FOXA1 Binding Research Articles

TET1 dioxygenase is required for FOXA2-associated chromatin remodeling in pancreatic beta-cell differentiation

Existing knowledge of the role of epigenetic modifiers in pancreas development has exponentially increased. However, the function of TET dioxygenases in pancreatic endocrine specification remains obscure. We set out to tackle this issue using a human embryonic stem cell (hESC) differentiation system, in which TET1/TET2/TET3 triple knockout cells display severe defects in pancreatic β-cell specification. The integrative whole-genome analysis identifies unique cell-type-specific hypermethylated regions (hyper-DMRs) displaying reduced chromatin activity and remarkable enrichment of FOXA2, a pioneer transcription factor essential for pancreatic endoderm specification. Intriguingly, TET depletion leads to significant changes in FOXA2 binding at the pancreatic progenitor stage, in which gene loci with decreased FOXA2 binding feature low levels of active chromatin modifications and enriches for bHLH motifs. Transduction of full-length TET1 but not the TET1-catalytic-domain in TET-deficient cells effectively rescues β-cell differentiation accompanied by restoring PAX4 hypomethylation. Taking these findings together with the defective generation of functional β-cells upon TET1-inactivation, our study unveils an essential role of TET1-dependent demethylation in establishing β-cell identity. Moreover, we discover a physical interaction between TET1 and FOXA2 in endodermal lineage intermediates, which provides a mechanistic clue regarding the complex crosstalk between TET dioxygenases and pioneer transcription factors in epigenetic regulation during pancreas specification.

Abstract 1458: Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues

Abstract Single cell mRNA atlases of multiple organs have been reported using scRNA-seq. We had previously reported single cell mRNA atlas of the breast. In this study, we performed scATAC-seq and scRNA-seq of breast tissues from 22 women of Ashkenazi (A) ancestry (19,935 nuclei), 20 women of European (non-A) ancestry (19,090 nuclei), six BRCA1 (6,261 nuclei) and five BRCA2 (3,400 nuclei) mutation carriers to correlate cell type-specific gene expression with chromatin accessibility. An integrated analysis revealed 17 cell clusters including basal, luminal progenitor, and mature luminal epithelial cells, two endothelial subtypes and two fibroblast/mesenchymal stem-like cell types. Basal cells could be further subdivided into two subclusters that differentially expressed genes such as TSHZ2, POSTN and ETV6. Similarly, there were four luminal progenitor subclusters that differentially expressed genes such as MALAT1, SOX13, and MDM4. Although ESR1 expression was observed predominantly in mature luminal cells, binding sites for ESR1 were accessible in multiple epithelial and non-epithelial cells with the exception of basal cells. FOXA1 expression and binding site enrichment were observed mostly in mature luminal cells. GATA3 expression was observed in all epithelial cell types with highest expression in mature luminal cells but binding sites for GATA3 were accessible in multiple cell types. Expression and binding site enrichment of alveolar differentiation-associated EHF were predominant in luminal progenitor cells. TP63 expression and binding site enrichment were restricted to basal cells, which is consistent with the proposed role of TP63 in maintaining stem cells among the basal cell populations. EMT-associated ZEB1 showed an intriguing pattern. Similar to our published immunohistochemistry-based analysis of breast tissues, ZEB1 expression was detected in fibroblasts/mesenchymal cells, endothelial cells and adipocytes but not in epithelial cells. However, binding sites for this transcription factor were enriched in epithelial cell types and in T-cells. ZEB1 mRNA stability is tightly controlled by the epithelial cell-enriched miR-200 family of microRNAs, and it is possible that ZEB1 transcribed in epithelial cells is immediately targeted for degradation through miR-200. To gain insight into this possibility, we examined the chromatin accessibility status of ZEB1 regulatory regions in fibroblasts/mesenchymal cells and in epithelial cells. ZEB1 regulatory regions displayed open chromatin status in both epithelial cells and fibroblasts. With the exception of a few genes such as SIGLEC1 in macrophages and IL7R in T cells, cell type-specific expression of many genes is not controlled at the level of chromatin accessibility of their regulatory regions. Collectively, these results reveal complexities in gene expression in different cell types of the breast. Citation Format: Poornima Bhat-Nakshatri, Duojiao Chen, Hongyu Gao, Yunlong Liu, Anna Maria Storniolo, Harikrishna Nakshatri. Single cell resolution chromatin accessibility and transcriptomic atlas of genetic ancestry mapped healthy breast tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1458.

Dynamic nucleosome landscape elicits a noncanonical GATA2 pioneer model

Knowledge gaps remain on how nucleosome organization and dynamic reorganization are governed by specific pioneer factors in a genome-wide manner. In this study, we generate over three billons of multi-omics sequencing data to exploit dynamic nucleosome landscape governed by pioneer factors (PFs), FOXA1 and GATA2. We quantitatively define nine functional nucleosome states each with specific characteristic nucleosome footprints in LNCaP prostate cancer cells. Interestingly, we observe dynamic switches among nucleosome states upon androgen stimulation, accompanied by distinct differential (gained or lost) binding of FOXA1, GATA2, H1 as well as many other coregulators. Intriguingly, we reveal a noncanonical pioneer model of GATA2 that it initially functions as a PF binding at the edge of a nucleosome in an inaccessible crowding array. Upon androgen stimulation, GATA2 re-configures an inaccessible to accessible nucleosome state and subsequently acts as a master transcription factor either directly or recruits signaling specific transcription factors to enhance WNT signaling in an androgen receptor (AR)-independent manner. Our data elicit a pioneer and master dual role of GATA2 in mediating nucleosome dynamics and enhancing downstream signaling pathways. Our work offers structural and mechanistic insight into the dynamics of pioneer factors governing nucleosome reorganization.

A Transcriptional Link between HER2, JAM-A and FOXA1 in Breast Cancer.

Overexpression of the human epidermal growth factor receptor-2 (HER2) is associated with aggressive disease in breast and certain other cancers. At a cellular level, the adhesion protein Junctional Adhesion Molecule-A (JAM-A) has been reported to regulate the expression of HER3 via a transcriptional pathway involving FOXA1. Since FOXA1 is also a suggested transcription factor for HER2, this study set out to determine if JAM-A regulates HER2 expression via a similar mechanism. An integrated tripartite approach was taken, involving cellular expression studies after targeted disruption of individual players in the putative pathway, in silico identification of relevant HER2 promoter regions and, finally, interrogation of cancer patient survival databases to deconstruct functionally important links between HER2, JAM-A and FOXA1 gene expression. The outcome of these investigations revealed a unidirectional pathway in which JAM-A expression transcriptionally regulates that of HER2 by influencing the binding of FOXA1 to a specific site in the HER2 gene promoter. Moreover, a correlation between JAM-A and HER2 gene expression was identified in 75% of a sample of 40 cancer types from The Cancer Genome Atlas, and coincident high mean mRNA expression of JAM-A, HER2 and FOXA1 was associated with poorer survival outcomes in HER2-positive (but not HER2-negative) patients with either breast or gastric tumors. These investigations provide the first evidence of a transcriptional pathway linking JAM-A, HER2 and FOXA1 in cancer settings, and support potential future pharmacological targeting of JAM-A as an upstream regulator of HER2.

Abstract PD1-05: Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer

Abstract Background: Aberrant activation of the pioneer transcription factor (TF) FOXA1 contributes to endocrine resistance and metastasis in ER+ breast cancer (BC) by promoting genome-wide enhancer and transcriptional reprogramming that engages the AP-1 TF complex. Identification of central transcriptional nexuses in this deregulated network is key for developing new therapeutic interventions focusing on transcriptional programs. We previously identified FRA1, among the AP-1 components, as the top super-enhancer target forming a FOXA1/FRA1-centered transcriptional axis to activate genes enriched in luminal B-subtype BC and ER+ metastases. To further dissect the FOXA1/FRA1-centered transcriptional axis and its potential therapeutic role, we employed integrative multi-omics data analysis and functional studies targeting FRA1 using our MCF7-parental (P) and FOXA1-amplifed tamoxifen-resistant (TamR) preclinical models. Methods: RNA-seq data were obtained in MCF7-P and TamR cells with FOXA1 and/or AP-1 (FRA1 and c-Jun) perturbation via overexpression and/or si/shRNA knockdown (KD). Differential gene expression was analyzed using DESeq2 or the limma-voom R package. ChIP-seq-based genome-wide FOXA1 binding sites were further refined by intersection with promoter-tethered regions (PTRs) denoting Hi-C-mapped chromatin looping. Genomic binding of FOXA1/c-Jun and H3K27ac modification at target gene loci were aligned and visualized by IGV. Significance of gene set enrichment was determined using a chi-square test adjusted for multiple comparisons. Clinical relevance was examined using an RNA-seq dataset of ER+ metastatic BC (SABCS19-GS2-02). Clonogenicity, soft agar, and wound-healing assays were performed using MCF7-TamR cell derivatives engineered for doxycycline (Dox)-inducible KD of FRA1 or NS shRNA. Significance of the KD effects in functional assays was determined using a linear mixed-effects model. Results: We found that FRA1 and the two embryonic TFs SOX9 and KLF4 that harbor FOXA1-bound PTRs were commonly down-regulated in TamR cells upon KD of FOXA1, FRA1, or c-Jun. We observed increased c-Jun binding at the FOXA1-bound super-enhancer and the PTR looping to the FRA1 gene locus in TamR vs. P cells, suggesting a feed-forward mechanism by which FRA1 transcription is strengthened by the AP-1-engaged super-enhancer in TamR cells. Among the FOXA1-activated genes in TamR cells, a subset of secretory protein-encoding genes was more enriched in the genes commonly dependent on both FRA1 and c-Jun, vs. the genes depending on either FRA1 or c-Jun alone. This FOXA1/FRA1/c-Jun-activated secretome is enriched for multiple biological processes engaged in tumor metastasis and associated microenvironmental niches, and was upregulated in clinical ER+ metastases vs. primary tumors. A larger proportion of this secretome, including CXCL8 and S100P, also relies on ER preferentially in TamR vs. P cells (40% vs. 16%, respectively). FRA1 KD using two different shRNA sequences in TamR cells reduced SOX9 and KLF4 expression levels, and significantly diminished clonogenicity, soft agar colony formation, and wound healing, compared to the control NS KD. Conclusions: Our integrative bioinformatics analyses reveal a feed-forward mechanism on FRA1 activation in amplifying high-FOXA1-induced transcriptional reprogramming in endocrine resistance. A prometastatic secretome activated by FRA1/c-Jun may represent a main transcriptional output of the FOXA1/FRA1-dependent nexus in promoting ER+ disease progression. These identified key transcriptional nexuses may present network hotspots susceptible to therapeutic interventions in which FRA1 inhibition could be used as a new transcriptional program-oriented therapy to treat advanced ER+ BC. Citation Format: Chia Chia Liu, Lanfang Qin, Carmine De Angelis, Sarmistha Nanda, Resel Pereira, Martin J. Shea, Agostina Nardone, Rinath Jeselsohn, Ofir Cohen, Nikhil Wagle, Zhijie Liu, Mothaffar F. Rimawi, C. Kent Osborne, Rachel Schiff, Xiaoyong Fu. Targeting the FRA1-dependent transcriptional nexus in high FOXA1-driven endocrine-resistant and metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD1-05.

Subtype-Independent ANP32E Reduction During Breast Cancer Progression in Accordance with Chromatin Relaxation

BackgroundChromatin state provides a clear decipherable blueprint for maintenance of transcriptional patterns, exemplifying a mitotically stable form of cellular programming in dividing cells. In this regard, genomic studies of chromatin states within cancerous tissues have the potential to uncover novel aspects of tumor biology and unique mechanisms associated with disease phenotypes and outcomes. The degree to which chromatin state differences occur in accordance with breast cancer features has not been established.MethodsWe applied a series of unsupervised computational methods to identify chromatin and molecular differences associated with discrete physiologies across human breast cancer tumors.ResultsChromatin patterns alone are capable of stratifying tumors in association with cancer subtype and disease progression. Major differences occur at DNA motifs for the transcription factor FOXA1, in hormone receptor-positive tumors, and motifs for SOX9 in Basal-like tumors. We find that one potential driver of this effect, the histone chaperone ANP32E, is inversely correlated with tumor progression and relaxation of chromatin at FOXA1 binding sites. Tumors with high levels of ANP32E exhibit an immune response and proliferative gene expression signature, whereas tumors with low ANP32E levels appear programmed for differentiation.ConclusionsOur results indicate that ANP32E may function through chromatin state regulation to control breast cancer differentiation and tumor plasticity. This study sets a precedent for future computational studies of chromatin changes in carcinogenesis.

Reduced NCOR2 expression accelerates androgen deprivation therapy failure in prostate cancer.

SUMMARYThis study addresses the roles of nuclear receptor corepressor 2 (NCOR2) in prostate cancer (PC) progression in response to androgen deprivation therapy (ADT). Reduced NCOR2 expression significantly associates with shorter disease-free survival in patients with PC receiving adjuvant ADT. Utilizing the CWR22 xenograft model, we demonstrate that stably reduced NCOR2 expression accelerates disease recurrence following ADT, associates with gene expression patterns that include neuroendocrine features, and induces DNA hypermethylation. Stably reduced NCOR2 expression in isogenic LNCaP (androgen-sensitive) and LNCaP-C4–2 (androgen-independent) cells revealed that NCOR2 reduction phenocopies the impact of androgen treatment and induces global DNA hypermethylation patterns. NCOR2 genomic binding is greatest in LNCaP-C4–2 cells and most clearly associates with forkhead box (FOX) transcription factor FOXA1 binding. NCOR2 binding significantly associates with transcriptional regulation most when in active enhancer regions. These studies reveal robust roles for NCOR2 in regulating the PC transcriptome and epigenome and underscore recent mutational studies linking NCOR2 loss of function to PC disease progression.

Sequence logic at enhancers governs a dual mechanism of endodermal organ fate induction by FOXA pioneer factors

FOXA pioneer transcription factors (TFs) associate with primed enhancers in endodermal organ precursors. Using a human stem cell model of pancreas differentiation, we here discover that only a subset of pancreatic enhancers is FOXA-primed, whereas the majority is unprimed and engages FOXA upon lineage induction. Primed enhancers are enriched for signal-dependent TF motifs and harbor abundant and strong FOXA motifs. Unprimed enhancers harbor fewer, more degenerate FOXA motifs, and FOXA recruitment to unprimed but not primed enhancers requires pancreatic TFs. Strengthening FOXA motifs at an unprimed enhancer near NKX6.1 renders FOXA recruitment pancreatic TF-independent, induces priming, and broadens the NKX6.1 expression domain. We make analogous observations about FOXA binding during hepatic and lung development. Our findings suggest a dual role for FOXA in endodermal organ development: first, FOXA facilitates signal-dependent lineage initiation via enhancer priming, and second, FOXA enforces organ cell type-specific gene expression via indirect recruitment by lineage-specific TFs.

Systematic functional interrogation of human pseudogenes using CRISPRi

BackgroundThe human genome encodes over 14,000 pseudogenes that are evolutionary relics of protein-coding genes and commonly considered as nonfunctional. Emerging evidence suggests that some pseudogenes may exert important functions. However, to what extent human pseudogenes are functionally relevant remains unclear. There has been no large-scale characterization of pseudogene function because of technical challenges, including high sequence similarity between pseudogene and parent genes, and poor annotation of transcription start sites.ResultsTo overcome these technical obstacles, we develop an integrated computational pipeline to design the first genome-wide library of CRISPR interference (CRISPRi) single-guide RNAs (sgRNAs) that target human pseudogene promoter-proximal regions. We perform the first pseudogene-focused CRISPRi screen in luminal A breast cancer cells and reveal approximately 70 pseudogenes that affect breast cancer cell fitness. Among the top hits, we identify a cancer-testis unitary pseudogene, MGAT4EP, that is predominantly localized in the nucleus and interacts with FOXA1, a key regulator in luminal A breast cancer. By enhancing the promoter binding of FOXA1, MGAT4EP upregulates the expression of oncogenic transcription factor FOXM1. Integrative analyses of multi-omic data from the Cancer Genome Atlas (TCGA) reveal many unitary pseudogenes whose expressions are significantly dysregulated and/or associated with overall/relapse-free survival of patients in diverse cancer types.ConclusionsOur study represents the first large-scale study characterizing pseudogene function. Our findings suggest the importance of nuclear function of unitary pseudogenes and underscore their underappreciated roles in human diseases. The functional genomic resources developed here will greatly facilitate the study of human pseudogene function.

Parallel Reporter Assays Identify Altered Regulatory Role of rs684232 in Leading to Prostate Cancer Predisposition.

Functional characterization of cancer risk-associated single nucleotide polymorphism (SNP) identified by genome-wide association studies (GWAS) has become a big challenge. To identify the regulatory risk SNPs that can lead to transcriptional misregulation, we performed parallel reporter gene assays with both alleles of 213 prostate cancer risk-associated GWAS SNPs in 22Rv1 cells. We disclosed 32 regulatory SNPs that exhibited different regulatory activities with two alleles. For one of the regulatory SNPs, rs684232, we found that the variation altered chromatin binding of transcription factor FOXA1 on the DNA region and led to aberrant gene expression of VPS53, FAM57A, and GEMIN4, which play vital roles in prostate cancer malignancy. Our findings reveal the roles and underlying mechanism of rs684232 in prostate cancer progression and hold great promise in benefiting prostate cancer patients with prognostic prediction and target therapies.

Abstract 2317: Targeting prostate cancer super enhancers by co-inhibiting LSD1 and BRD4

Abstract In prostate cancer (PCa) cells, “pioneer” factor FOXA1 resides at distal enhancers enriched with histone 3 lysine 4 methylation (H3K4me) and lysine 27 acetylation (H3K27ac). FOXA1 is capable of remodeling the compact chromatin to facilitate androgen receptor (AR) recruitment. Recent studies revealed overexpression of FOXA1 caused by frequent structural alterations in the lethal stage of PCa, namely castration-resistant PCa(CRPC), suggesting FOXA1 as a critical target in CRPC. However, a lack of targetable domain hinders the effective targeting of FOXA1. Our recent (epi)genomic profiling study revealed that lysine-specific demethylase (LSD1) and FOXA1 co-occupy at active enhancers in PCa cells and LSD1 demethylase activity is required for FOXA1 chromatin binding. LSD1 inhibition can quickly and drastically reduce FOXA1 global binding and concurrently impair the “openness” of enhancers, leading to disrupted AR cistrome. Mechanistically, we demonstrated that this activator function of LSD1 is mediated through directly demethylating K270 of FOXA1. By testing an array of adenocarcinoma CRPC in vivo models, we further showed that the efficacy of LSD1 inhibitors may be correlated with FOXA1 levels. Collectively, these results provide the molecular reasoning on co-targeting the epigenetic regulators that are important for promoting AR activity in treating CRPC. Furthermore, we have found that LSD1, FOXA1, and BRD4 (a Bromodomain and Extra-Terminal motif (BET) protein) co-occupy the super enhancers characterized by distinguishable high levels of H3K4me2 and H3K27ac in PCa cells. Super enhancers have been found to drive oncogenic transcription program in PCa. As an active enhancer marker and an important AR coactivator, BRD4 recognizes H3K27ac signal and promotes the initiation and elongation steps in gene transcription process. Herein, we focus on the potential synergism of combined targeting of LSD1 and BRD4. We found that FOXA1 interacted with BRD4 in different cell lines and patient-derived xenograft (PDX) tumor tissues. In line with this association, either LSD1 inhibitors or BET inhibitors could decrease FOXA1 and BRD4 chromatin binding. Characterization of the physical interaction is an ongoing study. We found that the combinational treatment lead to a more effective reduction of AR transcriptional activity than either single treatment. The PCa cell proliferation in vitro was also more profoundly inhibited by combined therapy. More importantly, we demonstrated this synergism in both 22RV1 xenograft and PDX LuCaP35CR in the castrated mice, with a lower dose of LSD1 inhibitor and BET inhibitor (both in clinical trials) which did not induce overt toxicity in the animals. Currently, we are performing RNA-seq studies to identify the convergent downstream pathways by this co-treatment. In summary, our study provides important pre-clinical insights for targeting the CRPC enhancers by combining inhibitors of LSD1 and BRD4. Citation Format: Shuai Gao, Muqing Li, Mingyu Liu, Dong Han, Zifeng Wang, Wanting Han, Changmeng Cai. Targeting prostate cancer super enhancers by co-inhibiting LSD1 and BRD4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2317.

PARP-1 Regulates Estrogen-Dependent Gene Expression in Estrogen Receptor α-Positive Breast Cancer Cells.

Poly(ADP-ribose) polymerase-1 (PARP-1) has gained considerable attention as a target for therapeutic inhibitors in breast cancers. Previously we showed that PARP-1 localizes to active gene promoters to regulate histone methylation and RNA polymerase II activity (Pol II), altering the expression of various tumor-related genes. Here we report a role for PARP-1 in estrogen-dependent transcription in estrogen receptor alpha (ERα)-positive (ER+) breast cancers. Global nuclear run-on and sequencing analyses functionally linked PARP-1 to the direct control of estrogen-regulated gene expression in ER+ MCF-7 breast cancer cells by promoting transcriptional elongation by Pol II. Furthermore, chromatin immunoprecipitation sequencing analyses revealed that PARP-1 regulates the estrogen-dependent binding of ERα and FoxA1 to a subset of genomic ERα binding sites, promoting active enhancer formation. Moreover, we found that the expression levels of the PARP-1- and estrogen-coregulated gene set are enriched in the luminal subtype of breast cancer, and high PARP-1 expression in ER+ cases correlates with poor survival. Finally, treatment with a PARP inhibitor or a transcriptional elongation inhibitor attenuated estrogen-dependent growth of multiple ER+ breast cancer cell lines. Taken together, our results show that PARP-1 regulates critical molecular pathways that control the estrogen-dependent gene expression program underlying the proliferation of ER+ breast cancer cells. IMPLICATIONS: PARP-1 regulates the estrogen-dependent genomic binding of ERα and FoxA1 to regulate critical gene expression programs by RNA Pol II that underlie the proliferation of ER+ breast cancers, providing a potential therapeutic opportunity for PARP inhibitors in estrogen-responsive breast cancers.

ATPase Effects on Pro-Nutrients-mTOR Release Long Fatty Acids Chains which Under Mitochondrial Phospholipase, Synthase, & Synthetase Effects form Three ROR-Alpha, Beta, Gamma Isoforms

ATPase Effects on Pro-Nutrients-mTOR Release Long Fatty Acids Chains which Under Mitochondrial Phospholipase, Synthase, & Synthetase Effects form Three ROR-Alpha, Beta, Gamma Isoforms

A Combination of RNA-Seq Analysis and Use of TCGA Database for Determining the Molecular Mechanism and Identifying Potential Drugs for GJB1 in Ovarian Cancer.

BackgroundThere has been increasing evidence for the vital role played by gap junction protein beta-1 (GJB1) in ovarian cancer (OC) and for the possibility of this protein serving as a therapeutic target. However, the detailed mechanism of GJB1 in OC has not yet been clearly determined. The current study aimed to establish the molecular mechanisms of the involvement of GJB1 in OC and to further predict potential drugs targeting this protein.MethodsTo better understand the molecular mechanisms of the involvement of GJB1 in OC, RNA-Seq transcriptome sequencing was performed. Then, we carried out an RNA-Seq analysis to determine the genes differentially co-expressed with GJB1. Subsequently, we carried out bioinformation methods to study the upstream regulatory transcriptional factor (TF) of GJB1. Further, the binding of FOXA1 and GJB1 promoter was tested using ChIP-qPCR. Moreover, we performed pathway enrichment to identify the downstream regulatory mechanisms of GJB1. Furthermore, potential drugs targeting GJB1 were screened using AutoDock 4.2.ResultsWe constructed the transcriptional factor FOXA1 regulatory network based on the AnimalTFDB, JASPAR, RNA-Seq, TCGA cohort and ChIP-qPCR to study the upstream regulation of GJB1. In addition, two key pathways for the involvement of GJB1 in OC—namely the “ECM–receptor interaction” and “focal adhesion” KEGG pathways—were identified. Furthermore, ZINC000005552022 was found in a screening to be a potentially promising drug targeting GJB1.ConclusionOur study results suggested that the transcriptional factor FOXA1 regulates the involvement of GJB1 in OC through ECM–receptor interaction and focal adhesion KEGG pathways, and that ZINC000005552022 may have promising potential as a drug targeting GJB1; this finding might be used to help accelerate drug development and improve the outcomes for patients with OC.

A noncanonical AR addiction drives enzalutamide resistance in prostate cancer

Resistance to next-generation anti-androgen enzalutamide (ENZ) constitutes a major challenge for the treatment of castration-resistant prostate cancer (CRPC). By performing genome-wide ChIP-seq profiling in ENZ-resistant CRPC cells we identify a set of androgen receptor (AR) binding sites with increased AR binding intensity (ARBS-gained). While ARBS-gained loci lack the canonical androgen response elements (ARE) and pioneer factor FOXA1 binding motifs, they are highly enriched with CpG islands and the binding sites of unmethylated CpG dinucleotide-binding protein CXXC5 and the partner TET2. RNA-seq analysis reveals that both CXXC5 and its regulated genes including ID1 are upregulated in ENZ-resistant cell lines and these results are further confirmed in patient-derived xenografts (PDXs) and patient specimens. Consistent with the finding that ARBS-gained loci are highly enriched with H3K27ac modification, ENZ-resistant PCa cells, organoids, xenografts and PDXs are hyper-sensitive to NEO2734, a dual inhibitor of BET and CBP/p300 proteins. These results not only reveal a noncanonical AR function in acquisition of ENZ resistance, but also posit a treatment strategy to target this vulnerability in ENZ-resistant CRPC.

Application of alternative de novo motif recognition models for analysis of structural heterogeneity of transcription factor binding sites: a case study of FOXA2 binding sites.

The most popular model for the search of ChIP-seq data for transcription factor binding sites (TFBS) is the positional weight matrix (PWM). However, this model does not take into account dependencies between nucleotide occurrences in different site positions. Currently, two recently proposed models, BaMM and InMoDe, can do as much. However, application of these models was usually limited only to comparing their recognition accuracies with that of PWMs, while none of the analyses of the co-prediction and relative positioning of hits of different models in peaks has yet been performed. To close this gap, we propose the pipeline called MultiDeNA. This pipeline includes stages of model training, assessing their recognition accuracy, scanning ChIP-seq peaks and their classification based on scan results. We applied our pipeline to 22 ChIP-seq datasets of TF FOXA2 and considered PWM, dinucleotide PWM (diPWM), BaMM and InMoDe models. The combination of these four models allowed a significant increase in the fraction of recognized peaks compared to that for the sole PWM model: the increase was 26.3 %. The BaMM model provided the main contribution to the recognition of sites. Although the major fraction of predicted peaks contained TFBS of different models with coincided positions, the medians of the fraction of peaks containing the predictions of sole models were 1.08, 0.49, 4.15 and 1.73 % for PWM, diPWM, BaMM and InMoDe, respectively. Thus, FOXA2 BSs were not fully described by only a sole model, which indicates theirs heterogeneity. We assume that the BaMM model is the most successful in describing the structure of the FOXA2 BS in ChIP-seq datasets under study.

Epigenetic Memory: Lessons From iPS Cells Derived From Human β Cells.

Incomplete reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) may be responsible for the heterogeneity in differentiation capacity observed among iPSC lines. It remains unclear whether it results from stochastic reprogramming events, or reflects consistent genetic or cell-of-origin differences. Some evidence suggests that epigenetic memory predisposes iPSCs to enhanced differentiation into the parental cell type. We investigated iPSCs reprogrammed from human pancreatic islet β cells (BiPSCs), as a step in development of a robust differentiation protocol for generation of β-like cells. BiPSCs derived from multiple human donors manifested enhanced and reproducible spontaneous and induced differentiation towards insulin-producing cells, compared with iPSCs derived from isogenic non-β-cell types and fibroblast-derived iPSCs (FiPSCs). Genome-wide analyses of open chromatin in BiPSCs and FiPSCs identified thousands of differential open chromatin sites (DOCs) between the two iPSC types. DOCs more open in BiPSCs (Bi-DOCs) were significantly enriched for known regulators of endodermal development, including bivalent and weak enhancers, and FOXA2 binding sites. Bi-DOCs were associated with genes related to pancreas development and β-cell function. These studies provide evidence for reproducible epigenetic memory in BiPSCs. Bi-DOCs may provide clues to genes and pathways involved in the differentiation process, which could be manipulated to increase the efficiency and reproducibility of differentiation of pluripotent stem cells from non-β-cell sources.

Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.

Chromatin binding of FOXA1 is promoted by LSD1-mediated demethylation in prostate cancer.

FOXA1 functions as a pioneer transcription factor by facilitating the access to chromatin for steroid hormone receptors, such as androgen receptor and estrogen receptor1-4, but mechanisms regulating its binding to chromatin remain elusive. LSD1 (KDM1A) acts as a transcriptional repressor by demethylating mono/dimethylated histone H3 lysine 4 (H3K4me1/2)5,6, but also acts as a steroid hormone receptor coactivator through mechanisms that are unclear. Here we show, in prostate cancer cells, that LSD1 associates with FOXA1 and active enhancer markers, and that LSD1 inhibition globally disrupts FOXA1 chromatin binding. Mechanistically, we demonstrate that LSD1 positively regulates FOXA1 binding by demethylating lysine 270, adjacent to the wing2 region of the FOXA1 DNA-binding domain. Acting through FOXA1, LSD1 inhibition broadly disrupted androgen-receptor binding and its transcriptional output, and dramatically decreased prostate cancer growth alone and in synergy with androgen-receptor antagonist treatment in vivo. These mechanistic insights suggest new therapeutic strategies in steroid-driven cancers.

Abstract 3645: Epigenetic regulation of oncogenic transcription mediated by aberrantly activated enhancers in prostate cancer

Abstract Since androgen receptor (AR) is a master transcription factor of prostate gland in both normal development and carcinogenesis, androgen deprivation therapy (ADT) is quite effective for primary prostate cancer (PC) even in advanced disease. During ADT, however, PC tumors generally acquire resistance through oncogenic activation independent from androgen-AR signaling, and progress to lethal castration-resistant PC (CRPC). Transcription factors (TFs) such as AR regulate gene expression through binding regulatory elements of genome called as enhancers. Aberrantly activated enhancers regulating oncogenes could be one of the mechanisms of cancer therapy resistance. In this study, to clarify how aberrant enhancer activation contribute to genesis of CRPC, we performed comprehensive transcriptome and epigenome analyses by RNA-seq and ChIP-seq for H3K4me1, H3K4me3, H3K27ac, H3K79me2, BRD4, AR, FOXA1 and RNA polymerase 2 (RNApol2). Using androgen dependent PC cells LNCaP and its derivative CRPC cells LNCaP95, we extracted BRD4(+)/AR(+)/FOXA1(+) enhancers and super enhancers (SEs) defined by cluster of H3K27ac regardless of BRD4, AR, FOXA1 binding. SE neighboring genes were mostly targeted by BRD4(+)/AR(+)/FOXA1(+) enhancers in LNCaP (57.5%), but markedly less in LNCaP95 (25.3%), suggesting a shift from AR-dependent to AR-independent regulation. In contrast, SE neighboring genes were mostly epigenetically modified with H3K79me2 in both LNCaP (77.4%) and LNCaP95 (76.2%). In addition, the mRNA expression levels of H3K79methyltansferase DOT1L was upregulated in CRPC clinical tissues. While BRD4 inhibition was quite effective for suppressing cell growth in LNCaP, but less effective in LNCaP95, DOT1L inhibition was markedly effective to both cells, inducing marked cell cycle arrest and apoptosis. Furthermore, precise analyses of RNApol2 behavior and transcriptome alteration revealed that BRD4 and DOT1L regulated different genes in a different manner. Mechanistically, while BRD4 inhibition suppressed AR target genes through reducing initiation of transcriptional elongation, DOT1L inhibition suppressed SE neighboring genes through reducing termination of transcriptional elongation. In conclusion, our results indicated that BRD4 and DOT1L played critical role in regulating transcription mediated by aberrantly activated enhancers, and could be targeted as an epigenetic therapeutic strategy for CRPC. Citation Format: Hiroaki Sato, Masahiro Sugiura, Manato Kanesaka, Atsushi Okabe, Masaki Fukuyo, Shinichi Sakamoto, Akira Komiya, Tomohiko Ichikawa, Atsushi Kaneda. Epigenetic regulation of oncogenic transcription mediated by aberrantly activated enhancers in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3645.