NLRP12 is a prognostic biomarker and correlated with immune infiltrates in epithelial ovarian cancer.

BACKGROUND: NLRP12 is a member of the intracellular Nod-like receptor (NLR) family, suggesting it is an innate immune receptor for the initiation and progression of several cancers. However, its role on prognosis and immune infiltrates in epithelial ovarian cancer (EOC) is still unknown. The present study aimed to evaluate its prognostic value and its association with immune infiltrates in EOC. METHODS: The mRNA expression of NLRP12 of EOC from The Cancer Genome Atlas (TCGA) was analyzed. The association between NLRP12 and clinicopathological characters was evaluated with logistic regression. The association between NLRP12 expression and survival was analyzed by Cox regression and Kaplan-Meier analyses. A nomogram was used to predict the impact of NLRP12 on prognosis. Gene Ontology term analysis and gene set enrichment analysis (GSEA) were performed to identify the signaling pathways related to NLRP12 expression. Immune cells infiltration for NLRP12 was analyzed using single-sample GSEA. The relationship between NLRP12 and tumor-infiltrating immune cells (TICs) was investigated by a Wilcoxon rank sum test. The expression of NLRP12 were also further verified in EOC tissues and cell lines. Additionally, we confirmed the biological function of NLRP12 in vitro. RESULTS: NLRP12 was highly expressed in patients with EOC from TCGA. High NLRP12 expression correlated with poor disease-specific survival (p < 0.001) and overall survival (p < 0.001). Multivariate analysis revealed that NLRP12 expression was an independent prognostic marker for overall survival (p = 0.042). The C-indexes and calibration plots of the nomogram based on multivariate analysis indicated an effective predictive performance for EOC patients. GSEA showed enrichment of cell adhesion, tumorigenesis and immune response in the NLRP12 high expression group. Increased NLRP12 expression correlated positively with several TICs, including macrophages, neutrophils, T effector memory cells and immature dendritic cells (p < 0.001). In addition, NLRP12 silencing inhibited cell proliferation and migration in EOC cells. CONCLUSIONS: In conclusion, increased NLRP12 expression correlated significantly with poor survival and immune infiltration in EOC.

PTTG1IP (PBF) is a prognostic marker and correlates with immune infiltrate in ovarian cancer.

OBJECTIVE: An oncogenic protein, pituitary tumor transforming gene 1 binding factor (PTTG1IP, also called PBF), has been found to be expressed in various cancers. However, few studies have explored its prognostic significance and biologic function in epithelial ovarian cancer (EOC). METHODS: Based on the Cancer Genome Atlas (TCGA) database, this study determined the differential expression of PBF at the mRNA level in EOC and normal tissues, which was then verified using real-time PCR and western blotting. Moreover, the Kaplan-Meier method and the Cox regression method were adopted to assess the clinical value of PBF in EOC. A nomogram model was constructed to evaluate the prognostic performance of PBF in EOC. Gene set enrichment analysis (GSEA) was employed to evaluate the signaling and pathway enrichment of PBF in EOC. The association between PBF expression and tumor-infiltrating immune cells (TIICs) in EOC was examined by single-sample GSEA and TIMER. RESULTS: PBF was significantly higher in EOC than normal tissues as shown through TCGA database, and this result was verified by qRT-PCR and western blotting of EOC tissues and different cell lines. High PBF was associated with tumor size and lymphatic metastasis status. Kaplan-Meier (KM) analysis indicated that high PBF expression correlated with poor prognosis in patients with EOC (P < 0.0001). Moreover, multivariate Cox regression analysis was used to verify that PBF is an independent prognostic factor for EOC. The nomogram model exhibited moderate predictive accuracy and clinical utility in predicting EOC prognosis. The GSEA revealed that the expression of signaling pathways, such DNA damage replication, p53 pathway, Akt phosphorylation pathway, and estrogen-dependent nuclear pathway, were increased in the phenotype with high PBF expression. PBF expression was associated with neutrophil cells, iDC cells, NK cells, and Tem cells. CONCLUSION: As a prognostic biomarker for EOC, PBF was found to be correlated with immune infiltration, and may therefore be a promising target for immunotherapy for EOC.

Single-Cell Dissection of the Multiomic Landscape of High-Grade Serous Ovarian Cancer.

High-grade serous cancer (HGSC) is the most common subtype of ovarian cancer. HGSC is highly aggressive with poor patient outcomes, and a deeper understanding of HGSC tumorigenesis could help guide future treatment development. To systematically characterize the underlying pathologic mechanisms and intratumoral heterogeneity in human HGSC, we used an optimized single-cell multiomics sequencing technology to simultaneously analyze somatic copy-number alterations (SCNA), DNA methylation, chromatin accessibility, and transcriptome in individual cancer cells. Genes associated with interferon signaling, metallothioneins, and metabolism were commonly upregulated in ovarian cancer cells. Integrated multiomics analyses revealed that upregulation of interferon signaling and metallothioneins was influenced by both demethylation of their promoters and hypomethylation of satellites and LINE1, and potential key transcription factors regulating glycolysis using chromatin accessibility data were uncovered. In addition, gene expression and DNA methylation displayed similar patterns in matched primary and abdominal metastatic tumor cells of the same genetic lineage, suggesting that metastatic cells potentially preexist in the subclones of primary tumors. Finally, the lineages of cancer cells with higher residual DNA methylation levels and upregulated expression of CCN1 and HSP90AA1 presented greater metastatic potential. This study characterizes the critical genetic, epigenetic, and transcriptomic features and their mutual regulatory relationships in ovarian cancer, providing valuable resources for identifying new molecular mechanisms and potential therapeutic targets for HGSC. SIGNIFICANCE: Integrated analysis of multiomic changes and epigenetic regulation in high-grade serous ovarian cancer provides insights into the molecular characteristics of this disease, which could help improve diagnosis and treatment.

FGA Controls VEGFA Secretion to Promote Angiogenesis by Activating the VEGFR2-FAK Signalling Pathway.

Background: Our previous work revealed the high expression of fibrinogen alpha chain (FGA) in patients with endometriosis (EM) and that it could promote the migration and invasion of endometrial stromal cells. Angiogenesis is the key condition for the development of EM. This study was aimed to elucidate the role of FGA in endometrial stromal cells involved in angiogenesis in EM. Methods: Immunohistochemistry was used to detect the microvessel density (MVD) and VEGF expression in the eutopic endometrium samples from EM and non-EM. The conditioned medium (CM) of human primary eutopic endometrial stromal cells (EuESC) and immortalized endometrial stromal cell line hEM15A with FGA knockdown were collected and used to treat human umbilical vein endothelial cells (HUVECs). Then, tube formation assay, EdU assay, wound assay, transwell assay and flow cytometry assays were performed to assess the function of HUEVCs in vitro. The angiogenic capability of HUVECs was further measured using a matrigel plug assay with BALB/c nude mice in vivo. Immunofluorescence was used to detect the expression of F-actin and VE-cadherin. RT-PCR and western blotting were used to detect the expression of angiogenesis-related factors in endometrial stromal cells and downstream signalling pathways in HUVECs. Results: MVD and VEGF expression in the eutopic endometrium of EM patients were significantly higher than those in the normal endometrium of non-EM patients, and the increased MVD in EM indicates an increased risk of recurrence. Functionally, we found that CM of endometrial stromal cells with FGA knockdown could inhibit HUEVCs migration and tube formation in vitro and in vivo, while having no significant effect on HUVECs proliferation, apoptosis and cell cycle. Mechanically, the expression of VEGFA, PDGF, FGF-B, VEGF, MMP-2 and MMP-9 was reduced in hEM15A cells with FGA knockdown. CM of hEM15A cells with FGA knockdown reduced the number of microfilaments and pseudopodia, as well as the expression of VE-cadherin, and inhibited the activity of VEGFR2 and the FAK signalling pathway in HUVECs. Conclusion: Our study demonstrated FGA could enhance the interaction between endometrial stromal cells and HUVECs via the potential VEGA-VEGFR-FAK signalling axis and promote EM angiogenesis, revealing a promising therapeutic approach for EM.

Identification of the Immune Signatures for Ovarian Cancer Based on the Tumor Immune Microenvironment Genes.

Ovarian cancer (OV) is a deadly gynecological cancer. The tumor immune microenvironment (TIME) plays a pivotal role in OV development. However, the TIME of OV is not fully known. Therefore, we aimed to provide a comprehensive network of the TIME in OV. Gene expression data and clinical information from OV patients were obtained from the Cancer Genome Atlas Program (TCGA) database. Non-negative Matrix Factorization, NMFConsensus, and nearest template prediction algorithms were used to perform molecular clustering. The biological functions of differentially expressed genes (DEGs) were identified using Metascape, gene set enrichment analysis (GSEA), gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The copy number variations (CNVs), single nucleotide polymorphisms (SNPs) and tumor mutation burden were analyzed using Gistic 2.0, R package maftools, and TCGA mutations, respectively. Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data and CIBERSORT were utilized to elucidate the TIME. Moreover, external data from the International Cancer Genome Consortium (ICGC) and ArrayExpress databases were used to validate the signature. All 361 samples from the TCGA OV dataset were classified into Immune Class and non-Immune Class with immune signatures. By comparing the two classes, we identified 740 DEGs that accumulated in immune-related, cancer-related, inflammation-related biological functions and pathways. There were significant differences in the CNVs between the Immune and non-Immune Classes. The Immune Class was further divided into immune-activated and immune-suppressed subtypes. There was no significant difference in the top 20 genes in somatic SNPs among the three groups. In addition, the immune-activated subtype had significantly increased proportions of CD4 memory resting T cells, T cells, M1 macrophages, and M2 macrophages than the other two groups. The qRT-PCR results indicated that the mRNA expression levels of RYR2, FAT3, MDN1 and RYR1 were significantly down-regulated in OV compared with normal tissues. Moreover, the signatures of the TIME were validated using ICGC cohort and the ArrayExpress cohort. Our study clustered the OV patients into an immune-activated subtype, immune-suppressed subtype, and non-Immune Class and provided potential clues for further research on the molecular mechanisms and immunotherapy strategies of OV.

Preliminary Safety and Potential Effect of 6B11-OCIK Adoptive Cell Therapy Against Platinum-Resistant Recurrent or Refractory Ovarian Cancer.

Ovarian cancer is a leading cause of death among gynecological malignancies, and novel therapies are urgently needed. Here we report preliminary findings on the potential safety and efficacy of 6B11-OCIK, an adoptive cell therapy of autologous T cells induced by the humanized anti-idiotypic antibody 6B11 minibody plus dendritic cells and cytokines, against platinum-resistant recurrent or refractory ovarian cancer in three patients. We found that 6B11-OCIK treatment was safe and well tolerated after five cycles of intravenous infusion with an initial dose of 1-2×109 cells and a dose-climbing strategy. Hemoglobin, platelets, white cell count, creatinine or liver enzyme values, coagulation function, kidney and heart function were not significantly affected over the duration of therapy. Two of the three enrolled patients showed potentially drug-related grade 1 and 2 weakness, and no other adverse events were observed. Of the three enrolled patients, one had stable disease and two showed disease progression. The patient with favorable clinical efficacy had better immune response as measured by 6B11-OCIK proliferation capacity, activation ability of CD3+CD8+ tumor-specific cytotoxic T lymphocytes and CD3+CD56+ cytokine-induced killer cells, and tumor cell killing efficiency. Changes in circulating tumor cells after treatment were consistent with serum level CA125 in the patient with stable disease (both decreased), while differences were observed in the two patients with disease progression (increased CA125 in both and decreased CTC in the patient with better immune response), suggesting that variation of circulating tumor cells was more consistent with immune response and reflected efficacy directly. This preliminary study suggested that autologous 6B11-OCIK treatment was safe and had potential clinical efficacy against ovarian cancer. Patients with better immune response had more favorable efficacy. In addition to imaging, CA125 and immunophenotypes, CTC monitoring may represent a potential indicator of immunotherapy response.

Ovarian endometrioid carcinoma and clear cell carcinoma: A 21-year retrospective study.

OBJECTIVE: This study aimed to identify the clinical characteristics of Chinese patients with ovarian endometrioid carcinoma (EC) and clear cell carcinoma (CCC) and to assess the impact of concurrent endometriosis on this group. METHODS: The present study reviewed the medical records of patients who received initial treatment and a postoperative pathological diagnosis of EC or CCC at our center in China between 1998 and 2018. RESULTS: Of 211 patients, 73 had pure EC, and 91 had pure CCC, and the remaining 47 had mixed cancer. The proportion of EC and CCC remained stable over past 21 years. The proportion of EC declined with aging and the age of EC onset to incline to the young. And the age of CCC onset had two peaks, namely, 36 and 77 years. After review by the pathologist, the number of endometriosis cases found in the pathological section of the analysis increased to 114, accounting for 54% of patients. As the stage progressed, the appearance of endometriosis became increasingly scarce in pathological sections(p = 0.001). Compared with CCC, EC had a higher frequency of concurrent endometrial cancer (independent endometrial lesions) and estrogen and progesterone receptor expression(p = 0.000). And more patients were in premenopausal state in EC group(p = 0.040). In the pure group, multivariate analysis showed that correlation existed between relevance to endometriosis and worse outcomes(p = 0.041). In patients with mixed cancer, mixed endometrioid histology was associated with better survival than other subtypes, even with stage III or poorly differentiated tumors(p = 0.001). CONCLUSIONS: CCC and EC which are common in ovarian cancer patients who have associated with endometriosis have distinct clinicopathological characteristics. Attention should be paid to ovarian cancer patients with a history of endometriosis and those with concurrent endometriosis in pathological sections.

Tumor-derived exosomal circRNA051239 promotes proliferation and migration of epithelial ovarian cancer.

Recent studies have shown the involvement of exosomes in intercellular communication during tumor progression. Circular RNAs (circRNAs) can be packaged into exosomes for extracellular communication, however, the possible effects of exosomal circRNAs in epithelial ovarian cancer (EOC) cells with high metastatic potential have been rarely studied. In this study, we identified exosomal circRNA051239 from high-metastatic ovarian cancer SKOV3.ip cells and subsequently analyzed circRNA051239 levels in both EOC tissues and exosomes derived from plasma and cells by qRT-PCR. A variety of in vitro assays were employed to observe the effects of exosomal circRNA051239 derived from high-metastatic ovarian cancer SKOV3.ip cells on low-metastatic ovarian cancer SKOV3 cells. Bioinformatics analysis and luciferase activity assays were further utilized to confirm the relationship between circRNA051239, miR-509-5p and PRSS3. As a result, circRNA051239 expression was increased in tissues and plasma exosomes from EOC patients. Moreover, si-circRNA051239-Exo (exosomes derived from circRNA051239 knockdown SKOV3.ip cells) inhibited the proliferation, migration as well as invasion of SKOV3 cells. Mechanistically, circRNA051239 functioned as a competitive endogenous RNA (ceRNA) by sponging miR-509-5p to facilitate PRSS3 expression. Exosomal circRNA051239 derived from high-metastatic ovarian cancer SKOV3.ip cells promoted the progression of low-metastatic ovarian cancer SKOV3 cells. Collectively, these outcomes implicated that higher metastatic EOC cells can confer this potential to lower metastatic potential via exosomal circRNA051239, causing enhanced proliferative, migratory and invasive capacities in recipient cells.

Fibrinogen alpha chain promotes the migration and invasion of human endometrial stromal cells in endometriosis through focal adhesion kinase/protein kinase B/matrix metallopeptidase 2 pathway†.

Fibrinogen alpha chain (FGA), a cell adhesion molecule, contains two arginyl-glycyl-aspartic acid (RGD) cell adhesion sequences. Our previous study demonstrated that FGA, as an up-regulated protein in endometriosis (EM), was closely related to disease severity and involved in the development of EM. However, the biological functions and underlying mechanism of FGA in EM have not been fully understood. To explore the roles of FGA in EM, we analyzed the effects of FGA on the biological behaviors of human primary eutopic endometrial stromal cells (EuESC). The results indicated FGA knockdown suppressed the migration and invasion ability of EuESC, which also altered the distribution of cytoskeletal filamentous and cell morphology. Western blot analysis demonstrated that knockdown of FGA attenuated the migration-related protein levels of vimentin and matrix metallopeptidase 2 (MMP-2), but not integrin subunit alpha V (ITGAV) and integrin subunit beta 3 (ITGB3). Meanwhile, integrin-linked transduction pathways were detected. We found FGA knockdown significantly suppressed the expression of focal adhesion kinase (FAK) level and protein kinase B (AKT) phosphorylation, without extracellular-signal-regulated kinase (ERK) dependent pathways. Treatment with the AKT inhibitor MK2206 or RGD antagonist highly decreased the effects of FGA on the migration and invasion of EuESC. RGD antagonist treatment strongly inhibited FAK- and AKT-dependent pathways, but not ERK pathways. Our data indicated that FGA may enhance the migration and invasion of EuESC through RGD sequences binding integrin and activating the FAK/AKT/MMP-2 signaling pathway. This novel finding suggests that FGA may provide a novel potential approach to the treatment of EM, which provides a new way to understand the pathogenesis of EM.

Near-infrared dye-labeled antibody COC183B2 enables detection of tiny metastatic ovarian cancer and optimizes fluorescence-guided surgery.

OBJECTIVE: We aimed to evaluate the ability of the fluorescent monoclonal antibody probe COC183B2-Cy7 (Cy7-conjugated COC183B2 antibody) to detect tiny metastatic lesions of ovarian cancer and thus guide precise tumor resection. METHODS: The expression of the tumor-associated antigen OC183B2 in lymph nodes and SKOV3-Luc cells was detected using immunohistochemistry and immunofluorescence. A subcutaneous mouse tumor model and an intraperitoneal ovarian cancer metastasis model were constructed using SKOV3-Luc cells. Near-infrared fluorescence (NIRF) imaging was performed to determine the imaging parameters and evaluate the ability of COC183B2-Cy7 to detect tiny metastatic lesions. RESULTS: OC183B2 was expressed in metastatic lymph nodes and SKOV3-Luc cells. NIRF imaging of the subcutaneous mouse tumor model showed that the tumor background ratio was significantly higher in the COC183B2-Cy7 group than in the control group at different time points postinjection. Biodistribution study showed that COC183B2-Cy7 did not accumulate in other organs. COC183B2-Cy7 can detect tiny metastatic lesions of ovarian cancer. The smallest intraperitoneal metastatic tumor detected by COC183B2-Cy7 was approximately 1 mm. CONCLUSIONS: COC183B2-Cy7 probe has relatively high specificity and sensitivity. Our study suggests that COC183B2-Cy7 probe is a promising diagnostic tool for the complete and accurate resection of malignant lesions in fluorescence-guided surgery.

Age and menopausal status are important factors influencing the serum human epididymis secretory protein 4 level: a prospective cross-sectional study in healthy Chinese people.

BACKGROUND: Human epididymis secretory protein 4 (HE4) is a new ovarian cancer biomarker. The factors influencing HE4 levels are not clear, and the reference data in China are limited. Here, we aim to evaluate the effects of menopause and age on HE4 levels and to provide a possible reference value for HE4 in healthy Chinese people. METHODS: A total of 2493 healthy females aged 40 years or older were recruited from March 2013 to March 2017 with the cooperation of four medical institutions across Beijing, China. The serum levels of HE4 and cancer antigen 125 (CA125) were measured by enzyme-linked immunosorbent assay. The Wilcoxon rank-sum test of variance and a stratified analysis were used to analyze the relationships among age, menopausal status, and levels of HE4 or CA125. Confidence intervals (5%-95%) were determined for reference ranges in different populations. RESULTS: There was a statistically significant difference in median HE4 levels between the post-menopausal (n = 2168) and pre-menopausal groups (n = 325) (36.46 vs. 24.04 pmol/L, Z = -14.41, P < 0.001). HE4 increased significantly with age in the post-menopausal groups (H = 408.18, P < 0.001) but not in the pre-menopausal subjects (Z = -0.43, P = 0.67). The upper 95th percentile of HE4 levels were 44.63 pmol/L for pre-menopausal women, 78.17 pmol/L for post-menopausal women, and 73.3 pmol/L for all women. In the post-menopausal population, the HE4 reference ranges were 13.15 to 47.31, 14.31 to 58.04, 17.06 to 73.51, 24.50 to 115.25, and 35.71 to 212.37 pmol/L for different age groups from forty divided by decade. The CA125 level was affected mainly by menopausal status and not age. CONCLUSIONS: Menopausal status and age were both important factors influencing the level of HE4, and age affected HE4 levels mainly in post-menopausal women. The HE4 level was higher in the post-menopausal population than in the pre-menopausal population and increased with age.

Evaluation of a novel ovarian cancer-specific fluorescent antibody probe for targeted near-infrared fluorescence imaging.

BACKGROUND: To meet clinical needs, fluorescence-guided surgery has emerged as a new technique that guides surgeons in the resection of cancerous tissue by highlighting tumour lesions during surgery. We aimed to evaluate the novel ovarian cancer-specific antibody fluorescent probe COC183B2-800 (COC183B2 conjugated with IRDye800CW) in tumour-specific imaging to determine if it can help surgeons remove malignant lesions under fluorescence guidance. METHODS: The expression of OC183B2 antigen in epithelial ovarian cancer (EOC) tissues and cell lines was determined using immunohistochemistry (IHC). Western blotting was used to verify the expression of OC183B2 in SKOV3-Luc tumours. Antibodies against OC183B2 and mouse immunoglobulin G1 (IgG1) were conjugated with IRDye800CW to develop the antibody fluorescent probes COC183B2-800 and IgG-800 (immunoglobulin G1 conjugated with IRDye800CW). A subcutaneous mouse tumour model of SKOV3-Luc cells was constructed. Bioluminescent imaging (BLI) was conducted to detect the tumour location. Near-infrared fluorescence (NIRF) imaging was performed after the mice were injected with imaging agents. The mice were sacrificed 96 h postinjection, and the biodistribution assays were performed using NIRF imaging. RESULTS: In 69 EOC patients, the total positive rate of OC183B2 in EOC tissues was 89.9% (62/69). Expression of the OC183B2 antigen was positive in SKOV3-Luc, 3AO, ES2 and A2780 cells. The OC183B2 antigen could be detected in SKOV3-Luc tumours. NIRF imaging of the COC183B2-800 probe at different doses showed a high fluorescent signal at the tumour location that was in line with the site detected by bioluminescent imaging. The tumour background ratio (TBR) was significantly higher in the COC183B2-800 group than in the IgG-800, IRDye800CW and PBS groups. The fluorescent probe COC183B2-800 is metabolized mainly through the liver and does not accumulate in other organs. CONCLUSIONS: COC183B2-800 shows effective tumour-specific targeting of EOC and is a promising diagnostic and therapeutic tool for fluorescence-guided surgery.

Evaluation of COC183B2 antibody targeting ovarian cancer by near-infrared fluorescence imaging.

OBJECTIVE: To evaluate the imaging potential of a novel near-infrared (NIR) probe conjugated to COC183B2 monoclonal antibodies (MAb) in ovarian cancer (OC). METHODS: The expression of OC183B2 antigen in OC was determined by immunohistochemical (IHC) staining using tissue microarrays with the H-score system and immunofluorescence (IF) staining of tumor cell lines. Imaging probes with the NIR fluorescent dye cyanine 7 (Cy7) conjugated to COC183B2 Mab were chemically engineered. OC183B2-positive human OC cells (SKOV3-Luc) were injected subcutaneously into BALB/c nude mice. Bioluminescent imaging (BLI) was performed to detect tumor location and growth. COC183B2-Cy7 at 1.1, 3.3, 10, or 30 µg were used for in vivo fluorescence imaging, and phosphate-buffered saline (PBS), free Cy7 dye and mouse isotype immunoglobulin G (IgG)-Cy7 (delivered at the same doses as COC183B2-Cy7) were used as controls. RESULTS: The expression of OC183B2 with a high H-score was more prevalent in OC tissue than fallopian tube (FT) tissue. Among 417 OC patients, the expression of OC183B2 was significantly correlated with the histological subtype, histological grade, residual tumor size, relapse state and survival status. IF staining demonstrated that COC183B2 specifically expressed in SKOV3 cells but not HeLa cells. In vivo NIR fluorescence imaging indicated that COC183B2-Cy7 was mainly distributed in the xenograft and liver with optimal tumor-to-background (T/B) ratios in the xenograft at 30 µg dose. The highest fluorescent signals in the tumor were observed at 96 h post-injection (hpi). Ex vivo fluorescence imaging revealed the fluorescent signals mainly from the tumor and liver. IHC analysis confirmed that xenografts were OC183B2 positive. CONCLUSIONS: COC183B2 is a good candidate for NIR fluorescence imaging and imaging-guided surgery in OC.

Aberrant expression of CHL1 gene and long non-coding RNA CHL1-AS1, CHL1-AS2 in ovarian endometriosis.

OBJECTIVE(S): CHL1 (close homologue of L1 or cell adhesion molecule L1 like), also referred as CALL, is a member of the L1 gene family of neural cell adhesion molecules and belongs to immunoglobulin superfamily. This study aims to investigate the potential correlation of the CHL1 gene and the long non-coding RNAs (lncRNAs), i.e., CHL1-AS1 and CHL1-AS2, and to validate the expression patterns of CHL1 and CHL1-AS2 in ovarian endometriosis (EM). STUDY DESIGN: Our previous microarray analyses (GSE86534) of 4 patients with ovarian EM indicated that CHL1 was the most upregulated mRNA in ectopic endometrium (EC) compared with eutopic endometrium (EU) tissues, and that its two antisense lncRNAs CHL1-AS1 and CHL1-AS2, exhibited the same expression pattern. We used a bioinformatics-based strategy to calculate the correlation among CHL1, CHL1-AS1 and CHL1-AS2. Gene set enrichment analysis (GSEA) was performed to analyze commonly enriched gene sets for CHL1-AS1 and CHL1-AS2. Using quantitative real-time polymerase chain reaction (qPCR), we examined the expression levels of CHL1 mRNA and lncRNA CHL1-AS2 in paired tissues of EC and EU from 30 EM patients and normal endometrium (NE) tissues from 27 controls using quantitative real-time polymerase chain reaction (qPCR). We also examined the expression of CHL1 protein in EC, EU and NE tissues using western blotting and immunohistochemistry (IHC). RESULTS: CHL1, CHL1-AS1 and CHL1-AS2 were significantly correlated with each other given that the Pearson correlation values were > 0.9 using bioinformatic calculation. GSEA revealed that CHL1-AS1 and CHL1-AS2 were negatively associated with the same gene set "WAMUNYOKOLI_OVARIAN_CANCER_LMP". qPCR confirmed that the CHL1 and CHL1-AS2 expression levels were significantly higher in EC tissues than in EU and NE tissues, while they were not significantly different in EU compared with NE tissues. The relative expression levels of CHL1 and CHL1-AS2 in EC compared with EU tissues were positively significantly correlated (Pearson correlation coefficient = 0.421 and P value = 0.02). Elevated expression of CHL1 protein in EC tissues was detected by western blotting. IHC revealed that CHL1 protein expression levels enhanced in ectopic endometrial glands and stroma. CONCLUSION(S): Our results indicate a significant correlation among CHL1, CHL1-AS1 and CHL1-AS2, which might be involved in the development of ovarian EM and serve as novel targets for future research.

Knockdown of long noncoding RNA CCDC144NL-AS1 attenuates migration and invasion phenotypes in endometrial stromal cells from endometriosis†.

Endometriosis (EM) is a mysterious and complicated disease that has been found to be multifactorial. Recent studies demonstrated that long noncoding RNAs (lncRNAs) play an important role in the pathogenesis of EM. However, the functional and biological mechanisms of lncRNAs in EM remain unknown. Here, we performed microarray analyses to compare the lncRNA expression profiles of four paired ectopic endometrial (EC) tissues and eutopic endometrial (EU) tissues from patients with ovarian EM. A novel lncRNA, CCDC144NL-AS1, was identified as being potentially functional. CCDC144NL-AS1 expression was upregulated in EC tissues compared to EU and normal endometrial (NE) tissues. Its expression was higher in EC tissues than in EU tissues in 86.7% (26/30) of patients with EM. Despite the lack of a significant increase according to revised American Fertility Society (rAFS) stages, approximately 60% of stage VI EM cases exhibited higher CCDC144NL-AS1 levels, many more than in the stage II-III cases. Subcellular fractionation demonstrated that CCDC144NL-AS1 was localized in the cytoplasm and nucleus of the human EM-derived immortalized endometrial stromal cell line hEM15A. CCDC144NL-AS1 depletion suppressed the migration and invasion of hEM15A cells, but exerted no effects on cell adhesion, proliferation, apoptosis, or cell cycle. Knockdown of CCDC144NL-AS1 dramatically altered the distribution of cytoskeletal filamentous actin (F-actin) stress fibers compared to the negative control group treatment. Western blot analysis revealed that knockdown of CCDC144NL-AS1 attenuated the protein levels of vimentin filaments and MMP-9, but not N-cadherin or ß-catenin. Collectively, our results suggest that CCDC144NL-AS1 might be involved in the pathogenesis of EM and provide a novel target for ovarian EM.

Low levels of ADAM23 expression in epithelial ovarian cancer are associated with poor survival.

BACKGROUND: ADAM23, a member of the disintegrin and metalloprotease (ADAM) family, has been reported to be expressed in several types of tumours. Nevertheless, the exact role of ADAM23 in epithelial ovarian cancer (EOC) remains unclear. The aim of this study was to investigate ADAM23 expression in EOC and evaluate its clinicopathological and prognostic significance. METHODS: Immunohistochemistry (IHC), western blot and real-time PCR (RT-PCR) were used to analyse ADAM23 expression in 133 EOC, 42 benign ovarian tumour and 35 healthy control samples. Moreover, we evaluated the expression of ADAM23 in both public database (Oncomine and Kaplan-Meier plotter). The association between ADAM23 expression and various clinicopathological parameters was analysed. RESULTS: The levels of ADAM23 mRNA and protein expression were significantly lower in EOC tissues than in corresponding control tissues and benign ovarian tumours, verifying results from the Oncomine databases. The loss of ADAM23 expression was significantly correlated with an advanced International Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. The IHC data in the EOC samples correlated with the RT-PCR data. Furthermore, patients with low ADAM23 expression had shorter progression-free survival (PFS) and overall survival (OS) than patients with high ADAM23 expression. The multivariate analysis indicated that ADAM23 was an independent predictor in patients with EOC. CONCLUSIONS: Our results demonstrate that ADAM23 expression is likely involved in the progression of EOC and may provide potential diagnostic and prognostic information regarding EOC.

IGHG1 promotes motility likely through epithelial-mesenchymal transition in ovarian cancer.

OBJECTIVE: Ovarian cancer (OC) is one of the leading causes of death for female cancer patients. COC166-9 is an OC-specific monoclonal antibody and we have identified immunoglobulin γ-1 heavy chain constant region (IGHG1) as its antigen. We explore the function of IGHG1 in proliferation, apoptosis and motility of OC cells further in this research. METHODS: IGHG1 expression in OC specimens was detected through immunohistochemistry. Real-time quantitative polymerase chain reaction (RT-qPCR) or western blotting assay was used to test IGHG1 expression in OC cells. Viability of OC cells was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry or western blotting assay was used to detect cell cycle and apoptosis. Cellular motility was analyzed by using transwell assay and the markers of epithelial-mesenchymal transition (EMT) were tested through immunoblots. RESULTS: Although it exerts negligible effect on the viability and apoptosis of OC cells, IGHG1 could promote migration and invasion of malignant cells in vitro. Mechanistically, IGHG1 increases the expression of N-cadherin and Vimentin while decreases E-cadherin expression. Additionally, IGHG1 expression in OC specimens is higher relative to the paired normal counterparts. Further analysis demonstrates that the increased IGHG1 expression correlates positively with the lymph node metastasis of OC. CONCLUSIONS: IGHG1 promotes the motility of OC cells likely through executing the EMT program. Increased IGHG1 expression in OC specimens is associated with the lymph node metastasis.

Diagnostic value of HE4+ circulating tumor cells in patients with suspicious ovarian cancer.

Lacking a satisfactory screening test, ovarian cancer is frequently diagnosed at a late stage, leading to poor patient outcomes. This study investigated the diagnostic value of circulating tumor cells (CTCs) in peripheral blood from patients with suspected ovarian tumors. Sixty-one women suspected of having an ovarian mass were prospectively enrolled in this study. CTCs were identified and counted using microfluidic isolation and immunofluorescent staining of CD45, HE4, and epithelial and mesenchymal (E&M) markers (epithelial cell adhesion molecule, cytokeratins, and vimentin). Thirty (49%) of the patients were diagnosed with ovarian cancer. DAPI+/E&M+/CD45-/HE4+ CTC counts were higher in these patients than in patients with benign tumors (p = 0.016). The receiver operating characteristic (ROC) curve showed that the sensitivity of CTCs was 73.3%, which was superior to that of CA125 (56.7%). In patients with elevated CA125 levels (≥35 U/ml), CTC counts still showed good specificity (86.7%). Our findings suggest the DAPI+/E&M+/CD45-/HE4+ CTC count is a useful diagnostic indicator in patients with suspected ovarian cancer.

Overexpression of GPNMB predicts an unfavorable outcome of epithelial ovarian cancer.

OBJECTIVE: Glycoprotein non-metastatic protein B (GPNMB) is a transmembrane glycoprotein that is expressed at higher levels in several malignant human tissues than those in matched normal tissues. Thus, GPNMB may serve as an attractive therapeutic target of cancer treatment. In this study, the prognostic value of GPNMB expression was examined in tumors derived from a cohort of patients with epithelial ovarian cancer (EOC). METHODS: GPNMB expression in matched formalin-fixed and paraffin-embedded tissue samples was evaluated by immunohistochemistry (IHC), whereas GPNMB mRNA expression in fresh-frozen biopsy tissues was detected using real-time quantitative PCR (qPCR). Meanwhile, the correlations of GPNMB expression with the clinical characteristics of EOC were assessed. Besides, survival data were analysed using Kaplan-Meier and Cox regression analyses, respectively. RESULTS: GPNMB expression was remarkably upregulated in EOC tissues compared with that in normal ovarian controls at both mRNA and protein levels. In addition, abundant GPNMB expression in EOC was correlated with the International Federation of Gynecology and Obstetrics (FIGO) stage (P < 0.001), residual tumor (P = 0.036), and lymph node metastasis (P = 0.004). Furthermore, results of univariate and multivariate analyses indicated that GPNMB expression level was an independent prognostic factor of the progression-free survival (PFS) and overall survival (OS) (P < 0.001 and P < 0.001, respectively) for EOC patients. CONCLUSION: Upregulated GPNMB levels in EOC patients are associated with dismal prognosis. Moreover, findings in the current study indicate that GPNMB is a potentially useful prognostic predictor of the therapeutic approaches for EOC.