Treatment with 17β-estradiol (E2) activated phosphorylation of ERK and FAK in RL95-2 endometrial cancer cells.

<p>(<b>A</b>) ERα and GPR30 expression in endometrial cancer cells. In RL95-2 cells, GPR30 was highly expressed where ERα was not expressed after several cell passages with estrogen deprivation. In contrast, Ishikawa cells expressed both ERα and GPR30. (<b>B</b>) Phosphorylation of both ERK and FAK was increased after treatment of ERα-negative RL95-2 cells with E2 (1 μM) by using a set of phosphorylation arrays. (<b>C</b>) Fold changes of phosphorylation of ERK and FAK on the phosphorylation arrays (B) are shown. DMSO was used as control in comparison with E2 effects. Results shown were obtained from three independent experiments and are presented as mean ± standard deviation (SD).</p

Estradiol and Tamoxifen Induce Cell Migration through GPR30 and Activation of Focal Adhesion Kinase (FAK) in Endometrial Cancers with Low or without Nuclear Estrogen Receptor α (ERα)

<div><p>Estrogens and tamoxifen (an antiestrogen) exert their actions by activation of estrogen receptor (ER) through genomic and non-genomic mechanisms and are implicated in the development of endometrial cancer. Previous reports have demonstrated that estradiol and tamoxifen induce proliferation of human endometrial cancer cells through GPR30 (non-genomic ER) signaling pathway. Herein, we demonstrate that phosphorylation of focal adhesion kinase (FAK) is involved in cell migration induced by estradiol, tamoxifen and G1 (a GPR30 agonist) through the transmembrane ER (GPR30) in endometrial cancer cell lines with or without ERα (Ishikawa and RL95-2). Additionally, the GPR30-mediated cell migration was further abolished by administration of either specific RNA interference targeting GPR30 or an FAK inhibitor. Moreover, we have validated that the signaling between GPR30 and phosphorylated FAK is indeed mediated by the EGFR/PI3K/ERK pathway. Clinically, a significant correlation between levels of GPR30 and phophorylated FAK (pFAK) observed in human endometrial cancer tissues with low or without ERα further suggested that estrogen-induced phosphorylation of FAK and cell migration were most likely triggered by GPR30 activation. These results provided new insights for understanding the pathophysiological functions of GPR30 in human endometrial cancers.</p></div

17β-estradiol (E2), GPR30 agonist (G1) and hydroxytamoxifen (OHT) triggered FAK Y397 phosphorylation through GPR30.

<p>RL95-2 (<b>A, C, E</b>) and Ishikawa cells (<b>B, D, F</b>) were treated with 1 μM E2, G1 and OHT for 5 mins. Phosphorylated ERK1/2 (pERK) (<b>A, B</b>) and FAK at Y397 (pFAK) (<b>C, D</b>) were detected by immunoblotting. Increased levels of pERK and pFAK were shown following treatment with E2, G1 and OHT as compared to control (DMSO). Endogenous GPR30 was knocked-down by shRNA in RL95-2 (<b>E</b>) or by siRNA in Ishikawa cells (<b>F</b>). Following treatment with 1 μM E2 and OHT for 5 mins, levels of pFAK were repressed in both endometrial cancer cells with knockdown of GPR30 (<b>E, F</b>). (<b>G</b>) In Ishikawa cells, knockdown of ERα using siRNA partially abolished phosphorylation of FAK induced by E2 and OHT. Similarly, E2- and OHT-induced phosphorylation of FAK was also blocked in the presence of ICI 182780 (an antagonist of ERα) (<b>H</b>). Results shown were obtained from three independent experiments and are presented as mean ± standard deviation (SD).</p

Histoscores of estrogen receptor (ER), GPR30 and phosphorylated FAK (pFAK) in human endometrial cancers.

<p>Data are presented as mean ± standard deviation (SD).</p>*<p>Student t-test.</p

Cell migration induced by 17β-estradiol (E2) and tamoxifen (OHT) is mediated through GPR30 and phosphorylation of FAK at Y397 (pFAK) in endometrial cancer cells.

<p>RL95-2 cells (<b>A</b>) and Ishikawa cells (<b>B</b>) were pre-treated with FAK inhibitor 14 (15 μM) for 2 hrs, then treated with 1 μM E2 or OHT for 5mins, and cell lysates were analyzed via Western blot using antibody against pFAK Y397. The expression levels of pFAK at Y397 induced by E2 or OHT were suppressed by FAK inhibitor14. E2- and OHT- induced cell migration were also abrogated in the presence of FAK inhibitor 14 in RL95-2 (<b>C</b>) and Ishikawa cells (<b>D</b>). After knockdown of endogenous GPR30 in RL95-2 cells (<b>E</b>) and Ishikawa cells (<b>F</b>), cell migration was inhibited as compared with mock depletion. Results shown were obtained from three independent experiments and are presented as mean ± standard deviation (SD).</p

Expression of ERα, GPR30 and pFAK (at Y397) in human endometrial cancers with low ERα.

<p>(<b>A</b>) In clinical samples of human endometrial cancers with low expression of ERα (histoscore  = 10), relatively high expression of GPR30 (histoscore  = 270) and pFAK (histoscore  = 225) were observed (40× magnification of the object lens). (<b>B</b>) The histoscores of both GPR30 and pFAK were lower in human endometrial cancers with high ER (histoscore >45, left panel) as compared to those with low ER (histoscore ≤45, right panel). (<b>C</b>) There was a positive correlation of histoscores between pFAK and GPR30 in human endometrial cancers with low ERα. (<b>D</b>) No association was found in histoscores between pFAK and ERα in human endometrial cancers. NS: not significant.</p

Positive associations between upregulated levels of stress-induced phosphoprotein 1 and matrix metalloproteinase-9 in endometriosis/adenomyosis

<div><p>Stress-induced phosphoprotein-1 (STIP1), an adaptor protein that coordinates the functions of HSP70 and HSP90 in protein folding, has been implicated in the development of human gynecologic malignancies. This case-control study investigates STIP1 serum levels and tissue expression in relation to endometriosis/adenomyosis in Taiwanese population. Female patients with surgically confirmed endometriosis/adenomyosis were compared with women free of endometriosis/adenomyosis. Serum STIP1 levels were measured using an enzyme-linked immunosorbent assay and surgical tissues were analyzed by immunohistochemistry. Both epithelial and stromal cells in surgical tissues of endometriosis and adenomyosis expressed STIP1 and MMP-9. Notably, MMP-9 expression was significantly decreased when STIP1 expression was knocked-down. <i>In vitro</i> experiments revealed that STIP1 was capable of binding to the MMP-9 promoter and enhanced its transcriptional expression. The preoperative serum STIP1 levels of patients with endometriosis/adenomyosis were significantly higher than those of the controls. In brief, our data suggest an association between STIP1 levels and endometriosis/adenomyosis.</p></div

Tumor Stress-Induced Phosphoprotein1 (STIP1) as a Prognostic Biomarker in Ovarian Cancer

<div><p>Stress-induced phosphoprotein 1 (STIP1) has been recently identified as a released biomarker in human ovarian cancer. In addition, STIP1 secreted by human ovarian cancer cells has been shown to promote tumor cell proliferation by binding to ALK2 (activin A receptor, type II-like kinase 2) and activating the SMAD-ID3 signaling pathways. In this study, a total of 330 ovarian cancer tumor samples were evaluated for STIP1 expression by immunohistochemistry and analyzed for a possible correlation with patient characteristics and survival. The quantification of immunoreactivity was accomplished by applying an immunohistochemical scoring system (histoscore). Patients with high-level STIP1 expression (histoscore ≥169) had a significantly worse survival (high STIP1, mean survival time = 76 months; low STIP1, mean survival time = 112 months; <i>P</i><0.0001). Moreover, STIP1 histoscores were significantly higher in high-grade tumors (grade 3) than in low-grade (grade 1–2) malignancies (<i>P</i><0.0001), suggesting that STIP1 may be a proxy for tumor aggressiveness. The results of multivariable analysis revealed that high STIP1 histoscores, advanced stages, histologic types, and the presence of residual disease (≥2 cm) were independent predictors of poor prognosis. The addition of STIP1 histoscores improved the prediction of overall and progression-free survival rates in the multivariable Cox proportional hazard model. The treatment of ovarian cancer cells with recombinant STIP1 stimulated cell proliferation and migration, but co-treatment with anti-STIP1 antibodies abrogated this effect. Our findings suggest that STIP1 expression may be related to prognosis and that the STIP1 pathway may represent a novel therapeutic target for human ovarian cancer.</p> </div

Decreased MMP-9 protein expression induced by STIP1 knockdown is independent of common signaling pathways.

<p>(A) STIP1 knockdown by siRNA in ARK2 cells decreased MMP-9 protein expression without affecting AKT, ERK, and IkB phosphorylation levels. (B) Decreased MMP-9 protein expression induced by siRNA knockdown of STIP1 was unaffected by exposure either to the DNA methylation inhibitor 5-aza-2’-deoxycytidine (AZA; 10 μM) or the histone deacetylase inhibitor trichostatin A (TSA; 20 nM). (C) Suppression of MMP-9 protein levels by siRNA knockdown of STIP1 was unaffected by treatment with either the proteasome inhibitor MG-132 (25 μM) or a JAK2 inhibitor AG490 (35 μM). (D) Treatment with an STIP1 inhibitor, peptide 520, suppressed MMP-9 protein levels in a dose-dependent manner.</p

STIP1 binds to the MMP-9 promoter and is required for MMP-9 expression.

<p>(A) ARK2 cells were transfected with the control (scrambled) or STIP1 siRNAs. The protein levels of MMP-9, STIP1, and actin were analyzed using western blot. The bar graph summarizes the decreased protein levels of MMP9 that were caused by STIP1 knockdown in three independent siRNA experiments. A representative zymograph (lower panel) exemplifies the decreased MMP-9 activity that was caused by STIP1 knockdown. (B) At 72 h after transfection of the control or STIP1 siRNA, real-time quantitative polymerase chain reaction (RT-QPCR) was used to quantify MMP-9 RNA levels in the ARK2 and MDAH2774 cells. (C) ARK2 cells were transfected with various MMP-9 reporter constructs (described in the “Methods” section) in the presence of the control or STIP1 siRNA. Reporter activity was measured using the luciferase assay. Individual control (the empty bar for each reporter assay) indicated reporter activity in the cells cotransfected with the designated reporter construct and a control STIP1 siRNA, the activities of which were set as 1. Results are expressed as mean ± standard error from three independent experiments. Statistical differences were calculated with Student’s <i>t</i>-test. *p < 0.05, **p < 0.01, and ***p < 0.001. (D) Identification of STIP1 potential binding sites on the MMP-9 gene promoter (from −1 kb to −2 kb upstream of the transcriptional start site). Two commercially available antibodies (each obtained from Santa Cruz and Abnova) were used to pull-down STIP1. Three independent ChIP experiments were performed. Quantification was performed by RT-QPCR, with the results being expressed as mean ± standard error of the mean. (E) Nuclear/cytosolic fractionation of ARK2 cells was performed. HSP90, STIP1, tubulin, and B23 protein levels were quantified with western blot. Tubulin and B23 were used as cytosolic and nuclear markers, respectively. (F) Cell migration ability was blocked when STIP1 expression was silenced (quantification shown in the lower panel). Results are expressed as mean ± standard error of the mean from three independent experiments. Statistical differences were calculated using Student’s <i>t</i>-test.</p