- Introduction
Already in 1896 Thomas Beatson described that removal of the ovaries in advanced breast cancer patients, often resulted in remarkable improvement. With that he had discovered the stimulating effect of the female ovarian hormone (estrogen) on breast cancer, even before the hormone itself was discovered. His work provided a foundation for the modern use of hormone therapy for the treatment and prevention of breast cancer. Only much later the cellular counterpart that mediated the described effects was discovered, the estrogen receptor. As it turned out this receptor plays a key role in the development and maintenance of the sexual reproductive tissues, and therefore, as Beatson had discovered, in breast cancer as well.
- Estrogen receptor
The estrogen receptor acts as a hormone dependent nuclear transcription factor. Upon entering the cell by passive diffusion, estrogen binds the receptor which subsequently dimerizes and translocates to the nucleus (see figure). There, ER binds specific sequences in the genome, called Estrogen Responsive Elements or EREs, and recruits a number of cofactors that facilitate gene transcription. Most estrogen receptor-positive breast cancers are fully dependent on this process for their growth, therefore these tumors are commonly treated with anti-estrogens that induce a conformational change in the receptor and prevent the recruitment of the cofactors essential for gene transcription. Unfortunately, resistance to these drugs forms a major problem in the clinic however. To study the mechanism by which the receptor can become drug-resistant, we set out to develop a sensitive assay that would allow us to directly visualize conformational changes in the receptor.- Approach
We coupled the human estrogen receptor to two color variants of the green fluorescent protein, YFP (yellow) at its N- and CFP (cyan) at its C-terminus, and created a stable cell line with this chimeric receptor. Next, we performed Fluorescence Resonance Energy Transfer (FRET) measurements on single nuclei. FRET can occur when the fluorophores on both ends of the receptor come in close proximity, for instance due to a conformational change after ligand binding (see figure). Where no detectable FRET change could be detected after the addition of the natural ligand estradiol, a rapid increase in FRET was observed after administration of the anti-estrogen tamoxifen, indicating that the receptor had undergone a conformational change and had been inactivated (see example trace). Using this assay we studied the effects of different factors that had previously been associated with anti-estrogen resistance.- Results
Our experiments revealed that phosphorylation of serine-305 in the hinge region of the estrogen receptor by Protein Kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERα-dependent transactivation instead (see figure). PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIα, was associated with tamoxifen-resistance in breast tumors prior to treatment. Enforced downregulation of this inhibitory subunit of PKA using RNAi kept the receptor in its active conformation and impairs tamoxifen action, ultimately resulting in tamoxifen resistant cell growth. Activation of PKA thus converts tamoxifen from an estrogen receptor inhibitor into an activator, a situation highly unwanted in breast cancer of course. Importantly, this form of resistance was not observed for a different anti-estrogen, Fulvestrant. Therefore, it is crucial to develop clinical assays that can determine resistance to tamoxifen beforehand, as these patients can then be treated with more strigent anti-estrogens.- Outlook
- The current focus of our research continues in two directions. First, we aim to develop a routine test for predicting tamoxifen resistance in breast cancer. Second, using our assay we further characterize different anti-estrogens and profile the requirements to generate resistance.
- Reference
- R. Michalides and A. Griekspoor, et al. Cancer Cell 2004 vol.5 pp.597-605. |
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Breast cancer is the most common form of cancer in women in the western world. Sadly, in the Netherlands 1 out of 9 women will ultimately develop breast cancer. Little is known about the factors that cause breast tumors to arise, although in 5-10% of the cases the disease is inherited. Except for women from these high-risk families, breast cancer is predominantly a disease of elderly, with increased risk when over 75 years of age. If possible, surgical removal of the cancer is the preferred choice of treatment. In addition, tumor growth is often dependent on the presence of hormones and a functional estrogen receptor in the tumor cells. Therefore, anti-estrogen treatment is commonly used as adjuvant therapy after surgery.
One of the most potent and widely used anti-estrogens is tamoxifen, a drug that can be considered to be one of the most successful anti-cancer compounds ever. Both in preventive and adjuvant therapy, as well as treatment of metastases, tamoxifen is effective in approximately 50% of the cases. Importantly, tamoxifen has relatively mild side-effects, and can be taken orally. One downside of tamoxifen however, is the fact that this drug has to taken for a long period, generally 5 years or longer.