FOXA1 Mutations Reveal Distinct Chromatin Profiles and Influence Therapeutic Response in Breast Cancer.

Mutations in the pioneer transcription factor FOXA1 are a hallmark of estrogen receptor-positive (ER+) breast cancers. Examining FOXA1 in ∼5,000 breast cancer patients identifies several hotspot mutations in the Wing2 region and a breast cancer-specific mutation SY242CS, located in the third ß strand. Using a clinico-genomically curated cohort, together with breast cancer models, we find that FOXA1 mutations associate with a lower response to aromatase inhibitors. Mechanistically, Wing2 mutations display increased chromatin binding at ER loci upon estrogen stimulation, and an enhanced ER-mediated transcription without changes in chromatin accessibility. In contrast, SY242CS shows neomorphic properties that include the ability to open distinct chromatin regions and activate an alternative cistrome and transcriptome. Structural modeling predicts that SY242CS confers a conformational change that mediates stable binding to a non-canonical DNA motif. Taken together, our results provide insights into how FOXA1 mutations perturb its function to dictate cancer progression and therapeutic response.

Chromatin Structure and the Pioneering Transcription Factor FOXA1 Regulate TDG-Mediated Removal of 5-Formylcytosine from DNA.

Although a functional relationship between active DNA demethylation and chromatin structure is often implied, direct experimental evidence is lacking. We investigated the relationship between chromatin structure and thymine DNA glycosylase (TDG) using chemically defined nucleosome arrays containing site-specifically positioned 5-formylcytosine (5fC) residues. We show that the extent of array compaction, as well as nucleosome positioning, dramatically influence the ability of TDG to excise 5fC from DNA, indicating that the chromatin structure is likely a key determinant of whether 5fC is removed from the genome or retained as an epigenetic mark. Furthermore, the H2A.Z/H3.3 double-variant nucleosome and the pioneering transcription factor forkhead box A1 (FOXA1), both of which are implicated in shaping the chromatin landscape during demethylation of tissue-specific enhancers, differentially regulate TDG activity on chromatin. Together, this work provides the first direct evidence that the higher order chromatin structure regulates active DNA demethylation through TDG and provides novel insights into the mechanism of 5fC turnover at enhancers.

FOXA1 mutations alter pioneering activity, differentiation and prostate cancer phenotypes.

Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression.

Distinct structural classes of activating FOXA1 alterations in advanced prostate cancer.

ABTRACT: Forkhead box A1 (FOXA1) is a pioneer transcription factor that is essential for the normal development of several endoderm-derived organs, including the prostate gland1,2. FOXA1 is frequently mutated in hormone-receptor-driven prostate, breast, bladder and salivary-gland tumours3-8. However, it is unclear how FOXA1 alterations affect the development of cancer, and FOXA1 has previously been ascribed both tumour-suppressive9-11 and oncogenic12-14 roles. Here we assemble an aggregate cohort of 1,546 prostate cancers and show that FOXA1 alterations fall into three structural classes that diverge in clinical incidence and genetic co-alteration profiles, with a collective prevalence of 35%. Class-1 activating mutations originate in early prostate cancer without alterations in ETS or SPOP, selectively recur within the wing-2 region of the DNA-binding forkhead domain, enable enhanced chromatin mobility and binding frequency, and strongly transactivate a luminal androgen-receptor program of prostate oncogenesis. By contrast, class-2 activating mutations are acquired in metastatic prostate cancers, truncate the C-terminal domain of FOXA1, enable dominant chromatin binding by increasing DNA affinity and-through TLE3 inactivation-promote metastasis driven by the WNT pathway. Finally, class-3 genomic rearrangements are enriched in metastatic prostate cancers, consist of duplications and translocations within the FOXA1 locus, and structurally reposition a conserved regulatory element-herein denoted FOXA1 mastermind (FOXMIND)-to drive overexpression of FOXA1 or other oncogenes. Our study reaffirms the central role of FOXA1 in mediating oncogenesis driven by the androgen receptor, and provides mechanistic insights into how the classes of FOXA1 alteration promote the initiation and/or metastatic progression of prostate cancer. These results have direct implications for understanding the pathobiology of other hormone-receptor-driven cancers and rationalize the co-targeting of FOXA1 activity in therapeutic strategies.

Resistance to bortezomib in breast cancer cells that downregulate Bim through FOXA1 O-GlcNAcylation.

Bortezomib (BTZ), a well-established proteasome inhibitor used in the clinical therapy, leads the modulation of several biological alterations and in turn induces apoptosis. Although clinical trials with BTZ have shown promising results for some types of cancers, but not for some others, including those of the breast. The molecular basis of BTZ resistance in breast cancer remains elusive. In the present study, we found that cellular O-GlcNAc modification was dramatically elevated by BTZ treatment in intrinsic resistant MCF-7 and T47D cells, but not in sensitive MDA-MB-231 cells. A progressive increase in O-GlcNAcylation characterized the increased acquired resistance of MDA-MB-231-derived cells. We showed that elevated O-GlcNAc subsequently modified breast cancer related pioneer factor FOXA1 and reduced its protein stability. Further, we demonstrated that FOXA1 attenuation was involved in transcriptional downregulation of proapoptotic Bim and thus suppressed breast cancer cell apoptosis. Finally, the combination of O-GlcNAc inhibitor L01 to BTZ sensitized resistant cells. Our results have revealed a new regulatory mechanism that involves O-GlcNAc elevation mediated Bim deficiency, which plays a key role in the apoptotic dysregulation and BTZ resistance in breast cancer cells.

Neural differentiation of fibroblasts induced by intracellular co-delivery of Ascl1, Brn2 and FoxA1 via a non-viral vector of cationic polysaccharide.

Direct reprogramming of other somatic cells into neurons is an alternative strategy for the recovery of an injured nervous system. In this work, we developed a new non-viral gene carrier based on Porphyra yezoensis polysaccharide (PYP). After modification with ethylenediamine, the cationized PYP (Ed-PYP) was combined with plasmids encoding Ascl1, Brn2 and FoxA1 to form spherical nanoscale particles (Ed-PYP-pABF nanoparticles). Cytotoxicity assays proved that Ed-PYP-pABF nanoparticles had a better safety profile than Lipofectamine 2000 and polyetherimide. Characterization tests illustrated that the Ed-PYP-pABF nanoparticles at an Ed-PYP:pABF weight ratio of 40:1 is a potential candidate for gene delivery, which was further supported by Western blot and plasmid encoding enhanced green fluorescence protein transfection. Based on this transfection strategy, we co-delivered pABF to 3T6 cells using Ed-PYP. ELISA indicated that the levels of brain-derived neurotrophic factor, nerve growth factors and sonic hedgehog reached a maximum at 14 days after the last transfection. Immunofluorescence and Western blot further exhibited positive expression of neurofilament 200, Nestin, glial fibrillary acidic protein, growth associated protein-43, ß-3tubulin, and microtubule associated protein 2, proving the successful conversion of 3T6 cells into neurons. Taken together, these results illustrated that a natural polysaccharide-based gene co-delivery system is a promising strategy for neural reprogramming.

Comprehensive Molecular Portraits of Invasive Lobular Breast Cancer.

Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.

Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties.

ChIP-exonuclease (ChIP-exo) is a modified ChIP-seq approach for high resolution mapping of transcription factor DNA sites. We describe an Illumina-based ChIP-exo method which provides a global improvement of the data quality of estrogen receptor (ER) ChIP and insights into the motif structure for key ER-associated factors. ChIP-exo of the ER pioneer factor FoxA1 identifies protected DNA with a predictable 8 bp overhang from the Forkhead motif, which we term mesas. We show that mesas occur in multiple cellular contexts and exist as single or overlapping motifs. Our Illumina-based ChIP-exo provides high resolution mapping of transcription factor binding sites.

Nuclear mobility and mitotic chromosome binding: similarities between pioneer transcription factor FoxA and linker histone H1.

There exists a hierarchy by which transcription factors can engage their target sites in chromatin, in that a subset of factors can bind transcriptionally silent, nucleosomal DNA, whereas most factors cannot, and this hierarchy is reflected, at least in part, in the developmental function of the factors. For example, transcription factors possessing the Forkhead box (Fox) DNA-binding domain contain an overall fold resembling that of linker histone and thus are structured to bind DNA, site specifically, in a nucleosomal context. Where tested, Fox factors bind early in the developmental or physiological activation of target genes, thereby enabling the binding of other factors that cannot engage chromatin on their own. To investigate the basis for early chromatin binding, we have used fluorescence recovery after photobleaching (FRAP) to analyze the mobility, in the live cell nucleus, of FoxA factors in comparison to linker histone and other transcription factors. We have further analyzed the factors for their ability to bind to chromatin in mitosis and thereby serve as epigenetic marks. The results indicate that the "pioneer" features of FoxA factors involve various chromatin-binding parameters seen in linker histones and that distinguish the factors with respect to their regulatory and mechanistic functions.

SMAD3 prevents binding of NKX2.1 and FOXA1 to the SpB promoter through its MH1 and MH2 domains.

Mechanisms of gene repression by transforming growth factor-beta (TGF-beta) are not well understood. TGF-beta represses transcription of pulmonary surfactant protein-B gene in lung epithelial cells. Repression is mediated by SMAD3 through interactions with NKX2.1 and FOXA1, two key transcription factors that are positive regulators of SpB transcription. In this study, we found that SMAD3 interacts through its MAD domains, MH1 and MH2 with NKX2.1 and FOXA1 proteins. The sites of interaction on NKX2.1 are located within the NH2 and COOH domains, known to be involved in transactivation function. In comparison, weaker interaction of FOXA1 winged helix, and the NH(2)-terminal domains was documented with SMAD3. Both in vitro studies and in vivo ChIP assays show that interaction of SMAD3 MH1 and MH2 domains with NKX2.1 and FOXA1 results in reduced binding of NKX2.1 and FOXA1 to their cognate DNA-binding sites, and diminished promoter occupancy within the SpB promoter. Thus, these studies reveal for the first time a mechanism of TGF-beta-induced SpB gene repression that involves interactions between specific SMAD3 domains and the corresponding functional sites on NKX2.1 and FOXA1 transcription factors.

Specific interactions of the wing domains of FOXA1 transcription factor with DNA.

FOX (forkhead box) transcription factors have diverse regulatory roles in development, signaling, and longevity, as well as being able to bind stably to target sites in silent chromatin. Crystal structure analysis showed that the FOXA DNA binding domain folds into a helix-turn-helix (HTH) motif flanked on either side by "wings" of polypeptide chain. The wings have the potential to interact with the DNA minor groove along the long axis of the DNA helix, flanking the HTH interactions with the major groove. Diverse FOX family homologs exist, and structural studies with certain DNA target sites suggest that neither of the wing regions are well ordered or provide a stable contribution to DNA target site binding. However, FOXA1 binds certain DNA target sites with high affinity, and as a monomer. To determine whether the wing domains contribute to stable DNA binding, we assessed complexes of FOXA with high and lower affinity DNA target sites by hydroxyl radical footprinting and site-directed mutagenesis. The data revealed clear protections predicted for wing interactions at the high affinity target, but less so at the lower affinity target, indicating that the wing domains stably interact with high affinity DNA sites for FOXA proteins.

The Foxa family of transcription factors in development and metabolism.

The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors has been the subject of genetic and biochemical study for over 15 years. During this time its three members, Foxa1, Foxa2 and Foxa3, have been found to play important roles in multiple stages of mammalian life, beginning with early development, continuing during organogenesis, and finally in metabolism and homeostasis in the adult. Foxa2 is required for the formation of the node and notochord, and in its absence severe defects in gastrulation, neural tube patterning, and gut morphogenesis result in embryonic lethality. Foxa1 and Foxa2 cooperate to establish competence in foregut endoderm and are required for normal development of endoderm-derived organs such as the liver, pancreas, lungs, and prostate. In post-natal life, members of the Foxa family control glucose metabolism through the regulation of multiple target genes in the liver, pancreas, and adipose tissue. Insight into the unique molecular basis of Foxa function has been obtained from recent genetic and genomic data, which identify the Foxa proteins as 'pioneer factors' whose binding to promoters and enhancers enable chromatin access for other tissue-specific transcription factors.