Poly r(C) Binding Protein 1 Regulates Posttranscriptional Expression of the Ubiquitin Ligase TRIM56 in Ovarian Cancer.

Our earlier work has shown that the E3 ligase TRIM56 messenger RNA (mRNA) level and vimentin protein expression followed an inverse correlation in ovarian carcinoma patients; however, the regulatory mechanisms underlying TRIM56 expression is unclear. Steady state expression of TRIM56 mRNA expression in the normal ovarian cell line Moody and ovarian cancer cell lines SKOV-3, A2780, and Caov-3 were not significantly different; however, TRIM56 protein expression was significantly lower in the ovarian cancer cell lines compared to the Moody cell line. Polysome profiling showed significant increase in translation of TRIM56 messenger RNA in the Moody cells compared to the SKOV-3 cells. We performed RNA-affinity pulldown using biotinylated TRIM56 5 'and 3'-UTR and postnuclear extracts from Moody and SKOV-3 cells. Whereas no notable difference was observed in affinity pull-down obtained with the 5'-UTR, there was obvious difference in protein binding patterns with the 3'-UTR. Mass spectrometry was used to determine the most differentially binding protein as poly r (c) binding protein 1 (PCBP1). PCBP1 expression and binding to the 3'-UTR was both higher in SKOV-3 cells compared to the Moody cells. Silencing of TRIM56 in Moody cells cause an increase in in vitro migration and invasion, and a similar effect was mimicked by overexpression of PCBP1. Conversely, silencing of PCBP1 or overexpression of TRIM56 in SKOV-3 cells significantly decreased in vitro migration and invasion. In xenograft assays, SKOV-3 cells stably overexpressing shRNA targeting PCBP1 decreased metastasis, whereas shRNA-targeting TRIM56 potentiated detection of metastatic lesions, compared to the parental SKOV-3 cells themselves. Taken together our results reveal a yet undefined posttranscriptional regulatory mechanism underlying low expression of TRIM56 in ovarian cancer. © 2018 IUBMB Life, 71(1):177-182, 2019.

The ubiquitin ligase TRIM56 inhibits ovarian cancer progression by targeting vimentin.

Tumor metastasis is responsible for 90% of all cancer-related deaths. Epithelial to mesenchymal transition (EMT) is an important prerequisite for tumor metastasis. One of the important mediators of EMT and cancer progression in ovarian cancer is the vimentin protein. The objective of the current study was to evaluate the molecular mechanism that regulates vimentin expression in ovarian cancer cells. Vimentin was robustly induced in the ovarian cancer cell line SKOV-3 compared to normal ovarian epithelial cell line Moody and the induction was not due to transcriptional upregulation. Treatment with the proteasomal inhibitor MG-132 revealed that vimentin is actively degraded by the proteasome in Moody cells and stabilized in the SKOV-3 cell line. Mass spectrometric analysis of vimentin immunoprecipitate of MG-132 treated Moody cells revealed candidate ubiquitin ligases associated with vimentin. RNAi mediated silencing of the candidate ubiquitin in Moody cells and concurrent overexpression of the candidate ubiquitin ligases in SKOV-3 confirmed that TRIM56 is the ubiquitin ligase that is degrading vimentin in Moody cells. RNAi mediated silencing of TRIM56 in Moody cells and ectopic overexpression of TRIM56 in SKOV-3 cells, respectively, significantly up- and down-regulated in vitro migration and invasion in these cells. Analysis of TRIM56 transcript level and vimentin protein expression in 25 patients with ovarian carcinoma confirmed an inverse correlation between TRIM56 and vimentin expression. Cumulatively, our data reveals for the first time a novel post-translational regulatory mechanism of regulating vimentin expression, EMT, and metastatic progression in ovarian cancer cells.

Cytotoxic pregnane steroids from the seeds of Cipadessa baccifera (Roth.) Miq.

A chemical investigation of the 80% ethanol extract of the seeds of Cipadessa baccifera (Roth.) Miq. led to the isolation of five new pregnane steroids, 17α,18,20S-trihydroxy-pregn-4-en-3,16-dione (1), 18-hydoxy-pregn-4,17(20)-trans-dien-3,16-dione (2), 3ß,18-dihydroxy-pregn-5,17(20)-trans-dien-16-one (3), 2α,3ß,4ß,18-tetrahydroxy-pregn-5,17(20)-trans-dien-16-one (4), and 2α,3ß,4ß,17α,18,20S-hexahydroxy-pregn-5-en-16-one (5), along with two known compounds, 17α,20S-dihydroxy-pregn-4-en-3,16-dione (6) and 3ß-hydroxy-pregn-5,17-dien-16-one (7). Structural elucidation of all the compounds was accomplished by spectral methods such as 1D and 2D NMR, IR, UV, and HRESIMS. The isolated compounds were tested in vitro for cytotoxic activities against seven tumor cell lines. As a result, pregnane-type steroids 1, 5 and 6 exhibited cytotoxicity with IC50 values <20µM against all tested tumor cell lines except meningioma cells (BEN-MEN-1).