IMP1 promotes choriocarcinoma cell migration and invasion through the novel effectors RSK2 and PPME1.

OBJECTIVE: Oncofetal protein insulin-like growth factor II mRNA-binding protein 1 (IMP1) regulates cellular proliferation and migration. Expression of IMP1 is limited to a few adult human tissues. However, it commonly expresses in a variety of cancers. Our objective was to study the regulatory mechanism of IMP1 on the cellular functions of choriocarcinoma (CC) JAR cells. METHODS: IMP1 protein levels were measured in CC tissues via immunohistochemistry. Specific siRNAs were used to down-regulate gene expressions. The abilities of migration and invasion were estimated by wound-healing and Matrigel chamber assays. The profile of IMP1-binding genes was investigated with an Agilent microarray. RT-qPCR, RNA immunoprecipitation, and IMP1 rescue experiments were performed to confirm the association between IMP1 and its binding genes. Gene expression was further analyzed by using RT-PCR and Western blotting. RESULTS: Strong IMP1 expressions were frequently detected in CC tissues. Knockdown of IMP1 expression in JAR cells inhibited cell migration and invasion, but did not affect cellular proliferation and morphology. Microarray and RNA-immunoprecipitation results revealed several candidate genes regulated by IMP1. Among them, ribosomal protein S6 kinase (RSK2) and protein phosphatase methylesterase 1 (PPME1) were confirmed to be down-regulated in IMP1-depleted JAR cells. Re-expression of IMP1 into the cells restored the expressions of RSK2 and PPME1. Furthermore, the depletion of RSK2 or PPME1 decreased the migration and invasion of JAR cells. CONCLUSION: Our results suggest that IMP1 plays an essential role in the regulation of migration and invasion of human CC cells, possibly through the novel effectors RSK2 and PPME1.

Elevated amniotic fluid stromal cell-derived factor-1alpha (SDF-1alpha) concentration in mid-gestation as a predictor of adverse birth outcomes.

BACKGROUND: This study aimed to predict maternal and neonatal outcomes by measuring mid-trimester amniotic fluid stromal cell-derived factor-1alpha (SDF-1alpha) concentration in healthy women. METHODS: Mid-trimester amniotic fluid samples from healthy women with a singleton pregnancy were obtained at the time of genetic amniocenteses. SDF-1alpha concentrations were determined by enzyme-linked immunosorbent assay. Maternal and neonatal characteristics were recorded. RESULTS: A total of 210 samples were collected. According to the SDF-1alpha cutoff value established by the receiver operating characteristic curve analysis (< 6.42 vs. > or = 6.42 pg/mL), there was a trend toward higher preterm birth rate, lower birth weight and lower 1-minute and 5-minute Apgar scores when SDF-1alpha levels increased (p < 0.05). The pair comparison between normal and selected pregnancy disorders (gestational diabetes, pre-eclampsia, and abnormal placentation) showed no statistical significance (p > 0.05). Pearson's correlations of SDF-1alpha to gestational age at delivery (r = -0.151) and birth weight (r = -0.194) were significant (p < 0.05). In the multivariate analysis on mid-trimester SDF-1alpha levels, maternal age at sampling (regression coefficient = -0.163) and 1-minute Apgar score (< 7 vs. > or = 7, regression coefficient = 2.028) were both significant (p < 0.05). CONCLUSION: Increased SDF-1alpha levels in mid-trimester amniotic fluid suggest a possible role in predicting pregnant women at risk of adverse neonatal outcomes including higher preterm birth rate, lower birth weight, and lower Apgar scores.

Metastasis associated lung adenocarcinoma transcript 1 is up-regulated in placenta previa increta/percreta and strongly associated with trophoblast-like cell invasion in vitro.

Placenta previa increta/percreta (I/P) is a severe form of invasive placentation associated with massive peripartum hemorrhage, which often requires Cesarean hysterectomy. The pathogenesis of invasive placentation is multidimensional, involving decidual deficiency, endomyometrial damage and excessively deep trophoblast invasion into the uterus. In this study, annealing control primer-polymerase chain reaction (ACP-PCR) was used to identify differentially expressed genes, which may impair placentation resulting in placenta previa I/P. Placental tissues from I/P and non-increta/percreta (non-I/P) sites were concomitantly collected from patients undergoing Cesarean hysterectomy. After ACP-PCR experiments (three patients), the differentially expressed bands, consistently showing up- or down-regulated trends between each of the I/P and non-I/P tissue pairs, were cloned and sequenced. Human non-protein coding metastasis associated lung adenocarcinoma transcript 1 (MALAT-1) gene was identified. Real-time quantitative PCR (10 patients) confirmed significant overexpression of MALAT-1 in I/P samples (P = 0.005). To investigate the role of MALAT-1 gene in the regulation of trophoblast cell invasion, targeting of MALAT-1 mRNA expression with short interfering RNA (siRNA) in trophoblast-like BeWo, JAR and JEG-3 choriocarcinoma cells was performed. The invasion ability of these cells was significantly suppressed after siRNA silencing (P < 0.001), and this was not correlated with abnormal MMP-2 and MMP-9 enzyme activities. Our results suggest that MALAT-1 expression in placenta previa I/P is increased and its down-regulation inhibits trophoblast-like cell invasion in vitro. MALAT-1 might be involved in regulating trophoblast invasion during the development of advanced invasive placentation.

Expression analysis of apoptosis-related markers TP53, BCL-2, BAX and c-MYC in female genital tract sarcomas.

BACKGROUND: Most female genital tract sarcomas are highly malignant and fatal. Their aggressive growth pattern and poor response to chemotherapy are the major causes of death. Deregulation of the apoptosis pathway is related to tumorigenesis and chemodrug resistance. The purpose of this study was to investigate the expression status and relationship of the apoptosis-related markers TP53, BCL-2, BAX and c-MYC in this group of tumors. In addition, correlations of these markers with clinicopathologic findings and their prognostic significance were also examined. METHODS: Paraffin blocks of female genital tract sarcoma tissue from 54 patients were obtained after pathology review. Protein expression of TP53, BCL-2, BAX and c-MYC was examined using immunohistochemical staining with standard procedures. A semiquantitative method was used to assess the staining result where scoring 1-3 was negative and 4-9 was positive for expression. The mutual relationships between TP53, BCL-2, BAX and c-MYC were examined. Associations between expression of the apoptotic markers and tumor stage as well as outcome were also analyzed. RESULTS: We found that all 4 of the apoptosis-related markers were frequently expressed in female genital tract sarcomas. Of the 54 cases, 24 (44%) were positive for TP53, 23 (43%) for BCL-2, 25 (46%) for BAX, and 30 (56%) for c-MYC. A significant positive association was observed between BAX and c-MYC (p < 0.001). There was no significant difference for the expression status of the 4 markers in early and late stage tumors. In prognostic analysis, overexpression of TP53, late stage, and age were significant prognostic factors in both univariate and multivariate analyses. CONCLUSION: Since changes in TP53, BCL-2, BAX and c-MYC frequently occur in female genital tract sarcomas, deregulation of apoptosis appears to be involved in the pathogenesis of this group of tumors. This mechanism may occur early in tumorigenesis and include the c-MYC/BAX apoptotic pathway or BCL-2. However, TP53 mutation may play a crucial role in this process, and clinically, it could be used as a prognostic indicator.

Phosphorylation of adducin by protein kinase Cdelta promotes cell motility.

Protein kinase Cdelta (PKCdelta) has been implicated to play a crucial role in cell proliferation, differentiation and apoptosis. In this study, we have investigated the role of PKCdelta in cell motility using Madin-Darby canine kidney cells. Overexpression of PKCdelta promoted membrane protrusions, concomitant with increased cell motility. By contrast, suppression of PKCdelta expression by RNA interference inhibited cell motility. Moreover, a fraction of PKCdelta was detected at the edge of membrane protrusions in which it colocalized with adducin, a membrane skeletal protein whose phosphorylation state is important for remodeling of the cortical actin cytoskeleton. Elevated expression of PKCdelta correlated with increased phosphorylation of adducin at Ser726 in intact cells. In vitro, PKCdelta, but not PKCalpha, directly phosphorylated the Ser726 of adducin. Finally, we demonstrated that overexpression of both adducin and PKCdelta could generate a synergistic effect on promoting cell spreading and cell migration. Our results support a positive role for PKCdelta in cell motility and strongly suggest a link between PKCdelta activity, adducin phosphorylation and cell motility.

Loss of Smad4 protein expression occurs infrequently in endometrial carcinomas.

Smad4 is a member of the Smad proteins, which are needed for mediating signals of transforming growth factor beta from the cell surface to the nucleus. Smad4 is also a tumor suppressor gene for cancers of the pancreas, colon, and lung. The aim of this study was to investigate the expression and prognostic significance of this gene product in endometrial cancer. Immunohistochemical staining for Smad4 was performed on formalin-fixed, paraffin-embedded specimens of endometrial tumors with an anti-Smad4 monoclonal antibody (clone B8): 97 primary endometrial carcinomas, 20 cases of endometrial hyperplasia, and 26 cases of metastases from endometrial carcinoma. The immunoreactivity of each tumor was correlated with the clinical and histopathologic parameters of the patients. Diffusely positive expression of Smad4 protein was detected in all 20 cases of endometrial hyperplasia and in most of the primary and metastatic endometrial cancers. The frequency of positive expression decreased progressively with tumor grade. Clinically, however, it was not associated with tumor progression, nor did it predict patient outcome. Although loss of heterozygosity at chromosome 18q21 (the location of the Smad4 gene) is frequent in endometrial carcinomas, the authors show in this immunohistochemical study that inactivation of this gene occurs infrequently in this tumor.

KAI1 metastasis suppressor protein is down-regulated during the progression of human endometrial cancer.

PURPOSE: KAI1 is a metastasis suppressor gene located on human chromosome 11p11.2. It is a member of the structurally distinct family of cell surface glycoprotein, transmembrane 4 protein superfamily. KAI1 was initially isolated as a gene that suppressed metastasis of rat prostate tumor cells. Decreased KAI1 expression has been observed recently in various human cancers, including pancreatic, lung, hepatic, colorectal, breast, ovarian, esophageal, and cervical cancers. Frequent down-regulation of the KAI1 protein was also observed in endometrial cancer cell lines. The aim of this study was to determine whether this gene is altered in human endometrial carcinoma. In addition, its prognostic significance in this tumor was also evaluated. EXPERIMENTAL DESIGN: Tumor specimens from 18 cases with various degrees of endometrial hyperplasia, 97 primary endometrial carcinomas with various stages, and 28 metastatic lesions of this cancer were examined in this study. Using the method of immunohistochemistry, we characterized the KAI1 protein expression in the 143 endometrial tumors. Expression of KAI1 at RNA level was also examined in 35 of the 143 samples using a real-time quantitative PCR method. The data from immunohistochemical analysis were correlated with various clinicopathological factors. RESULTS: High levels of KAI1 protein expression were detected in almost all of the specimens with endometrial hyperplasia (17 of 18). In contrast, loss of KAI1 expression occurred in an increasing frequency (27.8-71.4%) from early stages of primary endometrial carcinomas to metastatic tumors (P < 0.001). In addition, more poorly differentiated tumors demonstrated significantly lower KAI1 expression as compared with the well-differentiated tumors (P < 0.001). It was also found that patients with KAI1-negative tumors had a lower survival rate than those with KAI1-decreased or positive tumors (P = 0.0042 and 0.0286, respectively). However, in multivariate analysis, the prognostic significance of KAI1 expression was inferior to tumor stage. CONCLUSION: These data suggest that KAI1 expression is down-regulated in advanced endometrial cancer. Clinically it may be a useful indicator of the tumor progression and may provide prognostic information on the outcome of this disease.