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

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

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

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

A genomic catalog of Earth’s microbiomes

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes.</p