Elucidating the role of intracellular cell-signaling pathways controlling FOXA1 functions in Breast Cancer

Oestrogen Receptor (ER) mediates the response to oestrogens and it is responsible for driving proliferation in approximately 70% of the breast cancer patients. Targeting ER is a very successful therapeutic approach at clinical practice for patients expressing ER. However, a significant number of patients respond poorly to anti-ER therapies. A key determinant of the functions of ER is the transcription factor FOXA1. Hence, by identifying the regulators of FOXA1 in breast cancer patients with resistance to anti ER therapies we can open new strategies for the treatment of those patients. The results so far have demonstrated that FOXA1 is positively regulated in resistant breast cancer cells by kinases and protein deacetylates. Among all the candidates, we have identified and further characterized that the cell cycle kinases CDK2/4 and histone deacetylases HDAC2/4 are key positive regulators of FOXA1 in hormone resistant cancers. Moreover, in this research we have identified the sites of phosphorylation or acetylation of FOXA1. In particular, mutation of the sites phosphorylated by CDK4 has an effect in the binding of FOXA1 to chromatin. When mutated, FOXA1 binds to genes involved in cancer pathways (Her2 and PTEN) and for evasion of apoptosis. Importantly, the inhibition of all the kinases and protein deacetylases identified had a negative impact into the proliferation of the cells resistant to anti ER therapies. Future experiments in animal models will validate whether the inhibition of those FOXA1 regulators can inhibit growth and FOXA1 function in resistant tumours.

We have identified cyclin dependent kinases (CDKs) as regulators of the activity of the transcription factor FOXA1. We have characterised the specific CDK complexes that impinge on its function (CDK4 and CDK2) and we have also identified the sites that have an impact on FOXA1 function. We have generated mutant versions of FOXA1 that cannot be phosphorylated and we have interrogated their genomic location. Importantly, they reprogram the binding of FOXA1 to chromatin, so that it interacts with genes involved in cancer pathways (e.g. Her2 and PTEN) and apoptosis evasion (e.g. Bcl2). Our results show that CDK negatively regulates FOXA1 on specific genomic locations involved in proliferation. This is particularly relevant since CDK inhibitors are currently used for advanced breast cancer treatment but resistance is starting to emerge. Here we show that a potential mechanism involves the reprogramming of FOXA1 binding to chromatin.

The present application is compatible with the Research Council of Norway?s call and priorities as set out in the FRIMEDBIO Program for Young Research Talents. Breast cancer is a heterogeneous disease. Tumors are generally classified into ER positive, HER2 positive and the triple negative (TN) subtype, which lacks hormone receptors and HER2. Considering the expression of these two markers, 70-75% of the tumors are ER positive; 20% fall in the group of HER2 positive and the remaining are the TN. Out of this 20%, half of them are also ER positive. Patients can be treated with therapies targeting these factors, which are known to induce proliferation. Unfortunately, a very significant number of patients become resistant to these therapies. One of the principal challenges in the field is the identification of alternative targets in the resistant tumors. The FOXA1 transcription factor is expressed in ER and HER2 tumors and its expression is associated with proliferation, metastasis and differentiation. Recently, my team has identified a new role for FOXA1 in breast cancers as a mediator of HER2 signal and as a potential therapeutic target for the treatment of resistant tumors to anti-ER therapies (abstract attached). Breast cancer cell proliferation results from many different factors that activate multiple intracellular signaling pathways. This work besides on the hypothesis that FOXA1 integrates input signals originating from multiple cell-signaling pathways to generate output responses that culminate in proliferation, differentiation or metastasis in HER2 as well as in ER breast cancer subtypes. By means of chemical biology, functional genomics and proteomic methods in combination with in vivo and in vitro models, this research aims to elucidate which cell-signaling pathways are required to control FOXA1 functions and how they execute its control. Identification of the key regulatory elements responsible for FOXA1 functions might be used as future therapeutic target.