An AR-ERG transcriptional signature defined by long-range chromatin interactomes in prostate cancer cells.

The aberrant activities of transcription factors such as the androgen receptor (AR) underpin prostate cancer development. While the AR cis-regulation has been extensively studied in prostate cancer, information pertaining to the spatial architecture of the AR transcriptional circuitry remains limited. In this paper, we propose a novel framework to profile long-range chromatin interactions associated with AR and its collaborative transcription factor, erythroblast transformation-specific related gene (ERG), using chromatin interaction analysis by paired-end tag (ChIA-PET). We identified ERG-associated long-range chromatin interactions as a cooperative component in the AR-associated chromatin interactome, acting in concert to achieve coordinated regulation of a subset of AR target genes. Through multifaceted functional data analysis, we found that AR-ERG interaction hub regions are characterized by distinct functional signatures, including bidirectional transcription and cotranscription factor binding. In addition, cancer-associated long noncoding RNAs were found to be connected near protein-coding genes through AR-ERG looping. Finally, we found strong enrichment of prostate cancer genome-wide association study (GWAS) single nucleotide polymorphisms (SNPs) at AR-ERG co-binding sites participating in chromatin interactions and gene regulation, suggesting GWAS target genes identified from chromatin looping data provide more biologically relevant findings than using the nearest gene approach. Taken together, our results revealed an AR-ERG-centric higher-order chromatin structure that drives coordinated gene expression in prostate cancer progression and the identification of potential target genes for therapeutic intervention.

Identification of a Wells-Dawson polyoxometalate-based AP-2γ inhibitor with pro-apoptotic activity.

AP-2 gamma (AP-2γ) is a transcription factor that plays pivotal roles in breast cancer biology. To search for small molecule inhibitors of AP-2γ, we performed a high-throughput fluorescence anisotropy screen and identified a polyoxometalate compound with Wells-Dawson structure K6[P2Mo18O62] (Dawson-POM) that blocks the DNA-binding activity of AP-2γ. We showed that this blocking activity is due to the direct binding of Dawson-POM to AP-2γ. We also provided evidence to show that Dawson-POM decreases AP-2γ-dependent transcription similar to silencing the gene. Finally, we demonstrated that Dawson-POM contains anti-proliferative and pro-apoptotic effects in breast cancer cells. In summary, we identified the first small molecule inhibitor of AP-2γ and showed Dawson-POM-mediated inhibition of AP-2γ as a potential avenue for cancer therapy.

Targeting the heparin-binding domain of fibroblast growth factor receptor 1 as a potential cancer therapy.

BACKGROUND: Aberrant activation of fibroblast growth factor receptors (FGFRs) deregulates cell proliferation and promotes cell survival, and may predispose to tumorigenesis. Therefore, selective inactivation of FGFRs is an important strategy for cancer therapy. Here as a proof-of-concept study, we developed a FGFR1 neutralizing antisera, IMB-R1, employing a novel strategy aimed at preventing the access of essential heparan sulfate (HS) co-receptors to the heparin-binding domain on FGFR1. METHODS: The mRNA and protein expression level of FGFR1 and other FGFRs were examined in several lines of breast cancer and osteosarcoma cells and corresponding normal cells using Taqman real-time quantitative PCR and Western blot analysis. The specificity of IMB-R1 against FGFR1 was assessed with various ELISA-based approaches and Receptor Tyrosine Kinase array. Proliferation assay and apoptosis analysis were performed to assess the effect of IMB-R1 on cancer cell growth and apoptosis, respectively, in comparison with known FGFR1 inhibitors. The IMB-R1 induced alteration of intracellular signaling and gene expression were analysed using Western blot and microarray approaches. Immunohistochemical staining of FGFR1 using IMB-R1 were carried out in different cancer tissues from clinical patients. Throughout the study, statistical differences were determined by Student's t test where appropriate and reported when a p value was less than 0.05. RESULTS: We demonstrate that IMB-R1 is minimally cross-reactive for other FGFRs, and that it potently and specifically inhibits binding of heparin to FGFR1. Furthermore, IMB-R1 blocks the interaction of FGF2 with FGFR1, the kinase activity of FGFR1 and activation of intracellular FGFR signaling. Cancer cells treated with IMB-R1 displayed impaired FGF2 signaling, were unable to grow and instead underwent apoptosis. IMB-R1-induced cell death correlated with a disruption of antioxidative defense networks and increased expression of several tumor suppressors and apoptotic proteins, including p53. Immunostaining with IMB-R1 was stronger in human cancer tissues in which the FGFR1 gene is amplified. CONCLUSION: Our study suggests that blocking HS interaction with the heparin-binding domains of FGFR1 inhibited cancer cell growth, which can be an attractive strategy to inactivate cancer-related heparin-binding proteins.

Fast screening of ligand-protein interactions based on ligand-induced protein stabilization of gold nanoparticles.

High throughput screening of small molecular weight (LMW) ligands for protein and sensitive determination of ligand-induced protein stabilization is an important task in drug discovery and in protein structural and functional genomics studies. In this study, gold nanoparticles (AuNPs) and their aggregation property are used to develop a rapid and less equipment intensive assay for screening the interactions between LMW ligands and transcription factors (TFs) and human serum albumin. The assay is based on the fact that the aggregation/discpersion status of AuNPs is very sensitive to the conformation of surrounding proteins, and when a LMW ligand binds to the proteins, it can enhance proteins' salt and thermal stability, and therefore the protective effect on AuNPs from aggregation. Two TFs, i.e. FoxA1 (Forkhead box A1) and AP-2γ (activating enhancer binding protein 2 gamma), and 14 compounds from an NCI compounds library and human serum albumin (HSA) and three known ligands (ibuprofen, warfarin, and phenytoin) are involved to demonstrate the concept and to prove its generality and robustness. With this AuNP method, two strong LMW binders are identified for FoxA1 and AP-2γ; ligand induced protein stabilization is determined. The results have been verified using surface plasmon resonance spectroscopy (SPR) and differential static light scattering (DSLS) techniques. Tryptophan fluorescent measurement is also conducted to provide further information on protein conformational change upon LMW ligand loading as can be observed from AuNPs' UV-vis spectra. FoxA1 and AP-2γ are pivotal in regulating the transcriptional activity of estrogen receptor alpha and controlling the expression of estrogen-responsive breast cancer cells. Identification of drug candidates targeting these two transcription factors could be an alternative in treating breast cancer, in particular those that have become endocrine resistance.

Studying forkhead box protein A1-DNA interaction and ligand inhibition using gold nanoparticles, electrophoretic mobility shift assay, and fluorescence anisotropy.

Forkhead box protein 1 (FoxA1) is a member of the forkhead family of winged helix transcription factors that plays pivotal roles in the development and differentiation of multiple organs and in the regulation of estrogen-stimulated genes. Conventional analytical methods-electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy (FA)-as well as a gold nanoparticles (AuNPs)-based assay were used to study DNA binding properties of FoxA1 and ligand interruption of FoxA1-DNA binding. In the AuNPs assay, the distinct ability of protein-DNA complex to protect AuNPs against salt-induced aggregation was exploited to screen sequence selectivity and determine the binding affinity constant based on AuNPs color change and absorbance spectrum shift. Both conventional EMSA and FA and the AuNPs assay suggested that FoxA1 binds to DNA in a core sequence-dependent manner and the flanking sequence also played a role to influence the affinity. The EMSA and AuNPs were found to be more sensitive than FA in differentiation of sequence-dependent affinity. With the addition of a spin filtration step, AuNPs assay has been extended for studying small molecular ligand inhibition of FoxA1-DNA interactions enabling drug screening. The results correlate very well with those obtained using FA.

A transcriptional repressor co-regulatory network governing androgen response in prostate cancers.

Transcriptional corepressors are frequently aberrantly over-expressed in prostate cancers. However, their crosstalk with the Androgen receptor (AR), a key player in prostate cancer development, is unclear. Using ChIP-Seq, we generated extensive global binding maps of AR, ERG, and commonly over-expressed transcriptional corepressors including HDAC1, HDAC2, HDAC3, and EZH2 in prostate cancer cells. Surprisingly, our results revealed that ERG, HDACs, and EZH2 are directly involved in androgen-regulated transcription and wired into an AR centric transcriptional network via a spectrum of distal enhancers and/or proximal promoters. Moreover, we showed that similar to ERG, these corepressors function to mediate repression of AR-induced transcription including cytoskeletal genes that promote epithelial differentiation and inhibit metastasis. Specifically, we demonstrated that the direct suppression of Vinculin expression by ERG, EZH2, and HDACs leads to enhanced invasiveness of prostate cancer cells. Taken together, our results highlight a novel mechanism by which, ERG working together with oncogenic corepressors including HDACs and the polycomb protein, EZH2, could impede epithelial differentiation and contribute to prostate cancer progression, through directly modulating the transcriptional output of AR.

AP-2γ regulates oestrogen receptor-mediated long-range chromatin interaction and gene transcription.

Oestrogen receptor α (ERα) is key player in the progression of breast cancer. Recently, the cistrome and interactome of ERα were mapped in breast cancer cells, revealing the importance of spatial organization in oestrogen-mediated transcription. However, the underlying mechanism of this process is unclear. Here, we show that ERα binding sites (ERBS) identified from the Chromatin Interaction Analysis-Paired End DiTag of ERα are enriched for AP-2 motifs. We demonstrate the transcription factor, AP-2γ, which has been implicated in breast cancer oncogenesis, binds to ERBS in a ligand-independent manner. Furthermore, perturbation of AP-2γ expression impaired ERα DNA binding, long-range chromatin interactions, and gene transcription. In genome-wide analyses, we show that a large number of AP-2γ and ERα binding events converge together across the genome. The majority of these shared regions are also occupied by the pioneer factor, FoxA1. Molecular studies indicate there is functional interplay between AP-2γ and FoxA1. Finally, we show that most ERBS associated with long-range chromatin interactions are colocalized with AP-2γ and FoxA1. Together, our results suggest AP-2γ is a novel collaborative factor in ERα-mediated transcription.

Dynamic localization of tripartite motif-containing 22 in nuclear and nucleolar bodies.

Tripartite motif-containing 22 (TRIM22) exhibits antiviral and growth inhibitory properties, but there has been no study on the localization and dynamics of the endogenous TRIM22 protein. We report here that TRIM22 is dramatically induced by progesterone in MDA-MB-231-derived ABC28 cells and T47D cells. This induction was associated with an increase in TRIM22 nuclear bodies (NB), and an even more prominent increase in nucleolar TRIM22 bodies. Distinct endogenous TRIM22 NB were also demonstrated in several other cell lines including MCF7 and HeLa cells. These TRIM22 NB resemble Cajal bodies, co-localized with these structures and co-immunoprecipitated with p80-coilin. However, IFNgamma-induced TRIM22 in HeLa and MCF7 cells did not form NB, implying the forms and distribution of TRIM22 are regulated by specific cellular signals. This notion is also supported by the observation that TRIM22 NB undergoes dynamic cell-cycle dependent changes in distribution such that TRIM22 NB started to form in early G0/G1 but became dispersed in the S-phase. In light of its potential antiviral and antitumor properties, the findings here provide an interesting gateway to study the relationship between the different forms and functions of TRIM22.