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Glucocorticoids are widely used in conjunction with chemotherapy for ovarian cancer to prevent hypersensitivity reactions. Here we reveal a novel role for glucocorticoids in the inhibition of ovarian cancer metastasis.

Restoring Rap1B expression reverts glucocorticoid-miR cascade-mediated suppression of ovarian cancer cell invasion and metastasis. Clinically, low miR and high Rap1B are found in late-state ovarian tumours, as compared with normal, and patients with high miR rob3 gram pdf995 significantly better survival. Overall, our findings reveal an opportunity for glucocorticoids and their downstream mediators, miR or Rap1B, as therapeutic modalities against metastatic ovarian epithelial cancer.

Ovarian cancer is the sixth most common cancer in women. Approximatelywomen worldwide are diagnosed with this disease annually, with estimatedassociated deaths 1. Most patients already harbour metastasis upon initial diagnosis, and prognosis is poor with current standard therapies 2udacity course. Studies that devise novel therapeutic strategies against ovarian cancer metastasis are urgently warranted to improve patient outcomes.

Glucocorticoids GCs are known to exert pronounced effects on metabolism, differentiation, proliferation and survival of cells. Synthetic GCs, such as dexamethasone DEXare widely used as a pre-medication during chemotherapy to prevent hypersensitivity reactions in many cancer types 4.

Some in vitro studies suggest that GCs inhibit chemotherapy-induced ovarian cancer cell apoptosis 567raising the rob3 gram pdf995 concerns.

However, when comparing ovarian cancer patients who received chemotherapy concurrently with GCs and those without, there was no significant difference in survival 8. Nevertheless, other than the use of GCs at the period of chemotherapy, currently rob3 gram pdf995 comprehensive studies suggest the other treatment possibilities rob3 gram pdf995 GCs, which might be beneficial to inhibit cancer metastasis as suggested by our current study.

To date, no prospective analysis has assessed the effect of synthetic GCs on the tumour growth or metastasis of ovarian cancers. More studies are needed to clarify the exact roles and optimal time of GC administration in ovarian cancer therapy. Expression of miR impaired breast rob3 gram pdf995 metastasis via modulation of neuronatin Despite these advances recognizing miR as a potential tumour suppressor, its role in ovarian cancer has not been reported and the detailed mechanism of how miR is under regulation remains elusive.

In addition, it is likely that there exist additional miR targets involved in the regulation of ovarian cancer progression and metastasis. Synthetic GC treatments are widely used to rob3 gram pdf995 chemotherapy. In the present study, we report the finding that GC treatments play a suppressive role in ovarian cancer metastasis via the induction of miR Furthermore, clinical data reveal that low miR and high Rap1B levels are associated with advanced-stage ovarian cancer, and high miR predicts significantly better survival.

Overall, our study reveals a potential role and underlying mechanism of GCs in the suppression of ovarian cancer metastasis. Several previously reported metastasis-related miRNAs were also identified in our array results, including miRa ref.

We then analysed the expression of miR in human ovarian cancer specimens from commercial tissue arrays. Cell migration assays were performed by transwell Boyden chambers and also by scratch wound-healing assays. Cell invasion assays were performed by transwell Boyden chambers pre-coated with matrigel. The Matrigel used for coating invasion chambers resembles the extracellular matrix, and this in vitro assay is widely used to mimic invasion through the basement membrane by metastatic cancer cells.

Samples were subgrouped by tumor stages or whether metastasis occurred. The representative images from different rob3 gram pdf995 were shown in c. Scale bars: Ten pictures were taken for each mouse. The dots represent the means of individual mice. SKOV-I6iv cells with miR expression rob3 gram pdf995 vector control were orthotopically injected into the bursa of the mouse ovary.

The kinetics of cancer metastasis to the abdomen cavity was monitored by BLI. Representative BLIs are shown at day 28 after implantation. The suppression of ovarian cancer metastasis by miR in mouse models was examined. Mice in the miRexpressing group demonstrated significantly reduced lung metastatic tumour areas relative to rob3 gram pdf995 control Fig.

No metastases were observed from organs other than the lung in this model. SKOV-I6iv cells expressing miR were also orthotopically injected into mouse ovarian bursa, and the metastasis status was monitored by bioluminescence imaging BLI facilitated by stable expression of the luciferase gene.

BLI analysis indicated that expression of rob3 gram pdf995 resulted in significantly reduced abdominal metastasis relative rob3 gram pdf995 the control Fig. Impairment of metastasis was observed in the miR expression group among various abdominal organs, including the liver, omentum and gastrointestinal GI tract, as compared with the control. Notably, the tumour sizes were significantly reduced in miRexpressing tumors Supplementary Fig.

A study searching for differential miRNA expression in acute lymphoblastic leukemia Alpha beats david guetta samples revealed that upregulation of miR was associated with in vivo GC response in childhood ALL 19 ; therefore, GC-mediated signalling may be involved in the regulation of miR expression.

A protein synthesis inhibitor, cycloheximide CHXwas then applied to inhibit de novo protein synthesis before treatment with DEX. The putative promoter regions tested for luciferase activities are indicated as short grey bars with assigned numbers promoter-1 to We then sought to identify the promoter region of miR responsible for GC-mediated signalling. Four potential promoter regions were identified promoter 1—4, Fig. To localize the promoter regions, we constructed reporter plasmids carrying promoter-1 to 4 Fig.

Results demonstrated that only the promoter-3 region among the CpG islands had an over fold higher promoter activity in SKOV-3 cells compared with that of the vector control Fig. This promoter activity was further enhanced by 1.

Our findings suggest that the promoter-3 region contains the miR promoter responsible for its GC-mediated regulation in ovarian cancer cells. Overall, our results demonstrate that miR, together with ODZ4are transcriptionally regulated by the GR-mediated signalling pathways.

To identify miRmediated downstream regulator in ovarian cancer metastasis, three target prediction algorithms PicTar, Targetscan and miRanda were applied. Genes predicted by more than one algorithm are listed in Supplementary Table 3.

Rap1B, a known regulator of adhesion processes through integrin-mediated signalling 212223thus stands as an excellent candidate involved in metastatic processes. The Rap1B inhibition by miR was also observed in other ovarian cancer cell lines Supplementary Fig. Co-expression of anti-miR fully reversed miRmediated inhibition, indicating that Rap1B is a direct downstream target of miR Although our data only reflected mRNA levels, and some genes might be affected translationally, we decided to focus on Rap1B as the major target of miR in ovarian cancer.

TargetScan, miRanda and PicTar. Nucleotides mutated in miRbinding site are shown in red. The Rap1 signalling axis has been shown to promote invasion and metastasis through facilitating integrin-mediated actin remodelling rob3 gram pdf995 human pancreatic carcinoma cells 26 and breast cancer cells Expression of miR or knockdown of Rap1B disrupted the spreading of cells Fig. The inhibitory effect by miR was reversed by anti-miR Rob3 gram pdf995 of FAK is the major phosphorylation site for Src 28and this phosphorylation is crucial for integrin signalling-mediated promotion of epithelial—mesenchymal transition and migration The Rob3 gram pdf995 were detected.

To determine the proportion of each cell consisting of FAs, the area of FAs was divided by the total spreading area of the cell e.

Percentage of adhered cells was counted. We then examined the effect of miR on adhesion ability. These data suggest that miRmediated depletion of Rap1B is sufficient to impair integrin-mediated cell adhesion through disruption of FA formation. These data tether GCs to the miRRap1B signalling pathway in its ability to modulate cell spreading and adhesion through regulation rob3 gram pdf995 FA formation. To determine whether Rap1B is a rob3 gram pdf995 important target of miR in ovarian cancer cells, rescue experiments were performed.

The impaired recruitments of p-Paxillin Supplementary Fig. Scale Bars: We next examined this pathway in the in vivo mouse model. These two cell lines presented similar proliferation rates Supplementary Fig.

Five days after implantation, we started intraperitoneal DEX administration three times a week until day In agreement with the results we found in the miRexpressing tumour model Fig. To further study gene regulation by DEX treatment in vivolevels of miR and its downstream molecules were examined from the orthotopically implanted primary ovarian tumours. Overall, we demonstrate for the first time that GC-mediated signalling inhibits ovarian cancer abdominal metastasis, and expression of Rap1B significantly restored metastasis from this inhibition.

The kinetics of rob3 gram pdf995 abdominal metastasis was monitored by BLI. Representative BLI images are shown on day 28 after implantation. The miR expression was significantly decreased in tumour samples, while expression of Rap1B was increased Fig.

Samples were rob3 gram pdf995 subgrouped by tumor stages or whether metastasis occurred. To further evaluate the clinical significance of miR in ovarian cancer, 82 paraffin-embedded primary ovarian tumour specimens obtained from TVGH were analysed Supplementary Table 5. As compared with low miR, patients with high miR expression showed a significant better overall survival and relapse-free survival rates Fig.

In this study, we have unveiled a novel regulatory mechanism by steroid hormones, GCs, and its effect on ovarian cancer metastasis. Signalling mediated by GCs induced miR expression, leading to the suppression of Rap1B, the impairment of integrin-mediated FA formation, and suppression of ovarian cancer metastasis Fig. Our study implies the potential use of GCs or its downstream mediators to inhibit cancer metastasis. Commonwealth caribbean law and legal systems ebook studies presented controversial results in the use of GCs in ovarian cancers with respect to cancer cell apoptosis, survival and invasion as alluded to in the Introduction.

Here we found that GC treatments reduced ovarian cancer metastasis through miR, unveiling a new potential next model management instagram GC application in ovarian cancer therapy.

Future studies are needed to elucidate the optimal timing, duration, dosage, as well as the choice of appropriate GCs to improve the treatment regimen. In this study, we have identified the involvement of Rap1B in ovarian cancer metastasis.

Although the relative contribution of each isoform was not well discussed in earlier lines of work, several reports have revealed this nonredundant relationship between Rap1A and Rap1B. Isoform-specific knockout mice present different phenotypes 30 Rap1B-null mice are smaller in size and decreased integrin-mediated cell adhesion was observed. Moreover, Rap1A regulates endothelial cell junction through E-Cadherin 32while Rap1B determines neuronal polarity through sequential activation of Cdc42 ref.

Rap1A is predoominantly localized at the cell junctions, whereas Rap1B is more cytosolic and perinuclearly localized in endothelial cells Given the differential subcellular localizations, it is tempting to speculate that Rap1A and Rap1B may have distinct interacting complexes inside the cell.

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