Exosome-derived miRNAs and ovarian carcinoma progression.

The objective of this study was to analyze the expression, biological role and clinical relevance of exosomal microRNAs (miRNAs) from ovarian carcinoma (OC) effusion supernatants. Exosomal miRNA expression profiling was performed using miRNA Taqman arrays. Selected miRNAs were validated using quantitative PCR in 86 OC effusion supernatants. The role of exosomal miRNA in this cancer was further studied using in vitro and in vivo models. miRNA profiling identified 99 miRNAs with high expression levels in exosomes from OC effusion supernatants. Quantitative PCR validation of 11 miRNAs showed significant associations with effusion site (peritoneum versus pleura) and International Federation of Gynecology and Obstetrics stage. In univariate survival analysis, high levels of miRNAs 21, 23b and 29a were associated with poor progression-free survival (P = 0.01, P = 0.015 and P = 0.009, respectively), whereas high expression of miRNA 21 correlated with poor overall survival (P = 0.017). The latter association was retained in Cox multivariate analysis (P = 0.001). Exposure of LP9 mesothelial cells and ES2 OC cells to OC effusion-derived exosomes inhibited tumor spheroid expansion and reduced mesothelial clearance area. Treatment of severe combined immunodeficiency mice with exosomes from OC effusions prior to injection of tumor cells was associated with larger tumor load, more infiltrative tumors and shorter survival. Patient-derived OC effusion exosomes contain multiple miRNAs, of which some may have clinical relevance. In experimental models, OC exosomes affect both tumor cells and cells in the tumor microenvironment and induce more aggressive disease. Collectively, these data demonstrate the central role of miRNAs and their content in the biology of this cancer.

HOXB8 expression in ovarian serous carcinoma effusions is associated with shorter survival.

OBJECTIVE: HOX proteins are key transcription factors in embryogenesis. HOXB5 and HOXB8 were previously shown to be overexpressed in ovarian/primary peritoneal serous carcinoma compared to breast carcinoma using gene expression arrays. The present study investigated the clinical role of HOXB5 and HOXB8 in advanced-stage (FIGO III-IV) ovarian serous carcinoma. METHODS: HOXB5 and HOXB8 protein expression was analyzed in 286 effusions and 76 patient-matched solid lesions (27 primary carcinomas, 49 metastases) using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including survival. RESULTS: Cytoplasmic HOXB5 protein was detected in 268/286 (94%) effusions. HOXB8 was expressed at both the cytoplasm (252/286; 88%) and nucleus (131/286; 46%) of carcinoma cells. Cytoplasmic HOXB5, cytoplasmic HOXB8 and nuclear HOXB8 were found in 56/76 (74%), 76/76 (100%) and 30/76 (39%) solid lesions, respectively, with significantly higher HOXB5 expression in effusions (p=0.002) and higher cytoplasmic HOXB8 in solid lesions (p<0.001). HOXB5 expression was higher in post-chemotherapy disease recurrence effusions compared to pre-chemotherapy effusions tapped at diagnosis (p=0.04). In univariate survival analysis of the effusion cohort, higher expression of cytoplasmic HOXB8 was associated with significantly shorter progression-free survival (p=0.033), whereas higher nuclear HOXB8 expression was associated with significantly shorter overall survival in analysis limited to patients with post-chemotherapy effusions (p=0.036). Neither finding was independent prognostic factor in Cox multivariate analysis. CONCLUSIONS: HOXB5 and HOXB8 are frequently expressed in ovarian serous carcinoma, with anatomic site-related differences for cytoplasmic staining. HOXB5 may be affected by chemotherapy in effusions. HOXB8 expression is associated with shorter survival in metastatic serous carcinoma.

miRNA profiling along tumour progression in ovarian carcinoma.

MicroRNAs (miRNAs) are small non-coding RNAs that exert a regulatory effect post-transcriptionally by binding target mRNAs and inhibiting gene translation. miRNA expression is deregulated in cancer. The aim of this study was to characterize the differences in miRNA expression pattern and the miRNA-regulating machinery between ovarian carcinoma (OC) cells in primary tumours versus effusions. Using miRNA array platforms, we analysed a set of 21 tumours (13 effusions, 8 primary carcinomas) and identified three sets of miRNAs, one that is highly expressed in both primary carcinomas and effusions, one overexpressed in primary carcinomas and one overexpressed in effusions. Levels of selected miRNAs were analysed using quantitative PCR in an independent set of 45 additional tumours (30 effusions, 15 primary carcinomas). Reduced miR-145 and miR-214 and elevated let-7f, miR-182, miR-210, miR-200c, miR-222 and miR-23a levels were found in effusions in both sets. In silico target prediction programs identified potential target genes for some of the differentially expressed miRNAs. Expression of zinc finger E-box binding homeobox (ZEB)1 and c-Myc, targets of miR-200c, as well as of p21 protein (Cdc42/Rac)-activated kinase (PAK)1 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), predicted targets of miR-222, were analysed. Inverse correlations between expression levels of the indicated miRNAs and of the predicted target genes were found. In addition, higher expression of the miRNA-processing molecules Ago1, Ago2 and Dicer was observed in effusions compared to primary carcinomas. In conclusion, our data are the first to document different miRNA expression and regulation profiles in primary and metastatic OC, suggesting a role for these molecules in tumour progression.

Calreticulin expression is reduced in high-grade ovarian serous carcinoma effusions compared with primary tumors and solid metastases.

The objective of this study was to analyze the expression and clinical role of calreticulin, a multifunctional Ca(2+)-binding chaperone of the endoplasmic reticulum, in advanced-stage high-grade serous ovarian carcinoma. Cellular calreticulin messenger RNA (mRNA) and protein expression was investigated in 102 and 56 tumors, respectively, using reverse transcriptase polymerase chain reaction and Western blotting. Secreted calreticulin level was further analyzed in 31 effusion supernatants. Results were analyzed for association with anatomical site and clinicopathologic parameters, including survival. Calreticulin mRNA and protein were detected in 101 of 102 and 55 of 56 tumors, respectively. Calreticulin mRNA was overexpressed in solid metastases (n = 15) compared with effusions (n = 55) and primary carcinomas (n = 32; P = .009), whereas protein expression was significantly higher in solid metastases and primary carcinomas compared with effusion specimens (P = .007). Secreted calreticulin levels were higher in peritoneal compared with pleural effusions (P = .02). Higher cellular calreticulin protein expression in effusions was associated with better response to chemotherapy at diagnosis (P = .037). Calreticulin mRNA and protein expression was unrelated to patient survival. In conclusion, calreticulin is frequently expressed in serous ovarian carcinoma cells at all anatomical sites, but expression is reduced in effusions. Calreticulin protein levels in effusions may be predictive of chemotherapy response at diagnosis.

Alternatively spliced lysyl oxidase-like 4 isoforms have a pro-metastatic role in cancer.

We previously found LOXL4 to be alternatively spliced in an anatomic site-specific manner in tumors involving the serosal cavities. LOXL4 splice variants were predominantly or exclusively expressed in effusion specimens from ovarian and breast carcinoma patients, and were absent in primary carcinomas. In the present study, LOXL4 full-length or splice variants were overexpressed in ES-2 and MDA-MB-231 cells and their invasive and metastatic potential and microRNA expression profile were evaluated. ES-2 cells were further injected into SCID mice ovaries and the extent of tumor progression and metastases formation were compared. We show that both splice variants have a positive effect on the metastatic potential of cells in vitro and on tumor progression in vivo. In contrast, full-length LOXL4 is not pro-metastatic, and may even be considered as a tumor suppressor. In addition, we show that LOXL4 is a possible splicing target of the oncogenic splicing factors SRSF1 and hnRNP A1. In conclusion, our results point to a significant role for LOXL4 alternative splicing in tumor progression.

Argonaute, Dicer, and Drosha are up-regulated along tumor progression in serous ovarian carcinoma.

MicroRNAs are posttranscriptional regulators of messenger RNA synthesis that are intracellularly processed and transferred by the microRNA-regulating machinery consisting of Drosha, Dicer, and Argonaute. The present study analyzed the expression and clinical role of the microRNA-regulating machinery in advanced-stage ovarian carcinoma. Drosha, Dicer, Argonaute 1, and Argonaute 2 messenger RNA levels were analyzed in 144 specimens (82 effusions, 33 primary carcinomas, and 29 solid metastases) using quantitative polymerase chain reaction. Dicer, Argonaute 1, and Argonaute 2 protein levels were analyzed in 103 of the above specimens by Western blotting. Argonaute 1, Argonaute 2, and Drosha messenger RNAs were overexpressed in effusions compared with primary carcinomas and solid metastases (P<.001), whereas Argonaute 1 protein expression was highest in solid metastases (P=.004). Significantly higher expression of all 4 messenger RNAs was found in effusions compared with primary carcinomas (P<.001 to P=.006), whereas Argonaute 2 messenger RNA (P=.002), Drosha messenger RNA (P=.009), and Dicer protein (P=.006) were overexpressed in solid metastases compared with primary carcinomas. Drosha, Dicer, Argonaute 1, and Argonaute 2 messenger RNAs and protein levels in effusions were unrelated to clinicopathologic parameters. In primary carcinomas, higher levels of 3 messenger RNAs were significantly associated with high-grade histology (P=.003 for Dicer and P=.01 for Drosha and Argonaute 1). Higher Argonaute 2 messenger RNA levels in prechemotherapy effusions were related to shorter progression-free survival (P=.049), a finding that retained its significance in multivariate Cox analysis (P=.046). In conclusion, Drosha, Dicer, Argonaute 1, and Argonaute 2 are differentially expressed at different metastatic sites in ovarian carcinoma compared with primary carcinomas, suggesting a role for these molecules in tumor progression. Their clinical role in metastatic ovarian carcinoma merits further research.

Orally active, antimetastatic, nontoxic diphenyl ether-derived carbamoylphosphonate matrix metalloproteinase inhibitors.

Seven 4-phenoxybenzenesulfonamidopolymethylene carbamoylphosphonates (CPOs) bearing two to eight methylene units in the polymethylene chain were synthesized and evaluated as matrix metalloproteinase (MMP) inhibitors. The five lowest homologues [(CH2)2-6] are selective MMP-2 inhibitors, whereas the two with the longest linkers [(CH2)7,8] lack inhibitory activity. The most potent homologues are those with (CH2)5,6; these two were evaluated for antimetastatic activity in a murine melanoma model and showed good potency both by oral and intraperitoneal administration without any toxic--including musculoskeletal--side effects. In contrast to the previously reported cis-ACCP, which was shown to inhibit MMP-2 for ∼30 min, the new compounds inhibit MMP activity for the duration of measurement, lasting several hours. Pharmacokinetic evaluation revealed, on the one hand, low oral bioavailability; on the other hand, a relatively large calculated volume of distribution, consistent with the observed reversible absorption of CPO 5 to hydroxyapatite, as a model for bone.

Mesenchymal-to-epithelial transition determinants as characteristics of ovarian carcinoma effusions.

The present study investigated the intracellular regulation of E-cadherin in ovarian carcinoma. E-cadherin expression and regulation by Snail and Pak1 were studied in ES-2 and OVCAR-3 ovarian cancer cells in vitro. Twist1, Zeb1 and Vimentin mRNA expression and HIF-1alpha protein expression were analyzed in 80 and 189 clinical specimens, respectively. OVCAR-3 cells incubated with an anti-E-cadherin antibody formed smaller and looser spheroids compared to controls. Snail silencing using Small Hairpin RNA in ES-2 cells reduced invasion and MMP-2 activity, with unaltered cellular morphology. Using dominant negative (DN) and constitutively active (CA) Pak1 constructs, we found that DN Pak1 ES-2 and OVCAR-3 clones had reduced attachment to matrix proteins, invasion and MMP-2 activity compared to CA and wild-type cells. DN Pak1 ES-2 cells also bound less to LP9 mesothelial cells. DN Pak1 OVCAR-3 cells had lower Vimentin levels. Snail expression was lower in cultured effusions compared to primary carcinomas, and was cytoplasmic rather than nuclear. Twist1 (P < 0.001), Zeb1 (P = 0.003) and Vimentin (P = 0.03) mRNA expression was significantly higher in solid metastases compared to primary carcinomas and effusions. HIF-1alpha protein expression was lower in effusions compared to primary carcinomas and solid metastases (P = 0.033). Our data suggest that the previously reported E-cadherin re-expression in ovarian carcinoma effusions is regulated by Pak1. The transient nature of E-cadherin expression during ovarian carcinoma progression is probably the result of partial epithelial-to-mesenchymal transition (EMT) and the reverse process of mesenchymal-to-epithelial-like transition (MET). Expression of the EMT-related molecules Twist, Zeb1, Vimentin and HIF-1alpha is anatomic site-dependent in ovarian carcinoma.