PRP4K is a HER2-regulated modifier of taxane sensitivity

<p>The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified <i>in vitro</i>, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.</p

Potential Cross-Talk between Alternative and Classical NF-κB Pathways in Prostate Cancer Tissues as Measured by a Multi-Staining Immunofluorescence Co-Localization Assay

<div><p>Background</p><p>While the classical NF-κB/p65 pathway is known to be involved in prostate cancer progression and is associated with poor patient outcome, the role of the NF-κB /RelB alternative protein is not well defined. Here we analyzed the activation of both NF-κB pathways in prostate cancer tissues and correlate this activation with clinical features of the disease.</p><p>Methods</p><p>A multiple immunofluorescence technique was employed to concomitantly and quantitatively visualize the nuclear localization of p65 and RelB in 200 paraffin embedded samples. Epithelia were defined using appropriate fluorochrome markers and the resulting immunofluorescent signals were quantified with an automated scoring system.</p><p>Results</p><p>The nuclear frequency of p65 was found to be significantly increased in tumor tissues as compared with normal adjacent tissue, whereas the frequency for RelB was decreased (p < 0.001, Wilcoxon test). As previously reported, p65 nuclear frequency was associated with a risk of biochemical recurrence. Although, RelB nuclear frequency alone did not predict recurrence, the presence of activated RelB reduced the risk of recurrence associated with the activation of p65.</p><p>Conclusion</p><p>For the first time p65/RelB co-distribution was assessed in prostate cancer tissues and suggested a negative crosstalk between the two NF-κB pathways in prostate cancer progression.</p></div

Association between nuclear p65 and/or RelB and biochemical recurrence in prostate cancerpatients.

<p>Kaplan–Meier biochemical recurrence-free survival curves <b>A.</b> High (>20%) and low (<20%) frequency of nuclear p65 in the epithelia of cancer tissues. <b>B.</b> High (>5%) and low (<5%) frequency of nuclear RelB in the epithelia of cancer tissues. Significance (<i>p</i>) is indicated by log rank. <b>C.</b> Double nuclear epithelial staining of p65 and RelB. <b>D.</b> Epithelial and nuclear staining of p65 and RelB. The κB negative variable represents patients without positive tissue cores for p65 and RelB. The variable p65 alone or RelB alone represents patients positive for only nuclear p65 or nuclear RelB. The variable p65 + RelB are patients with presence of both nuclear subunits in the same core. <b>E.</b> Analysis of the epithelial proportion of nuclear p65 to RelB in tissue cores within both p65 and RelB staining. The ratio p65/RelB represents the frequency of p65 to the frequency of RelB. The variable p65 > RelB represents a ratio of at least 2 fold more p65 than RelB; p65-RelB represents a ratio between 0.50 and 2 fold, and RelB>p65 represents a ratio of at least 2 fold more RelB than p65.</p

Univariate and multivariate Cox regression analysis.

<p>Cox regression models: significance (p < 0.05) and Hazard ratio (HR) are indicated. CI: Confidence interval. PSA: Prostate Specific Antigen. NF-κB variables (p65 and RelB): status in cancer tissues. Significance is considered at p < 0.5.</p><p>Univariate and multivariate Cox regression analysis.</p

Example of immunofluorescence of p65 and RelB.

<p><b>A.</b> Magnification (40X) highlighting glandular structures in prostate cancer tissues. Merge: superimposed images of RelB (A488) in green, p65 (Cy5) in red and nuclei (DAPI) in blue, in normal adjacent (top) or cancer tissues (bottom). Arrows show stained nuclei. <b>B</b>. Comparative quantification from IHC (left) and IF (right) staining of p65. Graphs show the frequency distribution of nuclear p65 as evaluated visually (IHC) or automatically (IF). <b>C</b>. Frequency of nuclear p65 (left), RelB (middle) and double stained nuclei (right) in normal adjacent and tumor tissues. The comparison between adjacent normal and tumor cores was conducted using a Wilcoxon‘s test.</p

Nuclear co-localization of p65 and RelB by a quadruple multiple staining approach.

<p><b>A.</b> Epithelial staining with CK18, CK19 and PSA defines the epithelial mask using orange fluorochromes (A546, Cy3). <b>B.</b> Identification of epithelial area (presented in grey for optimal contrast in subsequent analyses) as ROI #1 (region of interest #1). Identification of nuclei (grey surrounded by red tracing) as ROI#2 from pre-defined ROI#1. P65 and RelB fluorescence were subsequently evaluated separately in ROI#1 and #2. <b>C.</b> RelB staining with green fluorescent dye (A488) and p65 staining with red fluorescent dye (Cy5). Steps 1, 2 and 3 correspond to the fluorescence analysis process followed using Visiomorph DP software.</p

Association between nuclear p65 and/or RelB and biochemical recurrence in prostate cancerpatients.

<p>Kaplan–Meier biochemical recurrence-free survival curves <b>A.</b> High (>20%) and low (<20%) frequency of nuclear p65 in the epithelia of cancer tissues. <b>B.</b> High (>5%) and low (<5%) frequency of nuclear RelB in the epithelia of cancer tissues. Significance (<i>p</i>) is indicated by log rank. <b>C.</b> Double nuclear epithelial staining of p65 and RelB. <b>D.</b> Epithelial and nuclear staining of p65 and RelB. The κB negative variable represents patients without positive tissue cores for p65 and RelB. The variable p65 alone or RelB alone represents patients positive for only nuclear p65 or nuclear RelB. The variable p65 + RelB are patients with presence of both nuclear subunits in the same core. <b>E.</b> Analysis of the epithelial proportion of nuclear p65 to RelB in tissue cores within both p65 and RelB staining. The ratio p65/RelB represents the frequency of p65 to the frequency of RelB. The variable p65 > RelB represents a ratio of at least 2 fold more p65 than RelB; p65-RelB represents a ratio between 0.50 and 2 fold, and RelB>p65 represents a ratio of at least 2 fold more RelB than p65.</p

Spearman correlation test between NF-κB and clinic-pathological parameters.

<p>Significant Spearman correlations are indicated in bold. Spearman correlations were considered significant at <i>p</i> < 0.05.</p><p>Spearman correlation test between NF-κB and clinic-pathological parameters.</p

BT3.2 expression in ovarian cancer cells.

<p><b>A</b>. Western-blot analysis of total protein extracts from TOV112D cell line infected with PLenti (vector control), BT3.1, BT3.2 or BT3.3 viral constructs. Extracts were loaded in triplicate on 10% SDS/PAGE gel and membranes were hybridized with anti-CD277 (eBioscience), anti-BT3.3 (Atlas Antibodies) and anti-BT3.2 (SDIX Inc.) as indicated at the bottom of the Figure. <b>B</b>. Immunohistochemistry analysis of paraffin-embedded cell pellets from TOV112D cell line infected with either PLenti (vector control), BT3.1, BT3.2 or the BT3.3 viral constructs. Only cells transfected with the BT3.2 construct stained positively with the anti-BT3.2 (SDIX Inc.). <b>C</b>. Immunohistochemistry of xenograft tumors from TOV112D transfected with either empty vector or BT3.2 construct. Only cells infected with the BT3.2 construct stained positively with the anti-BT3.2 (SDIX Inc.). <b>D</b>. Representative staining for immunohistochemistry of BT3.2 on a high-grade serous EOC TMA. From left to right: negative, low, moderate and high intensity.</p

Pearson correlation test (two-tailed) between intra-epithelial immune infiltrate and clinical parameters in EOC patients.

<p>Table showing the coefficient of correlation with a Pearson correlation test (Pearson Correlation) between clinical parameters and immune infiltrate density in the intra-epithelial ovarian tumor tissues. Grade and stage were evaluated according to the FIGO classification. Res. Dis. =  amount of residual disease at primary resection of the tumor as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038541#pone-0038541-t001" target="_blank">Table 1</a>. Recurrence is the first event of ovarian cancer recurrence after the primary resection of the tumor. Death is the event of death due to ovarian cancer. CA125 =  blood serum level CA125 at the date of primary resection of the ovarian tumor. P is the p value from Pearson correlation test. N is the number of cases included in the statistical analysis. C =  Pearson correlation coefficient. P = p value. N =  number of cases.</p