Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant

Dose-Related Estrogen Effects on Gene Expression in Fetal Mouse Prostate Mesenchymal Cells

Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from circulating estrogens and androgens into paracrine signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in morphological tissue development and sterol biosynthesis but suppress genes involved in growth factor signaling. Genes involved in cell adhesion were enriched among both up-regulated and down-regulated genes. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were enriched in the glycolytic pathway. At the highest dose (100 nM), E2 induced genes enriched for cell adhesion, steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF- β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas secreted growth factors and cytokines might play significant roles when estrogen level is high

HD CAGnome: A Search Tool for Huntingtin CAG Repeat Length-Correlated Genes

Background: The length of the huntingtin (HTT) CAG repeat is strongly correlated with both age at onset of Huntington’s disease (HD) symptoms and age at death of HD patients. Dichotomous analysis comparing HD to controls is widely used to study the effects of HTT CAG repeat expansion. However, a potentially more powerful approach is a continuous analysis strategy that takes advantage of all of the different CAG lengths, to capture effects that are expected to be critical to HD pathogenesis. Methodology/Principal Findings We used continuous and dichotomous approaches to analyze microarray gene expression data from 107 human control and HD lymphoblastoid cell lines. Of all probes found to be significant in a continuous analysis by CAG length, only 21.4% were so identified by a dichotomous comparison of HD versus controls. Moreover, of probes significant by dichotomous analysis, only 33.2% were also significant in the continuous analysis. Simulations revealed that the dichotomous approach would require substantially more than 107 samples to either detect 80% of the CAG-length correlated changes revealed by continuous analysis or to reduce the rate of significant differences that are not CAG length-correlated to 20% (n = 133 or n = 206, respectively). Given the superior power of the continuous approach, we calculated the correlation structure between HTT CAG repeat lengths and gene expression levels and created a freely available searchable website, “HD CAGnome,” that allows users to examine continuous relationships between HTT CAG and expression levels of ∼20,000 human genes. Conclusions/Significance: Our results reveal limitations of dichotomous approaches compared to the power of continuous analysis to study a disease where human genotype-phenotype relationships strongly support a role for a continuum of CAG length-dependent changes. The compendium of HTT CAG length-gene expression level relationships found at the HD CAGnome now provides convenient routes for discovery of candidates influenced by the HD mutation

Genome-wide analysis of YY2 versus YY1 target genes

Yin Yang 1 (YY1) is a critical transcription factor controlling cell proliferation, development and DNA damage responses. Retrotranspositions have independently generated additional YY family members in multiple species. Although Drosophila YY1 [pleiohomeotic (Pho)] and its homolog [pleiohomeotic-like (Phol)] redundantly control homeotic gene expression, the regulatory contributions of YY1-homologs have not yet been examined in other species. Indeed, targets for the mammalian YY1 homolog YY2 are completely unknown. Using gene set enrichment analysis, we found that lentiviral constructs containing short hairpin loop inhibitory RNAs for human YY1 (shYY1) and its homolog YY2 (shYY2) caused significant changes in both shared and distinguishable gene sets in human cells. Ribosomal protein genes were the most significant gene set upregulated by both shYY1 and shYY2, although combined shYY1/2 knock downs were not additive. In contrast, shYY2 reversed the anti-proliferative effects of shYY1, and shYY2 particularly altered UV damage response, platelet-specific and mitochondrial function genes. We found that decreases in YY1 or YY2 caused inverse changes in UV sensitivity, and that their combined loss reversed their respective individual effects. Our studies show that human YY2 is not redundant to YY1, and YY2 is a significant regulator of genes previously identified as uniquely responding to YY1

RNAi knockdown of CSK does not affect MCF-7 cell sensitivity to tamoxifen or paclitaxel.

<p>Cells were infected with empty lentivirus vector (pLKO.1) or two independent clones of lentiviruses expressing different shRNA species targeting CSK shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060889#pone-0060889-g001" target="_blank">Figure 1</a> (CSK KD#1 and #2) and then exposed to 1 µM 4-hydroxytamoxifen (4-OHT) for 10 days (A) or 1–1000 nM paclitaxel for 2 days (B). Cell viability was determined by crystal violet staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060889#pone.0060889.s003" target="_blank">Fig. S3</a>) and quantified by spectrophotometry (mean±SEM of three or more independent experiments).</p

CSK is required for fulvestrant-induced ERα protein degradation in MCF-7 cells.

<p>(A, B) RNAi knockdown of CSK protein expression caused resistance of intracellular ERα protein to fulvestrant-induced degradation: Western blotting. Cells were infected with control (pLKO.1) or two CSK-knockdown shRNA lentivirus clones and subjected to exposure to fulvestrant. Expression of ERα protein was determined by Western blotting at varying time points of exposure (A). Intensities of ERα protein bands were determined by densitometry (B, mean±SEM of three independent experiments. Asterisk indicates statistical significance, p<0.05). (C) Similar experiments as shown in panels (A, B) were performed, but amounts of ERα protein in total cellular protein were determined by ELISA (mean±SEM of three independent experiments; *, p<0.05 to vehicle control; #, p<0.05 to pLKO.1-infected cells exposed to fulvestrant for the same period).</p

Both fulvestrant and 17β-estradiol (E2) enhance proteasomal degradation of ERα protein in MCF-7 cells.

<p>(A–C) Fulvestrant (A) and E2 (B) caused time-dependent reduction in ERα protein expression: Western blotting. Intensities of ERα protein bands were determined by densitometry (C, mean±SEM of three independent experiments. Asterisks indicate statistical significance, p<0.05 to vehicle control). (D, E) E2 dose-dependent reduction in ERα protein expression. Cells were exposed to varying concentrations of E2 for 6 hours and subjected to Western blotting analysis of ERα protein (D). Intensities of ERα protein bands were determined by densitometry (E, mean±SEM of three independent experiments. Asterisk indicates t-test significance p<0.05 to vehicle control). (F–H), Pre-exposure to MG132 dose-dependently prevented reduction in ERα protein expression caused by fulvestrant (F) and E2 (G). Con, vehicle control (0.1% ethanol). Cells were exposed to varying concentrations of MG132 for 30 minutes and then exposed additionally to fulvestrant or E2 for 6 hours. Intensities of ERα protein bands were determined by densitometry (H, mean±SEM of three independent experiments. Asterisks indicate statistical significance, p<0.05).</p