Understanding the development of hormone-resistance mediated by the transcription factor FOXA1
We have identified kinases as potential regulators of FOXA1 function. Importantly, FOXA1 is regulated by exclusive kinases in hormone-resistant cell lines. among of these kinases we have identified drugs targeting FGFR, HER2, CDK4/6 and mTOR (the last two are already on clinical trials studies for patients resistant to hormono-therapy). Resistance to endocrine therapy is complex, heterogeneous and may differ from patient to patient. The majority of clinical trials thus far have focused on combining or alternating endocrine therapy agents, or intercalating targeted therapies against kinase inhibitors such as CDK, PI3K-AKT- mTOR, EGF or HER2. However, it seems clear that given the heterogeneity of resistance mechanisms and the toxicity and side effects associated with some alternative treatments, biomarker selection to stratify patients into clinically meaningful high and low risk groups in a move towards personalized therapy will be a crucial part of successfully combatting resistance to endocrine therapy. The current research of our group and previous findings support the notion that FOXA1 plays a key role in regulating tumor growth in hormone resistant patients. We believe that FOXA1 mediates the proliferation triggered by the above mentioned kinases and that are currently therapies in hormone-resistant patients. To test our hypothesis, we performed a drug screening with 550 compounds targeting kinases and phosphatases to analyze which of these proteins might be potential regulators of FOXA1. We first constructed a FOXA1 responsive luciferase reporter plasmid. The promoter of the TFF1 gene which is FOXA1 regulated, was inserted upstream of the luciferase open reading frame, and the FOXA1 activity is therefore reflected by the luciferase signal. Subsequently we performed the chemical screening in hormone- sensitive and hormone-resistant cell lines, which were positive for the expression of FOXA1. From the readout of the reporter signal, we got an indication of the role of the different drug targets regarding regulation of FOXA1 activity. In our first screening we identified 35 drugs that significantly inhibited the reporter signal in hormone sensitive cell lines and 144 drugs in hormone-resistant cell lines. Interestingly, only kinase inhibitors were identified in our screening. To select exclusively kinases that might control directly FOXA1, we performed an in silico FOXA1 phosphorylation prediction analysis (Group-based prediction system 3.0) to distinguish potential FOXA1 phosphorylation sites and kinases for FOXA1. Then by crossing the results from the first targeting FOXA1. Then, we repeated the drug screening with 45 inhibitors targeting these potential kinases. To avoid off-target effects and cell toxicity, cells were exposed with lower (5µM and 1µM) concentrations than for the first screening. From the second screening, we now identified 20 kinases and some of them are already used as targets in breast cancer therapy. In addition, we also identified other interesting targets, which have already been shown to be associated with hormone resistance: FGFR, mTOR, PI3K, CDK, HER2, FAK, JAK, PLK, PKC and JNK. Taken together, our preliminary results suggest that FOXA1 might be mediating the signals of these kinases in the control of proliferation for hormone-resistant patients. The future research of our group will focus on FGFR, mTOR and CDK kinases.