Research interest
Structural and functional analysis of the Estrogen Receptor to identify the mechanism of resistance to anti-estrogens used in the clinic. Our group is interested in resolving the mechanism of resistance to anti-estrogens in breast cancer. Estrogen Receptor (ER) positive breast cancer patients are being treated either with withdrawal of estrogen via aromatase inhibitors or with anti-estrogens that inhibit the transcriptional activity of the ER. Still, in approximately 50% of the cases resistance to anti-estrogens occurs. Anti-estrogens act by altering the protein conformation of the ER, thereby preventing the full assembly of a functional transcription complex. The various anti-estrogens alter the protein conformation of ER to different extents. We found that phosphorylation of ER at specific amino acids affects the conformation of ER as such that the inhibitory interaction with anti-estrogens is abrogated. These phosphorylations are the results of activation of other signaling pathways, including the MAPkinase and Protein Kinase A pathways. The combination of these phosphorylation events renders the ER resistant to anti-estrogens, where specific combinations of phosphorylations of ER are characteristic for resistance to particular anti-estrogens. Aim of our research is to reveal the mechanism of this kind of resistance to anti-estrogens and to disclose the relevance of the modifications of ER for resistance to endocrine treatment in breast cancer patients.
To identify the conformational alterations in ER, we use combinations of biophysical and biochemical assays, where we follow changes in energy transfer and mobility of fluorescently tagged ER proteins in life cells when they are exposed to different anti-estrogens. By making use of mutants of ER that mimic specific phosphorylation sites in ER, we study the effect of specific phosphorylations or combinations thereof, on resistance to anti-estrogens. We verify these effects in traditional reporter read-out assays and in growth assays where we can examine the effects of each phospho-mutant separately. For this purpose we are currently generating breast cancer cells infected with retroviruses that contain the particular phospho-mutant of ER, and eliminate expression of the endogenous ER by miRNA. These cells are also used to investigate transcripts that are specifically associated with resistance to particular anti-estrogens via microarray and CHIP-on chip analysis.
Recently, we have revealed the mechanism of resistance to one of these anti-estrogens, tamoxifen, by Protein Kinase A (PKA)-mediated phosphorylation of ER at Serine 305. This phosphorylation renders tamoxifen from an antagonist under normal conditions to an agonist when ER is phosphorylated at Serine 305 by PKA. This specific phosphorylation alters the orientation between ER and a cofactor in the transcription complex as such that now RNA polymerase II can bind to the complex in the presence of tamoxifen, whereas it normally does not bind to ER under these conditions.
Furthermore, we have generated an antibody that specifically recognizes the phosphorylated Serine 305 in ER, and use this antibody to investigate a possible predictive role for phosphorylation of Serine 305 in ER in human breast cancer (in collaboration with dr Sabine Linn, dept of Molecular Pathology).
Together, these observations illustrate specific effects of other signaling pathways on ER that alter sensitivity towards anti-estrogens. In this way, we aim to investigate which specific modifications of ER are indicative for resistance to a particular anti-estrogen and to develop screening assays to predict resistance to anti-estrogens in vitro, and from there on to come to prediction of resistance to particular anti-estrogens in the clinic.
key publications
Wilbert Zwart, Alexander Griekspoor, Valeria Berno, Kim Lakeman, Kees Jalink, Michael Mancini, Jacques Neefjes, Rob Michalides. PKA-induced resistance to tamoxifen is associated with an altered orientation of ERa towards co-activator SRC-1. EMBO J, accepted for publication.
Wilbert Zwart, Alexander Griekspoor, Mariska Rondaij, Desiree Verwoerd, Jacques Neefjes, Rob Michalides. Classification of anti-estrogens according to intramolecular FRET effects on phospho-mutants of estrogen receptor a. Mol Cancer Ther 2007;6(5):1526-1533.
Rob Michalides, Alexander Griekspoor, Astrid Balkenende, Desiree Verwoerd, Lennert Janssen, Kees Jalink, Arno Floore, Laura van ‘t Veer, and Jacques Neefjes. Tamoxifen resistance by a conformational arrest of the estrogen receptor a after PKA activation in breast cancer. Cancer Cell 5, 597-605, 2004.
Eric Bindels, Francois Lallemand, Astrid Balkenende, Desiree Verwoerd. Rob Michalides. Overexpression of G1/S cyclins affects oestrogen independent growth by sequestration of inhibitors. Oncogene 21, 8158-8165, 2002.
Zwijsen RML, E Wientjens, R Klompmaker, J van der Smam, R Bernards, RJAM Michalides. CDK-independent activation of estrogen receptor by cyclin D1. Cell 88, 405-415, 1997.
More publications by Rob Michalides on PubMed
Biographic sketch
Rob Michalides performed his graduate research in the laboratory of Jeffrey Schlom (Columbia University New York, and NIH, Washington) and obtained his Ph.D. in 1975. He has been connected with the Netherlands Cancer Institute since 1975, studying involvement of retroviruses and of adhesion molecules in cancer, and investigating the role of cell cycle regulators in cancer. His studies have been dealing with basal and translational aspects of human cancer. Currently, as an Assistant Professor at the Netherlands Cancer Institute, he is using molecular endocrinology to identify factors that contribute to resistance to endocrine treatment of breast cancer.
Co-workers
Rondaij, Mariska PhD Postdoctoral fellow
Zwart, Wilbert Graduate student
De Leeuw, Renee Graduate student
Verwoerd, Desiree MSc Technical staff
Bentin-Toaldo, Cristiane MSc Technical staff
Positions available
We have a vacancy for an undergraduate internship (“stage”) student. Please apply to r.michalides@nki.nl