Final Report Summary - ARKSS (A large-scale siRNA screen to study the role of kinases in Androgen Receptor activation)
Prostate cancer is extremely common disease that affects an estimated one out of every seven European men. Fortunately the majority of these patients can be effectively treated by surgery or radiotherapy, however patients with locally advanced, recurrent or metastatic forms of the disease are much more difficult to manage. These patients are typically treated with therapy designed to inhibit the activation of the androgen receptor (AR). While initially successful, the effectiveness of treatment is usually temporary and the surviving cancer almost always progress to a “castration-resistant” state (CRPC). Counter-intuitively extensive research has shown that AR signalling is directly involved in all stages of prostate cancer growth and disease progression, including CRPC. Given the recent clinical successes of Abiraterone and Enzalutamide, it is clear that the AR remains the best-validated therapeutic target to treat patients with CRPC.
Androgen dependent activation of the AR occurs through a well-characterized pathway. First the AR binds an androgenic ligand, such as testosterone or dihydrotestosterone, causing an allosteric modification that leads to the dissociation of chaperone and repressor proteins, homodimerization of the AR and then translocation of the receptor from the cytoplasm to the nucleus. Once in the nucleus, the AR binds various androgen response elements on the genome inducing an assembly with RNA polymerase II and other factors that lead to transcription of target genes. While this process appears simple, it is occurs through complex synchronized interactions with co-activators, co-repressors and epigenetic modifiers. Without these critical co-activators the AR cannot initiate transcription.
Therefore, we proposed in this project to identify essential co-activators required for AR-mediated transcription. Given their previously demonstrated role we initially focused on kinases. To allow comparison between the different kinases we proposed to conduct an siRNA screen to identify those kinases required for AR activation. Using a previously developed GFP reporter assay we conducted extensive optimization of the assay and transfection conditions. Following optimization we could see a moderate decrease in AR activation following siRNA knockdown with known kinase controls. However while the GFP reporter assay was suitable for small studies it was did not give the accuracy and reliability needed in such a large-scale screening project. In an effort to increase the dynamic range of the assay we conducted both fluorescent sorting and single-cell sorting. While this increased the signal this enriched assay could not reproducibly identify known kinases involved in AR-signalling. Therefore, to allow a more robust read-out we cloned, developed and validated additional AR specific assays. This has provided an inexpensive more robust AR readout that is being used in several additional projects. With this assay we will conduct a large-scale siRNA screen in multiple prostate cancer cell lines.
Concurrent to this work we also conducted a focused shRNA screen of epigenetic modifying enzymes in a model of castrate resistant prostate cancer to identify those essential co-activators in this lethal disease. From this we were able to identify a number of lysine demethylases that were essential for proliferation in CRPC. Interesting, our data suggests that that different epigenetic modifying enzymes are required in hormone sensitive and castrate resistant prostate cancer. Following extensive validation, we continued to study a poorly characterized H3K9 me1/2 lysine demethylase. Mechanistic studies demonstrated that down-regulation of this enzyme causes a decrease in AR mediated transcription. We are currently in collaboration with a major pharmaceutical company to test this enzyme as a potential pharmacological target in CRPC.
The Marie Curie CIG grant has been absolutely instrumental to my integration into both my University and the Turkish scientific community. It has allowed me to develop an independent research program that is currently supported by several national and international grants. Through these grants we have hired and trained graduate students that are making major contributions to our ongoing studies. Further, in an effort to disseminate information we have presented the results from this study at both national and international research conferences. The results from these studies will soon be prepared for publication. In addition to research I have also been able developed a strong teaching program and give well-received undergraduate and graduate level classes. Fundamentally, the Marie Curie CIG grant has been critical to establishing my professional independence in Turkey.
Androgen dependent activation of the AR occurs through a well-characterized pathway. First the AR binds an androgenic ligand, such as testosterone or dihydrotestosterone, causing an allosteric modification that leads to the dissociation of chaperone and repressor proteins, homodimerization of the AR and then translocation of the receptor from the cytoplasm to the nucleus. Once in the nucleus, the AR binds various androgen response elements on the genome inducing an assembly with RNA polymerase II and other factors that lead to transcription of target genes. While this process appears simple, it is occurs through complex synchronized interactions with co-activators, co-repressors and epigenetic modifiers. Without these critical co-activators the AR cannot initiate transcription.
Therefore, we proposed in this project to identify essential co-activators required for AR-mediated transcription. Given their previously demonstrated role we initially focused on kinases. To allow comparison between the different kinases we proposed to conduct an siRNA screen to identify those kinases required for AR activation. Using a previously developed GFP reporter assay we conducted extensive optimization of the assay and transfection conditions. Following optimization we could see a moderate decrease in AR activation following siRNA knockdown with known kinase controls. However while the GFP reporter assay was suitable for small studies it was did not give the accuracy and reliability needed in such a large-scale screening project. In an effort to increase the dynamic range of the assay we conducted both fluorescent sorting and single-cell sorting. While this increased the signal this enriched assay could not reproducibly identify known kinases involved in AR-signalling. Therefore, to allow a more robust read-out we cloned, developed and validated additional AR specific assays. This has provided an inexpensive more robust AR readout that is being used in several additional projects. With this assay we will conduct a large-scale siRNA screen in multiple prostate cancer cell lines.
Concurrent to this work we also conducted a focused shRNA screen of epigenetic modifying enzymes in a model of castrate resistant prostate cancer to identify those essential co-activators in this lethal disease. From this we were able to identify a number of lysine demethylases that were essential for proliferation in CRPC. Interesting, our data suggests that that different epigenetic modifying enzymes are required in hormone sensitive and castrate resistant prostate cancer. Following extensive validation, we continued to study a poorly characterized H3K9 me1/2 lysine demethylase. Mechanistic studies demonstrated that down-regulation of this enzyme causes a decrease in AR mediated transcription. We are currently in collaboration with a major pharmaceutical company to test this enzyme as a potential pharmacological target in CRPC.
The Marie Curie CIG grant has been absolutely instrumental to my integration into both my University and the Turkish scientific community. It has allowed me to develop an independent research program that is currently supported by several national and international grants. Through these grants we have hired and trained graduate students that are making major contributions to our ongoing studies. Further, in an effort to disseminate information we have presented the results from this study at both national and international research conferences. The results from these studies will soon be prepared for publication. In addition to research I have also been able developed a strong teaching program and give well-received undergraduate and graduate level classes. Fundamentally, the Marie Curie CIG grant has been critical to establishing my professional independence in Turkey.