Nuclear survivin expression is a positive prognostic factor in taxane-platinum-treated ovarian cancer patients

<p>Abstract</p> <p>Background</p> <p>Survivin is an inhibitor of apoptosis and a regulator of mitotic progression. TP53 protein is a negative transcriptional regulator of survivin. The aim of our study was to evaluate the clinical significance of survivin expression in advanced stages ovarian cancer with respect to the TP53 status.</p> <p>Methods</p> <p>Survivin and TP53 expression was evaluated immunohistochemically in 435 archival samples of ovarian carcinomas (244 patients were treated with platinum/cyclophosphamide-PC/PAC; 191-with taxane-platinum (TP) agents). Univariate and multivariate statistical analyses were performed in patients groups divided according to the administered chemotherapeutic regimen, and in subgroups with and without TP53 accumulation (TP53+ and TP53-, respectively).</p> <p>Results</p> <p>Nuclear and cytoplasmic survivin expression was observed in 92% and 74% of the carcinomas, respectively. In patients treated with TP, high nuclear survivin expression decreased the risk of disease recurrence and death, and increased the probability of high platinum sensitivity (p < 0.01), but only in the TP53(+) group, and not in the TP53(-) group.</p> <p>Conclusions</p> <p>It appears that TP53 status determines the clinical importance of nuclear survivin expression in taxane-platinum treated ovarian cancer patients.</p

Cytoplasmic HAX1 Is an Independent Risk Factor for Breast Cancer Metastasis

HAX1 is an antiapoptotic factor involved in the regulation of cell migration and calcium homeostasis, overexpressed in several cancers, including breast cancer. It has been suggested that HAX1 is also implicated in metastasis. Herein we report the results of meta-analysis of HAX1 expression, based on publicly available data, which confirms its significant overexpression in breast cancer and demonstrates copy number gain and prognostic value of HAX1 overexpression for metastatic relapse in ER+ tumors. IHC analysis reported here also reveals its significant overexpression in breast cancer samples from primary tumors, indicating significantly higher HAX1 protein levels in a group of patients who developed distant metastases in a disease course. Moreover, we demonstrate that HAX1 localization is important for the prediction of metastatic relapse and that cytoplasmic but not nuclear HAX1 is an independent risk factor for breast cancer metastasis

The Novel Gene <i>CRNDE</i> Encodes a Nuclear Peptide (CRNDEP) Which Is Overexpressed in Highly Proliferating Tissues

<div><p><i>CRNDE</i>, recently described as the lncRNA-coding gene, is overexpressed at RNA level in human malignancies. Its role in gametogenesis, cellular differentiation and pluripotency has been suggested as well. Herein, we aimed to verify our hypothesis that the <i>CRNDE</i> gene may encode a protein product, CRNDEP. By using bioinformatics methods, we identified the 84-amino acid ORF encoded by one of two <i>CRNDE </i>transcripts, previously described by our research team. This ORF was cloned into two expression vectors, subsequently utilized in localization studies in HeLa cells. We also developed a polyclonal antibody against CRNDEP. Its specificity was confirmed in immunohistochemical, cellular localization, Western blot and immunoprecipitation experiments, as well as by showing a statistically significant decrease of endogenous CRNDEP expression in the cells with transient shRNA-mediated knockdown of <i>CRNDE</i>. Endogenous CRNDEP localizes predominantly to the nucleus and its expression seems to be elevated in highly proliferating tissues, like the parabasal layer of the squamous epithelium, intestinal crypts or spermatocytes. After its artificial overexpression in HeLa cells, in a fusion with either the EGFP or DsRed Monomer fluorescent tag, CRNDEP seems to stimulate the formation of stress granules and localize to them. Although the exact role of CRNDEP is unknown, our preliminary results suggest that it may be involved in the regulation of the cell proliferation. Possibly, CRNDEP also participates in oxygen metabolism, considering our <i>in silico</i> results, and the correlation between its enforced overexpression and the formation of stress granules. This is the first report showing the existence of a peptide encoded by the <i>CRNDE</i> gene.</p></div

Molecular studies on CRNDEP.

<p>A) Simultaneous overexpression of the 6xHis-CRNDEP-EGFP and DsRed Monomer-6xHis-CRNDEP fusion proteins in HeLa cells, visualized under a fluorescence microscope. The former protein glows green and the latter glows red in these conditions. Yellow glow is caused by a co-localization of these two fusion proteins. Nuclei were stained blue with DAPI. The same shot with only the green (B) or the red (C) channel shown. D) Western blot-based verification of the size of the 6xHis-CRNDEP-EGFP fusion protein. M—Spectra Multicolor Low Range Protein Ladder (Thermo-Fisher Scientific), 1—the EGFP reporter protein (26.9 kDa), 2—6xHis-CRNDEP-EGFP (39.2 kDa). E) Western blot-based verification of the specificity of our custom-made polyclonal anti-CRNDEP antibody. M—Spectra Multicolor Low Range Protein Ladder; 1—DsRed Monomer-6xHis-CRNDEP (340 aas, 38.5 kDa); 2—purified 14 kDa protein containing the 6xHis tag, 1.4 μg (a negative control of the antibody's specificity, non-commercial); 3—6xHis-CRNDEP-EGFP (346 aas, 39.2 kDa); 4—empty; 5—EGFP (239 aas, 26.9 kDa, a negative control); 6—DsRed Monomer (232 aas, 26.2 kDa, a negative control). A loading control (the PVDF membrane used in this experiment, stained with Ponceau S) is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127475#pone.0127475.s013" target="_blank">S13 Fig</a>. F–G) Detection of the overexpressed 2xFLAG-CRNDEP protein (~14 kDa) in a total protein lysate from 0.25 million HeLa cells with either the anti-FLAG (F) or anti-CRNDEP (G) antibody. H) Immunoprecipitation of 2xFLAG-CRNDEP using the anti-CRNDEP antibody (2) and control IgG (1) (both from a rabbit). A total protein lysate before immunoprecipitation was loaded for comparison (3). After precipitation, the 2xFLAG-CRNDEP protein was detected on the PVDF membrane using the anti-FLAG antibody. The correct bands in Fig F–H are encircled.</p

Evaluation of shRNA-mediated knockdown of <i>CRNDE</i>.

<p>The effects of <i>CRNDE</i> gene silencing were evaluated at either mRNA (A) or protein level (B-C). The strongest decrease in the amount of the CRNDEP-coding transcript (by ~65%) was observed for the SH1 silencing construct (A). The effects of this knockdown were detectable at the protein level as well (B, C), leading to a statistically significant decline in the amount of CRNDEP (red signal) in the cells transfected with the silencing construct (green signal). As expected, such a correlation did not occur in the cells transfected with the construct encoding a control (scrambled) shRNA molecule (SH SCR). The transfected cells are marked with white arrows.</p

Primary and secondary antibodies used in the present study.

<p>¹) Western Blot.</p><p>²) Immunofluorescence.</p><p>³) IHC (Immunohistochemistry).</p><p>⁴) Two of three anti-CRNDEP antibodies were not purified (Western blots were performed using rabbit sera with relatively low concentration of specific antibodies). IHC—immunohistochemistry; HRP—horseradish peroxidase; N/A—not applicable.</p><p>Primary and secondary antibodies used in the present study.</p