Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth.

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity.

Normal progression through the cell cycle requires the sequential action of cyclin-dependent kinases CDK1, CDK2, CDK4, and CDK6. Direct or indirect deregulation of CDK activity is a feature of almost all cancers and has led to the development of CDK inhibitors as anticancer agents. The CDK-activating kinase (CAK) plays a critical role in regulating cell cycle by mediating the activating phosphorylation of CDK1, CDK2, CDK4, and CDK6. As such, CDK7, which also regulates transcription as part of the TFIIH basal transcription factor, is an attractive target for the development of anticancer drugs. Computer modeling of the CDK7 structure was used to design potential potent CDK7 inhibitors. Here, we show that a pyrazolo[1,5-a]pyrimidine-derived compound, BS-181, inhibited CAK activity with an IC(50) of 21 nmol/L. Testing of other CDKs as well as another 69 kinases showed that BS-181 only inhibited CDK2 at concentrations lower than 1 micromol/L, with CDK2 being inhibited 35-fold less potently (IC(50) 880 nmol/L) than CDK7. In MCF-7 cells, BS-181 inhibited the phosphorylation of CDK7 substrates, promoted cell cycle arrest and apoptosis to inhibit the growth of cancer cell lines, and showed antitumor effects in vivo. The drug was stable in vivo with a plasma elimination half-life in mice of 405 minutes after i.p. administration of 10 mg/kg. The same dose of drug inhibited the growth of MCF-7 human xenografts in nude mice. BS-181 therefore provides the first example of a potent and selective CDK7 inhibitor with potential as an anticancer agent.