Understanding the development of hormone-resistance mediated by the transcription factor FOXA1
- We have identified that the cell cycle regulator cyclin-dependent Kinase (CDK) 4 inhibits the binding of FOXA1 to the chromatin in hormone resistant cell lines. - Our biochemical data supports that FOXA1 interacts with CDK4 and that is likely phosphorylated by this kinase. - Our data might suggest that CDK4 inhibits FOXA1 and confers insensitivity to hormone therapy.The main goal of this study was to investigate kinases that control the function of the transcription factor FOXA1 in hormone resistant tumors. The results of our drug screening have revealed that one of the kinases controlling FOXA1 is the cell cycle regulator cyclin-dependent Kinase (CDK) 4. The use of a specific inhibitor for CDK4 increases the binding of FOXA1 to the chromatin in hormone resistant cell lines but not in hormone sensitive. Moreover, we have performed ChIP-sequencing of FOXA1 in hormone resistant cells that were treated with Palbociclib (an specific inhibitor against CDK4/6) to investigate how the inhibition of the kinase impacts the binding of FOXA1 to the chromatin. The results have revealed that the kinase prevents the binding of FOXA1 towards chromatin regions enriched with motifs for the transcription factor AP1 and the nuclear receptor Estrogen Receptor. Currently, we are validating our in vitro results with two PDX models: one hormone responsive and one hormone resistant. It is important to mention that Palbociclib is the first CDK4/6 selective inhibitor approved in February 2015 by the FDA for metastatic Estrogen receptor positive (ER+) breast cancers. Hence, our efforts are now focused to understand what is the role of CDK4 in the control of FOXA1 in hormone resistant patients. Our efforts are also focused to understand how CDK4 controls FOXA1. We have performed biochemical experiments that confirm an interaction between the CDK4 and FOXA1 in hormone resistant cells but not in hormone sensitive. Interestingly, the interaction of both proteins occurs when the kinase is active (when the kinase interacts with the activator Cyclin D1). Moreover, we have performed additional biochemical experiments revealing that FOXA1 is phosphorylated at the serine 234, which is located at the domain of interaction of the transcription factor with the DNA. Interestingly, this serine is a consensus phosphorylation site for CDK, which suggests that FOXA1 might be phosphorlated by CDK4. Previously, it has been reported that the phosphorylation of other forkhead proteins at the same domain has a negative impact to the transcription factor. The proposed mechanism of inactivation occurs as follows: the phosphorylated transcription factor facilitates the interaction with the protein 14-3-3 that culminates with the protein export to the cytoplasm and inactivation of the transcription factor. Importantly, clinical studies have reported mutations at the same aminoacid. Now, we are: (1) validating whether FOXA1 is directly phosphorylated by CDK4, (2) investigating whether such phosphorylation inactivates FOXA1 and (3) how the mutation contributes to the phosphorylation of FOXA1 and its function. As mentioned earlier, our efforts are now focused to elucidate the role of CDK4 in the control of FOXA1 and its implication in hormone resistant tumors. Our hypothesis of work is that in hormone resistant tumors, CDK4 might inactivating FOXA1 by phosphorylation and its nuclear export. Importantly, we previously reported that the function of ER is fully dependent of FOXA1. Hence, tumors with an increased activity of CDK4 might be less likely responsive to the hormone therapy. We believe that the inactivation of FOXA1 by CDK4 phosphorylation might impair the function of ER and therefore the tumor become less responsive to anti-ER drugs. The experiments that we are currently performing should validate our hypothesis.
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.