Main characteristics of study participants.

<p>PSA, prostate-specific antigen; CI, confidence interval; NA, not available.</p><p>Main characteristics of study participants.</p

SEPT9_i1 staining in prostate cancer metastases.

<p>Metastatic prostate cancer lesions from bone marrow, lymph node and bone were immunostained with SEPT9_i1. Left panels are low (x100) magnification (LM) (scale bar 100 μm) and right panel are high (x200) magnification (HM) (scale bar 50 μm). Note high level of SEPT9_i1 staining in all metastases.</p

Characterization of SEPT9_i1 antibodies and scoring of SEPT9_i1 staining intensity.

<p>HEK-293T embryonic human kidney cells were transiently transfected with Flag-SEPT9_i1 construct or empty vector (EV). (A) Whole cellular extracts were prepared and analyzed by 4–20% SDS-PAGE and immunoblotting (IB) with antibodies to Flag (1:2000), SEPT9_i1 (1:3000), preimmune serum (1:3000) or SEPT9_i1 antibody (1:3000) pre-incubated with 10 μM of the immunogen peptide for 4 hours. (B) The same cellular extracts were subjected to immunoprecipitation (IP) using anti-Flag antibody and the immuneprecipitates were subjected to 4–15% SDS-PAGE and then immunoblotted with anti-Flag or anti-SEPT9_i1 antibodies. (C) Representative SEPT9_i1 staining in human prostate cancer specimens. Score 0: no SEPT9_i1 staining, 1: low SEPT9_i1 staining 2: medium SEPT9_i1 staining and 3: high SEPT9_i1 staining. Magnification x200, scale bar 50 μm.</p

Correlation between "patients' characteristics" and SEPT9_i1 staining.

<p>PSA, prostate-specific antigen.</p><p>Correlation between "patients' characteristics" and SEPT9_i1 staining.</p

Gleason score correlation with SEPT9_i1 staining intensity.

<p>Mean SEPT9_i1 staining intensity ± SE of each Gleason score group (score 5: 3 patients, score 6: 8 patients, score 7: 19 patients, score 8: 3 patients, score 9: 6 patients and score 10: 2 patients) was calculated using either visual scoring (A) or automated image analysis with the ARIOL-SL50 system (B).</p

Vav1 Fine Tunes p53 Control of Apoptosis versus Proliferation in Breast Cancer

<div><p>Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.</p> </div

Vav1 is regulated by the Cbl-c ubiquitin ligase in breast cancer cells.

<p>(A) AU565Vector and AU565Vav1 were treated with10 mM of the proteasome inhibitor MG132 for four hours or left untreated. Cell lysates were subjected to immunoblotting with anti-Vav1 antibody. Anti β-catenin antibody was used as a control for an unstable protein with a short half-life. Anti-actin antibody served as a loading control. (B) The expression of Cbl isoforms (Cbl, Cbl-b and Cbl-c) was analyzed by Real-Time PCR in MCF-7Vector and AU565Vector cells. (C) Lysates of AU565Vav1 cells were incubated with bacterial GST fusion proteins expressing the SH2 domain of Vav1 (GST-Vav1SH2) or GFP (GST-GFP), and then immobilized on glutathione-Sepharose beads. Bound proteins were resolved on SDS-PAGE gels and immunoblotted with anti-Cbl-c or anti-pTyr antibodies. As loading control, total cell lysates were immunoblotted with anti-Cbl-c and actin antibodies. (D) Vav1 and Cbl-c expression in 50 breast cancer -originated cell lines using the same gene array as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054321#pone-0054321-g002" target="_blank">Figure 2A</a>. Red: greater than mean expression, Green: less than expression, Black: mean expression. (E) Cbl-c is silenced in AU565 cells following treatment with Cbl-c shRNA. Cells were infected with viral vectors containing shRNA for Cbl-c (shCbl-c 273) or control validated (shControl). cDNA was subjected to real time PCR analysis using primers against Cbl-c, Cbl and Cbl-b. GAPDH was used as a control. (F) Lysates from AU565 cells infected with shCbl-c 273 or control shScrambled were subjected to immunoblotting with anti-Vav1 antibody. As a control for Vav1 expression, lysates from H358 lung cancer cells infected with shVav1 or shControl were used. Anti-actin was used as loading control.</p

Vav1 pro-apoptotic effect in MCF-7 cells.

<p>(A) MCF-7Vector and MCF-7Vav1 were stained for detection of γ-H2AX foci. Three representative photographs of the foci from different areas are presented. (B) TUNEL assay of MCF-7Vector, MCF-7Vav1 cells was performed. Three representative photographs of cells from different areas are presented.</p

Vav1 is expressed in the majority of human breast tumors.

<p>An array of 70 specimens of human breast tumors was stained with anti-Vav1 monoclonal antibody. (A) Anti-Vav1 staining of three representative invasive ductal carcinoma grade II tumor specimens. (B) Vav1 expression correlated positively with expression of estrogen and progesterone receptors, but not with HER2 expression. Mean Vav1 expression values of groups were compared using the Unpaired Student's <i>t</i>-test, and normalized to each other. (*) indicates p<0.02 value.</p

The pro-apoptotic effect of Vav1 on MCF-7 cells is p53-dependant.

<p>(A) p53 is silenced in MCF-7Vav1 cells following treatment with p53 shRNA. Cells were infected with viral vectors containing shRNA for p53 (shp53) or control (pLKO). Cell lysates were subjected to immunoblotting with anti-p53 antibody. Anti-actin was used as loading control. (B) Real Time PCR analysis of selected apoptosis-related genes. The analysis was performed on the following cells: MCF-7Vector or MCF-7Vav1 infected with control viral vector and MCF-7Vav1 infected with shp53. (C) TUNEL assay of MCF-7Vav1 infected with pLKO or shp53 was performed. Three representative photographs of the cells from different areas are presented. (D) MCF-7Vav1 infected with pLKO or shp53 were suspended in RPMI medium containing 0.3% agar and 10% calf serum, and plated onto a bottom layer containing 0.8% agar. Cells were plated in triplicates, at a density of 1×10⁵/well in a 6-well plate, and colonies were photographed 14 days later. Three independent experiments were analyzed, and representative photographs of cells from different areas are presented.</p