CCAAT-displacement protein/cut homeobox transcription factor (CUX1) represses estrogen receptor-alpha (ER-α) in triple-negative breast cancer cells and can be antagonized by muscadine grape skin extract (MSKE).

Triple-Negative Breast Cancers (TNBCs) are the most difficult to treat subtype of breast cancer and are often associated with high nuclear expression of Snail and Cathepsin L (Cat L) protease. We have previously shown that Snail can increase Cat L expression/activity in prostate and breast cancer cells. This study investigated the role of CUX1 (a downstream substrate of Cat L) in TNBC. We showed that Cat L and CUX1 were highly expressed in TNBC patient tissue/cell lines, as compared to ER-positive samples, using cBioportal data and western blot/zymography analyses. Additionally, luciferase reporter and chromatin immunoprecipitation assays showed that CUX1 directly bound to estrogen receptor-alpha (ER-α) promoter in MDA-MB-468, a representative TNBC cell line, and that CUX1 siRNA could restore ER-α transcription and protein expression. Furthermore, Snail and CUX1 expression in various TNBC cell lines was inhibited by muscadine grape skin extract (MSKE, a natural grape product rich in anthocyanins) or Cat L inhibitor (Z-FY-CHO) leading to decreased cell invasion and migration. MSKE decreased cell viability and increased expression of apoptotic markers in MDA-MB-468 cells, with no effect on non-tumorigenic MCF10A cells. MSKE also decreased CUX1 binding to ER-α promoter and restored ER-α expression in TNBC cells, while both MSKE and CUX1 siRNA restored sensitivity to estradiol and 4-hydoxytamoxifen as shown by increased cell viability. Therefore, CUX1 activated by Snail-Cat L signaling may contribute to TNBC via ER-α repression, and may be a viable target for TNBC using natural products such as MSKE that targets cancer and not normal cells

Association of Epithelial Mesenchymal Transition with prostate and breast health disparities.

African Americans (AA) have higher death rates due to prostate and breast cancer as compared to Caucasian Americans (CA), and few biomarkers have been associated with this disparity. In our study we investigated whether epithelial-mesenchymal transition (EMT) with a focus on Snail and Cathepsin L (Cat L), could potentially be two markers associated with prostate and breast health disparities. We have previously shown that Snail can increase Cat L protein and activity in prostate and breast cancer. Western blot and real-time PCR analyses showed that mesenchymal protein expression (Snail, vimentin, Cat L) and Cat L activity (shown by zymography) was higher in AA prostate cancer cells as compared to CA normal transformed RWPE-1 prostate epithelial cells, and androgen-dependent cells, and comparable to metastatic CA cell lines. With respect to breast cancer, mesenchymal markers were higher in TNBC compared to non-TNBC cells. The higher mesenchymal marker expression was functionally associated with higher proliferative and migratory rates. Immunohistochemistry showed that both nuclear Snail and Cat L expression was significantly higher in cancer compared to normal for CA and Bahamas prostate patient tissue. Interestingly, AA normal tissue stained higher for nuclear Snail and Cat L that was not significantly different to cancer tissue for both prostate and breast tissue, but was significantly higher than CA normal tissue. AA TNBC tissue also displayed significantly higher nuclear Snail expression compared to CA TNBC, while no significant differences were observed with Luminal A cancer tissue. Therefore, increased EMT in AA compared to CA that may contribute to the more aggressive disease

"I Hate to be the Feminist Here"...Reading the Post-Epitaph Chick Flick

Numerous accounts in recent years have announced (often zealously) the death of the chick flick, a genre whose output has conspicuously slowed since a peak in the 1990s. We analyse here two recent hits from the “post-epitaph” phase in order to understand the kinds of generic updates such films are attempting and how they place women in regard to changing gender norms and the conditions of neoliberal capitalism. We find that in Trainwreck (2015) and The Intern (2015) female protagonists’ affiliation with patriarchal power structures is re-confirmed and both films ultimately retain a commitment to a rigid order unwilling to countenance female sexual agency or envision more flexible economic arrangements

Proteomics-Metabolomics Combined Approach Identifies Peroxidasin as a Protector against Metabolic and Oxidative Stress in Prostate Cancer

Peroxidasin (PXDN), a human homolog of Drosophila PXDN, belongs to the family of heme peroxidases and has been found to promote oxidative stress in cardiovascular tissue, however, its role in prostate cancer has not been previously elucidated. We hypothesized that PXDN promotes prostate cancer progression via regulation of metabolic and oxidative stress pathways. We analyzed PXDN expression in prostate tissue by immunohistochemistry and found increased PXDN expression with prostate cancer progression as compared to normal tissue or cells. PXDN knockdown followed by proteomic analysis revealed an increase in oxidative stress, mitochondrial dysfunction and gluconeogenesis pathways. Additionally, Liquid Chromatography with tandem mass spectrometry (LC-MS/MS)-based metabolomics confirmed that PXDN knockdown induced global reprogramming associated with increased oxidative stress and decreased nucleotide biosynthesis. We further demonstrated that PXDN knockdown led to an increase in reactive oxygen species (ROS) associated with decreased cell viability and increased apoptosis. Finally, PXDN knockdown decreased colony formation on soft agar. Overall, the data suggest that PXDN promotes progression of prostate cancer by regulating the metabolome, more specifically, by inhibiting oxidative stress leading to decreased apoptosis. Therefore, PXDN may be a biomarker associated with prostate cancer and a potential therapeutic target

Differentially expressed proteins in MSKE-treated and control cultures.

<p>Differentially expressed proteins in MSKE-treated and control cultures.</p

MSKE induces autophagy-mediated apoptosis.

<p>(A) C4-2 prostate cancer cells were treated with increasing concentrations of 0μ g/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL for 72 h. The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% sodium citrate and 0.1% Triton X. DNA fragmentation was determined by TdT-mediated dUTP nick end labeling (TUNEL). TUNEL assay (green channel). DAPI (blue channel) is used to locate the nuclei of the cells. (B) Cells were stained with Annexin V- Alexa Fluor 488 and PI and analyzed by flow cytometry following treatment with 5 μg/mL MSKE with or without 20 μM chloroquine. Percent of cells in the lower right quadrant that represent Annexin V ⁺/PI^- or early apoptotis and cells in the upper right quadrant that represent Annexin V ⁺/PI⁺ or late apoptotis was graphed. (C) Western blot analysis was performed to examine pro-apoptotic markers (Bax, cleaved caspase-3 and -7) and anti-apoptotic marker (BCL2) following treatment with MSKE in the presence or absence of 20 μM chloroquine. Actin was utilized as a loading control. (D) Results of western blot were analyzed using Image J and graphed. The experiments were performed in triplicate at least two times independently. Graphical data represents three independent experiments; * means 0.05 > <i>p</i> value > 0.01, ** means 0.01 > <i>p</i> value > 0.001, and *** means <i>p</i> value < 0.001.</p

MSKE treatment induces autophagy.

<p>(A) C4-2 prostate cancer cells were treated with MSKE (0 μg/mL, 5 μg/mL, 10 μg/mL, and 20 μg/mL) for 72 h. Fixation was performed with methanol/ethanol 1:1 volume followed by washes with 1× PBS. The cells were then exposed to acridine orange (5 μg/ml) for 15 min at 37°C, followed by washes with 1× PBS, prior to counterstaining with DAPI. We observed that treatment with higher concentrations of MSKE (10 and 20μg/ml) showed extensive acridine orange leakage into the cytosol, producing a diffuse yellow color and an increase in lysosomes indicating that MSKE induces cell death via autophagy compared to control. (B) C4-2 cells were treated with MSKE, with and without 20 μM chloroquine, for 72 h. The cells were then exposed to acridine orange (5 μg/ml) for 15 min at at 37°C, followed by washes with 1× PBS, prior to counterstaining with DAPI. Chloroquine treatment reversed the effects of MSKE. (C) Immunofluorescence staining for LC3B was performed on C4-2 cells treated with MSKE plus or minus chloroquine for 72 h. (D) Western blot analysis for LC3B was performed on C4-2 cells treated with MSKE plus/minus chloroquine. Image J analysis was performed to plot the ratios of LC3BI and LC3BII relative to actin loading control. The results are representative of experiments that have been performed in triplicate at least two times independently. Graphical data represents three independent experiments; * means 0.05 > <i>p</i> value > 0.01, ** means 0.01 > <i>p</i> value > 0.001.</p

Proposed model highlighting unfolded protein response pathway with pro-apoptotic protein signatures triggered by ER stress in MSKE treated C4-2 prostate cancer cells.

<p>MSKE treatment of C4-2 cells promoted an unfolded protein response (UPR) pathway in a mitochondria-specific stress response (UPRmt) with pro- and anti-apoptotic protein signature triggered by ER stress. Strong ER stress and activation of the UPR initiate apoptosis. In contrast, mild UPR activation induces a beneficial ER homeotic response by reducing the load of misfolded proteins and by activating cellular protective mechanisms like autophagy. PERK mediates phosphorylation of eIF2α and ATF4-dependent transcriptional activation of autophagy proteins. The model highlights insufficient folding or degradation capacity in the mitochondria, contributing to apoptosis.</p

Quantitative Western blot of key ER stress markers.

<p>Western blot analysis in C4-2 cells treated with 20 μg/ml MSKE as compared to ethanol-treated controls. As a loading control total protein from Ponceau S staining was assessed. Expression of ER stress markers IRE1-alpha and GRP78 and pro-apoptotic markers DFF45, PARP and caspase-12 was analyzed and quantification of western blot analysis performed using Image J, NIH. The standard deviation was used to assess data dispersion.</p