may play an important role in the development of ovarian cancer.

TGF-ß1 fucosylation enhances the autophagy and mitophagy via PI3K/Akt and Ras-Raf-MEK-ERK in ovarian carcinoma.

Transforming growth factor-ß, a cell secretion factor of the TGF-ß superfamily, is involved in the regulation of cell proliferation, differentiation, cytoskeleton formation, migration, invasion and other biological behaviors. Autophagy and mitophagy play an important role in tumor progression by regulating self-digestion, and degradation and reuse of cells and mitochondria. In this study, changes in autophagy and mitophagy processes in ovarian cancer cells under TGF-ß1 treatment were detected via Western blot and immunofluorescence, as well as the role of fucosylation modification. Changes in mitochondrial membrane potential in response to TGF-ß1 and fucosylation were detected via immunofluorescence. The effects of TGF-ß1 and its fucosylation on autophagic flux were further determined by transient transfection of cells with Ad-mRFP-GFP-LC3 adenovirus. TGF-ß1 clearly promoted autophagy and mitophagy in ovarian cancer cells. TGF-ß1 fucosylation stimulated these regulatory effects on ovarian cancer cells via modulation of PI3K/Akt and Ras-Raf-MEK-ERK pathways through TAK1. Our collective data support the physiological significance of TGF-ß1 and provide a novel direction for targeted therapy for ovarian cancer.

Interaction of HE4 and ANXA2 exists in various malignant cells-HE4-ANXA2-MMP2 protein complex promotes cell migration.

BACKGROUND: The interaction between human epididymis protein 4 (HE4) and annexin A2 (Annexin A2) has been found in ovarian cancer. However, it is dimness whether the interaction exists in other malignant tumors. METHODS: Real-time PCR, western blotting and immunocytochemistry were used to detect mRNA and proteins expression. Co-immunoprecipitation and double-labeling immunofluorescence were used to detect the interaction among HE4, ANXA2 and MMP2. MTS assay was used to test cell proliferation. Adhesion test was used to test cell adhesion. Flow cytometry was applied to examine cell cycle. The scratch test and Transwell assay was performed to detect the migration and invasion of various malignant cell lines. RESULTS: Here we show that the overexpression of HE4 and ANXA2 in various malignant cells is a common phenomenon. HE4 and ANXA2 are co-localized in the cytoplasm and membrane of various tumor cells. ES-2 cells which had both high expression of HE4 and ANXA2 were much stronger in proliferation, adhesion, invasion, and migration than other tumor cells. HE4-ANXA2-MMP2 could form a triple protein complex. HE4 could mediate the expression of MMP2 via ANXA2 to promote cell migration progress. CONCLUSIONS: The interaction of HE4 and ANXA2 exists in various types of cancer cells. HE4 and ANXA2 can promote the proliferation, adhesion, invasion, and migration of cancer cells. HE4-ANXA2-MMP2 form a protein complex and ANXA2 plays the role of "bridge". They performed together to promote cell migration.

Gene expression profile analysis in response to α1,2-fucosyl transferase (FUT1) gene transfection in epithelial ovarian carcinoma cells.

The aim of this study was to identify differentially expressed genes (DEGs) in response to α1,2-fucosyl transferase (FUT1) gene transfection in epithelial ovarian cancer cells. Human whole-genome oligonucleotide microarrays were used to determine whether gene expression profile may differentiate the epithelial ovarian cell line Caov-3 transfected with FUT1 from the empty plasmid-transfected cells. Quantitative real-time PCR and immunohistochemical staining validated the microarray results. Gene expression profile identified 215 DEGs according to the selection criteria, in which 122 genes were upregulated and 93 genes were downregulated. Gene Ontology (GO) and canonical pathway enrichment analysis were applied, and we found that these DEGs are involved in BioCarta mammalian target of rapamycin (mTOR) pathway, BioCarta eukaryotic translation initiation factor 4 (EIF4) pathway, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in cancer. Interaction network analysis predicted genes participating in the regulatory connection. Highly differential expression of TRIM46, PCF11, BCL6, PTEN, and FUT1 genes was validated by quantitative real-time PCR in two cell line samples. Finally, BCL6 and Lewis Y antigen were validated at the protein level by immunohistochemistry in 103 paraffin-embedded ovarian cancer tissues. The identification of genes in response to FUT1 may provide a theoretical basis for the investigations of the molecular mechanism of ovarian cancer.

Human epididymis protein 4 in association with Annexin II promotes invasion and metastasis of ovarian cancer cells.

BACKGROUND: The objective of the present study was to identify human epididymis protein 4 (HE4) interacting proteins and explore the mechanisms underlying their effect on ovarian cancer cell invasion and metastasis. METHODS: HE4 interacting proteins were identified by mass spectrometry and validated by co-immunoprecipitation and pull-down assays. The scratch test, the Transwell assay and animal experiments were used to assess the invasive and metastatic abilities of ovarian cancer cells before and after transfection and HE4 protein treatment. HE4 and annexin II protein expression in epithelial ovarian tissues was detected by immunohistochemistry, and the relation between their expression levels was examined. RESULTS: Annexin II was identified as an HE4 interacting protein. HE4 and annexin II binding interaction promoted ovarian cancer cell invasion and metastasis. HE4 and annexin II expression levels were significantly higher in malignant epithelial ovarian tissues than in benign and normal epithelial ovarian tissues, and they were higher in tissues with lymph node metastases than in those without. HE4 gene interference downregulated the expression of MAPK and the FOCAL adhesion signaling pathway-associated molecules MKNK2 and LAMB2, and HE4 protein supplementation reversed this effect. CONCLUSION: The binding interaction between HE4 and annexin II activates the MAPK and FOCAL adhesion signaling pathways, promoting ovarian cancer cell invasion and metastasis.

Beclin 1 expression in ovarian tissues and its effects on ovarian cancer prognosis.

Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox's regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p<0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p<0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p=0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.

Autophagy-independent enhancing effects of Beclin 1 on cytotoxicity of ovarian cancer cells mediated by proteasome inhibitors.

BACKGROUND: The ubiquitin-proteasome system and macroautophagy (hereafter referred to autophagy) are two complementary pathways for protein degradation. Emerging evidence suggests that proteasome inhibition might be a promising approach for tumor therapy. Accumulating data suggest that autophagy is activated as a compensatory mechanism upon proteasome activity is impaired. METHOD: Autophagy activation was measured using acridine orange staining and LC3 transition. Cell viability and apoptosis were measured using MTT assay and flow cytometry, respectively. Beclin 1 expression vectors or shRNA against Beclin 1 (shBeclin 1) were transfected to investigate the role of Beclin 1 in autophagy activation and cytotoxicity of ovarian cancer cells induced by proteasome inhibitors. RESULTS: Proteasome inhibitors suppressed proliferation and induced autophagy in ovarian cancer cells. Neither phosphoinositide 3-kinase (PI3K) inhibitors nor shRNA against Beclin 1 could abolish the formation of acidic vacuoles and the processing of LC3 induced by proteasome inhibitors. Moreover, Beclin 1 overexpression enhanced anti-proliferative effects of proteasome inhibitors in ovarian cancer cells. CONCLUSIONS: For the first time, the current study demonstrated that proteasome inhibitors induced PI3K and Beclin 1-independent autophagy in ovarian cancer cells. In addition, this study revealed autophagy-independent tumor suppressive effects of Beclin 1 in ovarian cancer cells.

Lewis y regulate cell cycle related factors in ovarian carcinoma cell RMG-I in vitro via ERK and Akt signaling pathways.

OBJECTIVE: To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells. METHODS: mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot. RESULTS: Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression. CONCLUSION: Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.

Elevated levels of Lewis y and integrin α5ß1 correlate with chemotherapeutic drug resistance in epithelial ovarian carcinoma.

OBJECTIVE: To measure Lewis y and integrin α(5)ß(1) expression in epithelial ovarian carcinoma and to correlate the levels of these molecules with ovarian carcinoma chemotherapy and prognosis. METHODS: The study population included 34 ovarian carcinoma patients with chemotherapeutic drug-resistance, six partially drug-sensitive cases, and 52 drug-sensitive cases (92 total). Immunochemistry was used to determine expression of Lewis y antigen and integrin α(5)ß(1) in ovarian carcinoma tissues, and correlation of these molecules with chemotherapy resistance was further investigated, Multi-factor logistic regression analysis was applied to investigate: age, surgical stage, grade, subtype of patient cases, metastasis of lymph nodes, residual tumor size, expression levels of Lewis y antigen and integrin α(5)ß(1) correlation with ovarian carcinoma chemotherapy resistance. RESULTS: The expression rates of Lewis y antigen and integrins α(5) and ß(1) were significantly greater in the drug-resistant group (91.17%, 85.29%, 88.24%) than the partially sensitive (50.00%, 33.33%, 50.00%) or sensitive groups (61.54%, 57.69%, 55.77%). Binary logistic regression analysis revealed that surgical stage, residual tumor size, and expression of integrin α(5) and Lewis y in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance. CONCLUSIONS: Overexpression of Lewis y and integrin α(5) are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.

The stimulation of IGF-1R expression by Lewis(y) antigen provides a powerful development mechanism of epithelial ovarian carcinoma.

OBJECTIVE: This study aimed to measure and correlate the expression of insulin-like growth factor receptor-1 (IGF-1R) and the Lewis(y) antigen in ovarian cancer cell lines and tissue samples. METHODS: Reverse transcriptase PCR (RT-PCR), Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence double-labeling techniques were applied to detect and measure the expression of Lewis(y) and IGF-1R. RESULTS: In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, IGF-1R expression was significantly upregulated compared with cells that do not overexpress α1,2-FT (P < 0.05). The amount of Lewis(y) expressed on IGF-1R increased 1.81-fold in α1,2-FT-overexpressing cells (P < 0.05), but the ratio of Lewis(y) expressed on IGF-1R to total IGF-1R was unaltered between two cells (P > 0.05). In malignant epithelial ovarian tumors, the positivity rates of Lewis(y) and IGF-1R detection were 88.3% and 93.33%, respectively, which is higher than the positivity rates in marginal (60.00% and 63.33%, all P < 0.05), benign (33.00% and 53.33%, all P < 0.01), and normal (0% and 40%, all P < 0.01) ovarian samples. No correlations were detected in positivity rates of Lewis(y) or IGF-1R expression with respect to clinicopathological parameters in ovarian cancers (all P > 0.05). Both IGF-1R and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P < 0.05). CONCLUSION: Overexpression of Lewis(y) results in overexpression of IGF-1R. Both IGF-1R and Lewis(y) are associated with the occurrence and development of ovarian cancers.

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.

miR-5193, regulated by FUT1, suppresses proliferation and migration of ovarian cancer cells by targeting TRIM11.

Ovarian cancer is the most lethal gynecological malignancy worldwide. A better understanding of the pathogenesis of ovarian cancer may help to improve the overall survival. Our previous studies have demonstrated that alpha-(1,2)-fucosyltransferase 1 (FUT1) is an oncogenic glycogene in ovarian cancer. However, the underlying mechanism is not fully clarified. In this study, we identified a microRNA as an important downstream regulator for the carcinogenic effect of FUT1 in ovarian cancer. miR-5193 was found down-regulated in ovarian cancer cells, FUT1-overexpression ovarian cancer cells and ovarian tumor samples. MTT, flow cytometry and Transwell assays demonstrated that miR-5193 inhibited the proliferation and migration, and induced the cell cycle arrest and apoptosis of ovarian cancer cells. Real-time PCR and western blot assays showed that miR-5193 downregulated the expression of TRIM11 and upregulated the expression of p53 and p21. Dual luciferase reporter assay indicated that TRIM11 was a direct target of miR­5193. Rescue experiments confirmed that miR-5193 functioned in ovarian cancer cells by directly targeting TRIM11. Moreover, transfection with miR-5193 mimic in FUT1-overexpression ovarian cancer cells reversed the carcinogenic effect of FUT1. Taken together, our results suggest that miR-5193 is an essential suppressor of human ovarian cancer development, and is an important downstream regulator regarding the carcinogenesis of FUT1 in ovarian cancer.

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) antigen promotes the progression of epithelial ovarian cancer by stimulating MUC1 expression.

MUC1 is a type I transmembrane glycoprotein and is overexpressed in various epithelial tumor tissues. Some researchers have demonstrated that the glycosylation status of MUC1 can affect MUC1-mediated tumor growth and cell differentiation. In our previous study, we proved that the abilities of cell proliferation, adhesion, invasion and metastasis, and drug resistance were enhanced in ovarian cancer cells stably expressing Lewis(y). Therefore, we hypothesized that Lewis(y) antigen may play a central role in regulating MUC1 expression, and MUC1-mediated cell growth and differentiation may be closely associated with Lewis(y) antigen. This study aimed to examine the correlation between MUC1 expression and Lewis(y) antigen levels in ovarian cancer cell lines and tissue samples. A series of techniques, including RT-qPCR, western blot anlaysis, immunoprecipitation, immunohistochemistry and double-labeling immunofluorescence were applied to detect the expression of Lewis(y) and MUC1. In malignant epithelial ovarian tumors, the positive expression rates of Lewis(y) antigen and MUC1 were 88.33 and 86.67%, respectively, which were markedly higher than those in borderline (60.00 and 53.33%, P<0.05), benign (33.33 and 30%, P<0.01) and normal (0 and 25%, P<0.01) ovarian samples. There was no correlation between the positive expression rates of Lewis(y) or MUC1 and clinicopathological parameters in ovarian cancers (P>0.05). The expression levels of Lewis(y) and MUC1 correlated with the clinical FIGO stage (P<0.05). Both MUC1 and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P<0.01). In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, the gene and protein expression levels of MUC1 were significantly upregulated compared with the cells that did not overexpress α1,2-FT (P<0.05). The ratio of Lewis(y) immunoprecipitated with MUC1 to total MUC1 increased 1.55-fold in α1,2-FT-overexpressing cells (P<0.05). The overexpression of Lewis(y) resulted in the upregulation of MUC1. On the whole, our data indicate that both MUC1 and Lewis(y) are associated with the occurrence and development of ovarian cancers.

c-Fos mediates α1, 2-fucosyltransferase 1 and Lewis y expression in response to TGF-ß1 in ovarian cancer.

FUT1 is a key rate-limiting enzyme in the synthesis of Lewis y, a membrane-associated carbohydrate antigen. The aberrant upregulation of FUT1 and Lewis y antigen is related to proliferation, invasion and prognosis in malignant epithelial tumors. A c-Fos/activator protein-1 (AP-1) binding site was found in the FUT1 promoter. However, the mechanisms of transcriptional regulation of FUT1 remain poorly understood. TGF-ß1 is positively correlated to Lewis y. In the present study, we investigated the molecular mechanism of FUT1 gene expression in response to TGF-ß1. We demonstrated that c-Fos was highly expressed in 77.50% of ovarian epithelial carcinoma cases and was significantly correlated with Lewis y. Using luciferase activity and chromatin immunoprecipitation (ChIP) assay, we further revealed that c-Fos interacted with the FUT1 promoter in ovarian cancer cells and transcriptional capacity of the heterodimer formed by c-Fos and c-Jun was stronger than that of the c-Fos or c-Jun homodimers. Then, we demonstrated that TGF-ß1 induced dose-dependent c-Fos expression, which was involved in TGF-ß1-induced ovarian cancer cell proliferation. In addition, inhibition of MAPK activation or TGF-ß1 receptor by pharmacological agents prevented TGF-ß1-induced c-Fos and Lewis y expression. Silencing of c-Fos prevented TGF-ß1-induced Lewis y expression. Collectively, the results of these studies demonstrated that TGF-ß1 regulated FUT1 and Lewis y expression by activating the MAPK/c-Fos pathway.

Lewis y antigen promotes p27 degradation by regulating ubiquitin-proteasome activity.

As a tumor-associated carbohydrate antigen, elevated expression of Lewis y promotes the malignant behaviors of tumor cells. Although our preliminary study showed that the increased expression of Lewis y antigen decreased the expression of cell cycle inhibitor protein p27, the relevant mechanism remains unclear. Autophagy and the ubiquitin-proteasome system are two main ways of intracellular protein degradation, whose abnormal activities are closely associated with progression of malignant tumors. In our present study, we constructed two stable transfected cell lines with high expression of Lewis y antigen, named CAOV3-FUT1 and SKOV3-FUT1. We showed that the proportion of cells at S phase was significantly increased after FUT1 transfection, whereas p27 protein was obviously decreased. The autophagy activity, the levels of ubiquitination, and chymotrypsin-like protease activity were increased remarkably in the transfected cells. Interestingly, Lewis y antigen promoted the degradation of p27 by increasing ubiquitin-proteasome activity. In the vivo studies, Lewis y antigen improved the tumorigenic ability of ovarian cancer cells in nude mice and reduced the expression of p27. These findings suggested that Lewis y antigen activated both the autophagy and ubiquitin-proteasome activity and promoted the degradation of p27 through the ubiquitin-proteasome pathway.

Lewis Y antigen modified CD47 is an independent risk factor for poor prognosis and promotes early ovarian cancer metastasis.

CD47 is a membrane receptor that belongs to the immunoglobulin superfamily and plays an important role in the mechanisms of tumor immune escape. CD47 participates in tumor immune escape by combining with SIRPα to reduce the phagocytic activity of macrophages. There are six potential N-glycosylation sites on CD47, and glycosylation is known to be necessary for its membrane localization. However, it is still unknown to what extent glycosylation influences CD47 ligand binding properties and subsequent signaling. By using immunoprecipitation and confocal laser scanning microscopy, we showed that CD47 contains Lewis y antigen. Immunohistochemical analysis demonstrated that both the positive expression and the overexpression of CD47 and Lewis y antigen in cancer tissues and borderline tumors were significantly higher than those in benign ovarian tumors and normal ovarian tissues (P < 0.05). A linear correlation between the expression patterns of CD47 and Lewis y antigen was evident (r = 0.47, P < 0.01). The high expression of CD47 and Lewis y antigen showed significant correlations with the clinical pathological parameters of ovarian cancer [International Federation of Gynecology and Obstetrics (FIGO) standards, lymph node metastasis, and degree of differentiation] (P < 0.05). The Cox model and Kaplan-Meier tests showed that high expression of CD47 was an independent adverse risk factor for the prognosis of ovarian cancer. Cases with both high CD47 and Lewis y antigen expression had poor prognoses. Our study demonstrates that Lewis y antigens of CD47 may play a crucial role in the development of ovarian cancer, and could be new targets for immunotherapy for ovarian cancer.

The expression and correlation between the transcription factor FOXP1 and estrogen receptors in epithelial ovarian cancer.

BACKGROUND: Estrogen plays an important role in the progression of ovarian cancer in humans. FOXP1 belongs to the forkhead/winged-helix transcription factor family, and previous research indicated that FOXP1 functioned as a tumor suppressor gene. FOXP1 may be similar to FOXA1 and is closely related to steroid hormone receptors, but the relationship between FOXP1 and ER currently remains unclear. METHODS: Ovarian tumors (60 malignant cases, 26 borderline cases, and 13 benign cases) and 14 normal ovarian tissues were collected retrospectively. Immunohistochemistry, western blotting and real-time PCR were used to characterize the expression patterns of FOXP1, ERα, and ERß both at the mRNA and protein levels. We also used co-immunoprecipitation and immunofluorescent colocalization to investigate whether a correlation exists between FOXP1 and ERα/ERß in ovarian cancer tissues. RESULTS: The mRNA level for FOXP1 and ERß in ovarian carcinoma tissues decreased, while the expression level of ERα mRNA increased compared with normal ovarian tissues. With an increase in the degree of ovarian carcinoma malignancy, the ERα expression level also increased. The expression pattern of ERß in ovarian neoplasms was similar to that of the FOXP1 protein; presenting nuclear staining decreased, while cytoplasmic expression increased. Colocalization of FOXP1, ERα, and ERß was present in the cytoplasm, with ERß specific co-localization with FOXP1 in the perinuclear area. While immunoprecipitates created with FOXP1 mouse anti-human monoclonal antibody showed a positive reaction to an anti-ER antibody, immunoprecipitates containing anti-ER antibody and react to anti-FOXP1 antibody. CONCLUSION: Interactions between FOXP1 and ER may play a pivotal role in the progression of ovarian cancer, and the activation or induction of FOXP1 and ERß expression in cancer cells may inhibit tumor proliferation.

Lewis y enhances CAM-DR in ovarian cancer cells by activating the FAK signaling pathway and upregulating Bcl-2/Bcl-XL expression.

Oligosaccharides on the surface of adhesion molecules may contribute to the process of CAM-DR. To investigate the role of the Lewis y antigen in this process, we established a cell adhesion model mediated by the integrin α5ß1-FN interaction in the ovarian cancer cell line, RMG-1-hFUT, which highly expresses Lewis y by transfection with α1,2-fucosyltransferase into RMG-1 cells. Our results indicate that the rates of carboplatin-induced apoptosis and necrosis are reduced in FN-adhered tumor cells, and carboplatin resistance is significantly decreased in the presence of anti-Lewis y antibody. CAM-DR in tumor cells has been correlated with elevated expression of the nuclear anti-apoptotic proteins Bcl-2 and Bcl-XL. Lewis y promotes the expression of the Bcl-2 and Bcl-XL genes by activating the focal adhesion kinase signaling pathway and accelerating their transcription. Thus, Lewis y leads to inhibition of apoptosis and enhancement of CAM-DR by activation of the FAK signaling pathway and upregulation of Bcl-2/Bcl-XL expression in ovarian cancer cell lines.

Overexpression of Lewis y antigen promotes human epididymis protein 4-mediated invasion and metastasis of ovarian cancer cells.

To study Human epididymis protein 4 (HE4) surface fucosylation and to determine the effects and significance of Lewis y antigen on HE4-mediated invasion and metastasis of ovarian cancer cells, we investigated four types of ovarian cancer cells and found that six fucosylated antigens (Lewis y, Lewis x, Lewis a, Lewis b, sLewis a, and sLewis x) were identified on HE4 in ovarian cancer cells. Moreover, modification of the type II sugar chain (Lewis y, Lewis x, and sLewis x) was significantly higher than the type I sugar chain (Lewis a, Lewis b, sLewis a) of the lactose series. To confirm the effects of Lewis y antigen on HE4-mediated invasion and metastasis of ovarian cancer cells, the CaoV-3 cells with high Lewis y antigen on the HE4 surface and ES-2 cells, with high Lewis x antigen but low Lewis y antigen, were investigated. We found that the expression levels of HE4 and Lewis y increased in both cell lines while the level of Lewis x didn't have any change after transfection. Furthermore, the high expression of Lewis y antigen significantly enhanced the HE4-mediated invasion and metastasis of ovarian cancer cells. The invasion and metastasis capacities were significantly decreased after Lewis y antibody blocking. This study demonstrates that overexpression of the Lewis y antigen on HE4 promotes ovarian cancer cell invasion and metastasis, which is likely to be used as a target for the clinical treatment of ovarian cancer.