Benjamin Sunkel

Three-tiered role of the pioneer factor GATA2 in promoting androgen-dependent gene expression in prostate cancer

In prostate cancer, androgen receptor (AR) binding and androgen-responsive gene expression are defined by hormone-independent binding patterns of the pioneer factors FoxA1 and GATA2. Insufficient evidence of the mechanisms by which GATA2 contributes to this process precludes complete understanding of a key determinant of tissue-specific AR activity. Our observations suggest that GATA2 facilitates androgen-responsive gene expression by three distinct modes of action. By occupying novel binding sites within the AR gene locus, GATA2 positively regulates AR expression before and after androgen stimulation. Additionally, GATA2 engages AR target gene enhancers prior to hormone stimulation, producing an active and accessible chromatin environment via recruitment of the histone acetyltransferase p300. Finally, GATA2 functions in establishing and/or sustaining basal locus looping by recruiting the Mediator subunit MED1 in the absence of androgen. These mechanisms may contribute to the generally positive role of GATA2 in defining AR genome-wide binding patterns that determine androgen-responsive gene expression profiles. We also find that GATA2 and FoxA1 exhibit both independent and codependent co-occupancy of AR target gene enhancers. Identifying these determinants of AR transcriptional activity may provide a foundation for the development of future prostate cancer therapeutics that target pioneer factor function.

Agonist and antagonist switch DNA motifs recognized by human androgen receptor in prostate cancer

Human transcription factors recognize specific DNA sequence motifs to regulate transcription. It is unknown whether a single transcription factor is able to bind to distinctly different motifs on chromatin, and if so, what determines the usage of specific motifs. By using a motif‐resolution chromatin immunoprecipitation‐exonuclease (ChIP‐exo) approach, we find that agonist‐liganded human androgen receptor (AR) and antagonist‐liganded AR bind to two distinctly different motifs, leading to distinct transcriptional outcomes in prostate cancer cells. Further analysis on clinical prostate tissues reveals that the binding of AR to these two distinct motifs is involved in prostate carcinogenesis. Together, these results suggest that unique ligands may switch DNA motifs recognized by ligand‐dependent transcription factors in vivo. Our findings also provide a broad mechanistic foundation for understanding ligand‐specific induction of gene expression profiles.

Ligand-dependent genomic function of glucocorticoid receptor in triple-negative breast cancer.

Glucocorticoids (GCs) have been widely used as coadjuvants in the treatment of solid tumours, but GC treatment may be associated with poor pharmacotherapeutic response or prognosis. The genomic action of GC in these tumours is largely unknown. Here we find that dexamethasone (Dex, a synthetic GC)-regulated genes in triple-negative breast cancer (TNBC) cells are associated with drug resistance. Importantly, these GC-regulated genes are aberrantly expressed in TNBC patients and are associated with unfavourable clinical outcomes. Interestingly, in TNBC cells, Compound A (CpdA, a selective GR modulator) only regulates a small number of genes not involved in carcinogenesis and therapy resistance. Mechanistic studies using a ChIP-exo approach reveal that Dex- but not CpdA-liganded glucocorticoid receptor (GR) binds to a single glucocorticoid response element (GRE), which drives the expression of pro-tumorigenic genes. Our data suggest that development of safe coadjuvant therapy should consider the distinct genomic function between Dex- and CpdA-liganded GR.

Computational analysis reveals a correlation of exon-skipping events with splicing, transcription and epigenetic factors

Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, ‘skipping’, exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the process of exon–intron boundary establishment leading to skipping events.

S100A14: Novel Modulator of Terminal Differentiation in Esophageal Cancer

Aberrant keratinocyte differentiation is a key mechanism in the initiation of cancer. Because activities regulating differentiation exhibit altered or reduced capacity in esophageal cancer cells, it is vital to pinpoint those genes that control epidermal proliferation and terminal differentiation to better understand esophageal carcinogenesis. S100A14 is a member of the S100 calcium-binding protein family and has been suggested to be involved in cell proliferation, apoptosis, and invasion. The present study used immunohistochemistry analysis of S100A14 in clinical specimens of esophageal squamous cell carcinoma (ESCC) to show that decreased S100A14 is strongly correlated with poor differentiation. Furthermore, both mRNA and protein expression of S100A14 was drastically increased upon 12-O-tetra-decanoylphorbol-13-acetate (TPA) and calcium-induced esophageal cancer cell differentiation. Overexpression of S100A14 resulted in a G1-phase cell cycle arrest and promoted calcium-inhibited cell growth. Conversely, decreasing S100A14 expression significantly promoted G1–S transition and prevented the morphologic changes associated with calcium-induced cell differentiation. Molecular investigation demonstrated that S100A14 altered the calcium-induced expression of late markers of differentiation, with the most prominent effect on involucrin (IVL) and filaggrin (FLG). Finally, it was determined that S100A14 is transcriptionally regulated by JunB and that S100A14 and JunB status significantly correlated in ESCC tissue. In summary, these data demonstrate that S100A14 is transcriptionally regulated by JunB and involved in ESCC cell differentiation. Implications: This study further differentiates the molecular mechanism controlling the development and progression of esophageal cancer. Mol Cancer Res; 11(12); 1542–53. ©2013 AACR.

Integrative analysis identifies targetable CREB1/FoxA1 transcriptional co-regulation as a predictor of prostate cancer recurrence

Identifying prostate cancer-driving transcription factors (TFs) in addition to the androgen receptor promises to improve our ability to effectively diagnose and treat this disease. We employed an integrative genomics analysis of master TFs CREB1 and FoxA1 in androgen-dependent prostate cancer (ADPC) and castration-resistant prostate cancer (CRPC) cell lines, primary prostate cancer tissues and circulating tumor cells (CTCs) to investigate their role in defining prostate cancer gene expression profiles. Combining genome-wide binding site and gene expression profiles we define CREB1 as a critical driver of pro-survival, cell cycle and metabolic transcription programs. We show that CREB1 and FoxA1 co-localize and mutually influence each others binding to define disease-driving transcription profiles associated with advanced prostate cancer. Gene expression analysis in human prostate cancer samples found that CREB1/FoxA1 target gene panels predict prostate cancer recurrence. Finally, we showed that this signaling pathway is sensitive to compounds that inhibit the transcription co-regulatory factor MED1. These findings not only reveal a novel, global transcriptional co-regulatory function of CREB1 and FoxA1, but also suggest CREB1/FoxA1 signaling is a targetable driver of prostate cancer progression and serves as a biomarker of poor clinical outcomes.

Synthesis of Apo-13-, and Apo-15-Lycopenoids, Cleavage Products of Lycopene that are Retinoic Acid Antagonists

Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene’s biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, β-apo-13-carotenone, the “first half” putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like β-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.

Genome-wide analysis reveals positional-nucleosome-oriented binding pattern of pioneer factor FOXA1

The compaction of nucleosomal structures creates a barrier for DNA-binding transcription factors (TFs) to access their cognate cis-regulatory elements. Pioneer factors (PFs) such as FOXA1 are able to directly access these cis-targets within compact chromatin. However, how these PFs interplay with nucleosomes remains to be elucidated, and is critical for us to understand the underlying mechanism of gene regulation. Here, we have conducted a computational analysis on a strand-specific paired-end ChIP-exo (termed as ChIP-ePENS) data of FOXA1 in LNCaP cells by our novel algorithm ePEST. We find that FOXA1 chromatin binding occurs via four distinct border modes (or footprint boundary patterns), with a preferential footprint boundary patterns relative to FOXA1 motif orientation. In addition, from this analysis three fundamental nucleotide positions (oG, oS and oH) emerged as major determinants for blocking exo-digestion and forming these four distinct border modes. By integrating histone MNase-seq data, we found an astonishingly consistent, ‘well-positioned’ configuration occurs between FOXA1 motifs and dyads of nucleosomes genome-wide. We further performed ChIP-seq of eight chromatin remodelers and found an increased occupancy of these remodelers on FOXA1 motifs for all four border modes (or footprint boundary patterns), indicating the full occupancy of FOXA1 complex on the three blocking sites (oG, oS and oH) likely produces an active regulatory status with well-positioned phasing for protein binding events. Together, our results suggest a positional-nucleosome-oriented accessing model for PFs seeking target motifs, in which FOXA1 can examine each underlying DNA nucleotide and is able to sense all potential motifs regardless of whether they face inward or outward from histone octamers along the DNA helix axis.

Diverse AR-V7 cistromes in castration-resistant prostate cancer are governed by HoxB13

Significance Mechanisms underlying androgen receptor (AR) splice variant 7 (AR-V7) oncogenic function at the genomic level remain poorly defined. Studies here found that AR-V7 cistromes are cell-context–dependent in castration-resistant prostate cancer (CRPC) cells and tissues, resulting in tremendous diversity in AR-V7–regulated transcriptomes across CRPC patients. Thus, few downstream targets of AR-V7 can universally account for CRPC progression, leaving us without adequate, common, viable therapeutic targets for this heterogeneous disease in which AR-V7 itself is not druggable by antiandrogens. Remarkably, we discovered that HoxB13 governs the diverse AR-V7 cistromes among CRPC, thus shifting focus from the previously characterized role of HoxB13 in androgen-dependent prostate cancer to a distinct role in CRPC. These findings will significantly impact therapeutic strategies for AR-V7–driven CRPC, for which there is no approved therapy. The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7–driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7–driven prostate tumors.

Abstract 1387: Convergent CREB1/FoxA1 transcriptional activity defines castration-resistant prostate cancer gene expression profile

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA To identify novel pathways and therapeutic targets in lethal, castration-resistant prostate cancer (CRPC), we extended our previous findings that the overexpression of several G1/S and G2/M phase cell cycle genes in CRPC vs. androgen-dependent prostate cancer (ADPC) cells relies on the cooperative activity of the transcription factors (TFs) FoxA1 and CREB1, and sought to comprehensively characterize their role in supporting CRPC growth. We have taken an integrated genomics approach to reveal the CREB1/FoxA1-coregulated gene expression profile of ADPC (LNCaP) and CRPC (LNCaP-abl) cell line models. Chromatin-immunoprecipitation combined with high-throughput sequencing (ChIP-seq) was performed using antibodies against CREB1 and FoxA1 to reveal genome-wide TF binding sites. Our results indicate that these factors adopt divergent binding patterns in each cell line and that the CREB1 and FoxA1 cistromes exhibit a higher degree of overlap in CRPC vs. ADPC. RNA-seq was also performed in each cell line following transfection with Control-, FoxA1-, or CREB1-targeting small interfering RNA (siRNA) molecules. We identified putative direct CREB1/FoxA1-coregulated genes by noting instances of TF binding near cooperatively coregulated genes (i.e. those up- or down-regulated by both CREB1 and FoxA1 knockdown). Unlike previous analyses, which found that cooperative TF activity occurs primarily in the event of TF co-occupancy of gene regulatory elements, we found that non-overlapping as well as overlapping CREB1/FoxA1 binding events were significantly enriched within cooperatively coregulated target gene loci compared to genes that were unresponsive to TF knockdown. Gene Ontology (GO) analysis of LNCaP-abl-coregulated genes found that CREB1/FoxA1 enhance the expression of critical CRPC pathways including “DNA Repair” and “Aurora Kinase,” while they repress pathways such as “Apoptosis” and, consistent with previous findings, “Androgen Receptor Signaling.” Importantly, CREB1/FoxA1-up-regulated and down-regulated pathway elements exhibit overexpression or under-expression, respectively, in LNCaP-abl vs. LNCaP as well as in public gene expression datasets of metastatic vs. primary prostate cancer. Finally, we performed several proof-of-concept analyses to demonstrate that the CREB1/FoxA1-coregulatory pathway is sensitive to compounds that target CREB1 phosphorylation, a critical event in the transactivation of CREB1 mediated in large part by the related AGC kinase family members PKA and Akt/PKB. Together these results reveal that CREB1 and FoxA1 assume overlapping roles in determining the expression of genes/networks relevant to experimental models and clinical cases of advanced prostate cancer, and though targeting transcription factors has proven challenging, this coregulatory pathway may in fact represent a therapeutic target owing to its kinase-dependent activation. Citation Format: Benjamin D. Sunkel, Dayong Wu, Xiangtao Liu, Zhenqing Ye, Victor Jin, Qianben Wang. Convergent CREB1/FoxA1 transcriptional activity defines castration-resistant prostate cancer gene expression profile. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1387. doi:10.1158/1538-7445.AM2014-1387