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Androgen receptor (AR) plays a central role in driving prostate cancer (PCa) progression. How AR promotes this process is still not completely clear. Herein, we used single-cell transcriptome analysis to reconstruct the transcriptional network of AR in PCa. Our work shows AR directly regulates a set of signature genes in the ER-to-Golgi protein vesicle-mediated transport pathway. The expression of these genes is required for maximum androgen-dependent ER-to-Golgi trafficking, cell growth, and survival. Our analyses also reveal the signature genes are associated with PCa progression and prognosis. Moreover, we find inhibition of the ER-to-Golgi transport process with a small molecule enhanced antiandrogen-mediated tumor suppression of hormone-sensitive and insensitive PCa. Finally, we demonstrate AR collaborates with CREB3L2 in mediating ER-to-Golgi trafficking in PCa. In summary, our findings uncover a critical role for dysregulation of ER-to-Golgi trafficking expression and function in PCa progression, provide detailed mechanistic insights for how AR tightly controls this process, and highlight the prospect of targeting the ER-to-Golgi pathway as a therapeutic strategy for advanced PCa.

Earlier detection of biomarkers responsible for cancer relapse facilitates more rational cancer treatment regimens to be designed. Herein, we develop a mass cytometry-based strategy for unbiased mining of cell subsets that potentially contribute to cancer recurrence through panoramic examination of the immunophenotypic features and multidrug resistance characteristics. The incorporation of metal tags enables multiplexed information of single cells to be interrogated based on metal fingerprint. Using acute lymphoblastic leukemia (B-ALL) as a showcase, we show overexpressed multidrug resistance biomarkers, i. e., BCRP, Bcl-2, MRP1, and P-gp in B-ALL cells compared with healthy control, and a positive correlation among different multidrug resistance biomarkers. Different cell subsets with multidrug resistance are well-defined, featured with CD34+ CD38+ CD10- and CD34+ CD38+/int CD10+ . Importantly, we uncovered that CD34 expression level is positively correlated to multidrug resistance, indicative of a higher potential of immature cells to induce B-ALL relapse. In addition, the cell subsets positively expressing CD73 and CD304 (CD34+ CD10+ CD304+ ; CD34+ CD38+/int CD10+ CD73+ ) also overexpress multidrug resistance biomarkers, suggesting that they may serve as additional new biomarkers for B-ALL stratification and prognosis. Our data provide the first evidence that highly expressed multidrug resistance biomarkers in certain cell subpopulations with specific immunophenotypes may potentially induce B-ALL recurrence. The incorporation of multidrug resistance features with cell phenotypes using mass cytometry proposed in this study provides a general strategy for risk assessment and the prediction of recurrence of different types of cancers.

We elucidated the anti-inflammatory mechanisms of IL-38 in allergic asthma. Human bronchial epithelial cells and eosinophils were cocultured upon stimulation with the viral RLR ligand poly (I:C)/LyoVec or infection-related cytokine TNF-α to induce expression of cytokines/chemokines/adhesion molecules. House dust mite (HDM)-induced allergic asthma and humanized allergic asthma NOD/SCID murine models were established to assess anti-inflammatory mechanisms in vivo. IL-38 significantly inhibited induced proinflammatory IL-6, IL-1β, CCL5, and CXCL10 production, and antiviral interferon-β and intercellular adhesion molecule-1 expression in the coculture system. Mass cytometry and RNA-sequencing analysis revealed that IL-38 could antagonize the activation of the intracellular STAT1, STAT3, p38 MAPK, ERK1/2, and NF-κB pathways, and upregulate the expression of the host defense-related gene POU2AF1 and anti-allergic response gene RGS13. Intraperitoneal injection of IL-38 into HDM-induced allergic asthma mice could ameliorate airway hyperreactivity by decreasing the accumulation of eosinophils in the lungs and inhibiting the expression of the Th2-related cytokines IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid (BALF) and lung homogenates. Histological examination indicated lung inflammation was alleviated by reductions in cell infiltration and goblet cell hyperplasia, together with reduced Th2, Th17, and innate lymphoid type 2 cell numbers but increased proportions of regulatory T cells in the lungs, spleen, and lymph nodes. IL-38 administration suppressed airway hyperreactivity and asthma-related IL-4 and IL-5 expression in humanized mice, together with significantly decreased CCR3+ eosinophil numbers in the BALF and lungs, and a reduced percentage of human CD4+CRTH2+ Th2 cells in the lungs and mediastinal lymph nodes. Together, our results demonstrated the anti-inflammatory mechanisms of IL-38 and provided a basis for the development of a regulatory cytokine-based treatment for allergic asthma.

Summary Pyruvate is a key metabolite in glycolysis and the tricarboxylic acid (TCA) cycle. Exogenous pyruvate modulates metabolism, provides cellular protection, and is essential for the maintenance of human preimplantation embryos and human embryonic stem cells (hESCs). However, little is known about how pyruvate contributes to cell-fate determination during epiblast stage. In this study, we used hESCs as a model to demonstrate that elevated exogenous pyruvate shifts metabolic balance toward oxidative phosphorylation in both maintenance and differentiation conditions. During differentiation, pyruvate potentiates mesoderm and endoderm lineage specification. Pyruvate production and its mitochondrial metabolism are required in BMP4-induced mesoderm differentiation. However, the TCA-cycle metabolites do not have the same effect as pyruvate on differentiation. Further study shows that pyruvate increases AMP/ATP ratio, activates AMPK, and modulates the mTOR pathway to enhance mesoderm differentiation. This study reveals that exogenous pyruvate not only controls metabolism but also modulates signaling pathways in hESC differentiation., Graphical Abstract, Highlights • Exogenous pyruvate enhances TCA-cycle metabolism in hESCs • Exogenous pyruvate promotes BMP4-induced mesoderm differentiation • Exogenous pyruvate influences differentiation through AMPK/mTOR pathways, In this report, Chen and colleagues demonstrate that pyruvate promotes mesoderm differentiation in human embryonic stem cells. Pyruvate elevates TCA-cycle metabolism and modulates AMPK and mTOR pathways to promote BMP4-induced mesoderm differentiation. This discovery highlights that pyruvate as a common metabolite can regulate signaling pathways in cell-fate determination.

The human genome is mostly transcribed, yielding a rich repository of noncoding transcripts that are involved in a myriad of biological processes including cancer. However, how many noncoding transcripts such as long noncoding RNAs (lncRNA) function in cancer is still unclear. This study identified a novel set of clinically relevant androgen-regulated lncRNAs in prostate cancer. Among this group, LINC00844 was demonstrated to be a direct androgen-regulated target that is actively transcribed in androgen receptor (AR)–dependent prostate cancer cells. The expression of LINC00844 is higher in normal prostate compared with malignant and metastatic prostate cancer clinical specimens, and patients with low expression had a poor prognosis and significantly increased biochemical recurrence, suggesting LINC00844 functions in suppressing tumor progression and metastasis. Indeed, in vitro loss-of-function studies revealed that LINC00844 prevents prostate cancer cell migration and invasion. Moreover, findings from gene expression profiling analysis indicated that LINC00844 functions in trans, affecting global androgen-regulated gene transcription. Mechanistic evidence reveals that LINC00844 is important in facilitating AR binding to the chromatin. Finally, LINC00844 mediates its phenotypic effects in part by activating the expression of NDRG1, a crucial cancer metastasis suppressor. Collectively, LINC00844 is a novel coregulator of AR that plays a central role in the androgen transcriptional network and the development and progression of prostate cancer. Implications: This study highlights the function of the lncRNA, LINC00844, in regulating global AR-regulated genes in prostate cancer by modulating AR binding to chromatin.

Identification of prognostic biomarkers helps facilitate the prediction of patient outcomes as well as guide treatments. Accumulating evidence now suggests that long non-coding RNAs (lncRNAs) play key roles in tumor progression with diagnostic and prognostic values. However, little is known about the biological functions of lncRNAs and how they contribute to the pathogenesis of cancer. Herein, we performed weighted correlation network analysis (WGCNA) on 380 RNA-seq samples from prostate cancer patients to create networks comprising of microRNAs, lncRNAs, and protein-coding genes. Our analysis revealed expression modules that associated with pathological parameters. More importantly, we identified a gene module that is involved in protein translation and is associated with patient survival. In this gene module, we explored the regulation axis involving GAS5, ZFAS1, and miR-940. We show that GAS5, ZFAS1, and miR-940 are up-regulated in tumors relative to normal prostate tissues, and high expression of either lncRNA is an indicator of poor patient outcome. Finally, we constructed a co-expression network involving GAS5, ZFAS1, and miR-940, as well as the targets of miR-940. Our results show that GAS5 and ZFAS1 are targeted by miR-940 via NAA10 and RPL28. Taken together, co-expression analysis of gene expression profiling from RNA-seq can accelerate the identification and functional characterization of novel prognostic markers in prostate cancer.

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.

The identification and functional characterization of novel biomarkers in cancer requires survival analysis and gene expression analysis of both patient samples and cell line models. To help facilitate this process, we have developed KM-Express. KM-Express holds an extensive manually curated transcriptomic profile of 45 different datasets for prostate and breast cancer with phenotype and pathoclinical information, spanning from clinical samples to cell lines. KM-Express also contains The Cancer Genome Atlas datasets for 30 other cancer types with matching cell line expression data for 23 of them. We present KM-Express as a hypothesis generation tool for researchers to identify potential new prognostic RNA biomarkers as well as targets for further downstream functional cell-based studies. Specifically, KM-Express allows users to compare the expression level of genes in different groups of patients based on molecular, genetic, clinical and pathological status. Moreover, KM-Express aids the design of biological experiments based on the expression profile of the genes in different cell lines. Thus, KM-Express provides a one-stop analysis from bench work to clinical prospects. We have used this tool to successfully evaluate the prognostic potential of previously published biomarkers for prostate cancer and breast cancer. We believe KM-Express will accelerate the translation of biomedical research from bench to bed. Database URL: http://ec2-52-201-246-161.compute-1.amazonaws.com/kmexpress/index.php

Objectives Drug combination in cancer therapy aims to achieve synergistic therapeutic effect, reduced drug dosage, reduced drug toxicity and minimizes or delays the induction of drug resistance. In the present study, we investigated the anticancer effects of the combination of two metabolic modulators, dichloroacetate (DCA) and bacillus caldovelox arginase (BCA) (or pegyated human arginase (HA)). Methods The combination treatments were evaluated in MCF-7 and MDA-MB 231 cells as well as in MDA-MB 231 breast cancer xenograft model. Key findings Dichloroacetate and BCA combination exhibited anti-proliferative effects on MCF-7 cells, which were found to be synergistic. Analysis of the gene expression upon drug treatments revealed that the synergistic anti-proliferative effect on MCF-7 cells was possibly in part due to the activation of the p53 pathway. A similar synergistic anti-proliferative effect was observed in the combined use of DCA and HA on MCF-7 and MDA-MB231 cells, which was due to induction of cell cycle arrest at G2/M phase. Moreover, the combination enhanced anti-tumour activity in a MDA-MB 231 xenograft mouse model. Conclusions Our results suggested that dichloroacetate and arginase combination exhibited enhanced anti-cancer effects in preclinical breast cancer models which may offer an additional treatment option for breast cancer.

Despite altered metabolism being an accepted hallmark of cancer, it is still not completely understood which signaling pathways regulate these processes. Given the central role of androgen receptor (AR) signaling in prostate cancer, we hypothesized that AR could promote prostate cancer cell growth in part through increasing glucose uptake via the expression of distinct glucose transporters. Here, we determined that AR directly increased the expression ofSLC2A12, the gene that encodes the glucose transporter GLUT12. In support of these findings, gene signatures of AR activity correlated withSLC2A12expression in multiple clinical cohorts. Functionally, GLUT12 was required for maximal androgen-mediated glucose uptake and cell growth in LNCaP and VCaP cells. Knockdown of GLUT12 also decreased the growth of C4-2, 22Rv1 and AR-negative PC-3 cells. This latter observation corresponded with a significant reduction in glucose uptake, indicating that additional signaling mechanisms could augment GLUT12 function in an AR-independent manner. Interestingly, GLUT12 trafficking to the plasma membrane was modulated by calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-5′-AMP-activated protein kinase (AMPK) signaling, a pathway we previously demonstrated to be a downstream effector of AR. Inhibition of CaMKK2-AMPK signaling decreased GLUT12 translocation to the plasma membrane by inhibiting the phosphorylation of TBC1D4, a known regulator of glucose transport. Further, AR increasedTBC1D4expression. Correspondingly, expression ofTBC1D4correlated with AR activity in prostate cancer patient samples. Taken together, these data demonstrate that prostate cancer cells can increase the functional levels of GLUT12 through multiple mechanisms to promote glucose uptake and subsequent cell growth.

Estrogen regulates diverse physiological effects and drives breast tumor progression by directly activating estrogen receptor α (ERα). However, due to the stochastic nature of gene transcription and the resulting heterogeneous cellular response, it is important to investigate estrogen-stimulated gene expression profiles at the single-cell level in order to fully understand how ERα regulates transcription in breast cancer cells. In this study, we performed single-cell transcriptome analysis on ERα-positive breast cancer cell lines following 17β-estradiol stimulation. Overall, we observed robust gene expression diversity between individual cells. Moreover, we found over two thirds of the genes in breast cancer cells displayed a bimodal expression pattern, which caused averaging artifacts and masked the identification of potential estrogen-regulated genes. We overcame this issue by reconstructing a dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Pathway analysis of the differentially expressed genes derived from the pseudotemporal analysis showed an estrogen-stimulated metabolic switch that favored biosynthesis and cell proliferation but reduced estrogen degradation. In addition, we identified folate-mediated one-carbon metabolism as a novel estrogen-regulated pathway in breast cancer cells. Notably, estrogen stimulation reprogramed this pathway through the mitochondrial folate pathway to coordinately fuel polyamine and de novo purine synthesis. Finally, we showed AZIN1 and PPAT, key regulators in the above pathways, are direct ERα target genes and essential for breast cancer cell survival and growth. In summary, our single-cell study illustrated a dynamic transcriptional heterogeneity in ERα-positive breast cancer cells in response to estrogen stimulation and uncovered a novel mechanism of an estrogen-mediated metabolic switch.

The majority of the human genome is transcribed, yielding a rich repository of non-coding transcripts that are involved in a myriad of biological processes including cancer. However, how non-coding transcripts such as long non-coding RNAs (lncRNAs) function in prostate cancer is still unclear. In this study, we have identified a novel set of clinically relevant androgen-regulated lncRNAs in prostate cancer. Among this group, we showed LINC00844 is a direct androgen-regulated target that is actively transcribed in androgen receptor (AR)-dependent prostate cancer cells. The expression of LINC00844 is higher in normal prostate compared to malignant and metastatic prostate cancer samples and patients with low expression demonstrate poor prognosis and significantly increased biochemical recurrence, suggesting LINC00844 may function in suppressing tumor progression and metastasis. Indeed,in-vitroloss-of-function studies revealed that LINC00844 prevents prostate cancer cell migration and invasion. Moreover, findings from gene expression analysis indicated that LINC00844 functions intrans, affecting global androgen-regulated gene transcription. Mechanistically, we provide evidence to show LINC00844 is important in facilitating AR binding to the chromatin. Finally, we demonstrated LINC00844 mediates its phenotypic effects in part by activating the expression of NDRG1, a crucial cancer metastasis suppressor. Collectively, our findings suggest LINC00844 is a novel coregulator of AR that plays a central role in the androgen transcriptional network and the development and progression of prostate cancer.

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.

Summary: Estrogen drives breast cancer (BCa) progression by directly activating estrogen receptor α (ERα). However, because of the stochastic nature of gene transcription, it is important to study the estrogen signaling pathway at the single-cell level to fully understand how ERα regulates transcription. Here, we performed single-cell transcriptome analysis on ERα-positive BCa cells following 17β-estradiol stimulation and reconstructed the dynamic estrogen-responsive transcriptional network from discrete time points into a pseudotemporal continuum. Notably, differentially expressed genes show an estrogen-stimulated metabolic switch that favors biosynthesis but reduces estrogen degradation. Moreover, folate-mediated one-carbon metabolism is reprogrammed through the mitochondrial folate pathway and polyamine and purine synthesis are upregulated coordinately. Finally, we show AZIN1 and PPAT are direct ERα targets that are essential for BCa cell survival and growth. In summary, our study highlights the dynamic transcriptional heterogeneity in ERα-positive BCa cells upon estrogen stimulation and uncovers a mechanism of estrogen-mediated metabolic switch. : Zhu et al. perform single-cell RNA-seq to reveal a dynamic transcriptional network in ERα+ breast cancer cells following estrogen stimulation and show that estrogen signaling promotes breast cancer cell survival and growth by mediating a metabolic switch in which folate-mediated one-carbon metabolism is reprogrammed via the mitochondrial folate pathway. Keywords: breast cancer, single-cell RNA sequencing, estrogen receptor α, metabolic switch

The progression of prostate cancer is largely dependent on the activity of the androgen receptor (AR), which in turn, correlates with the net output of the AR transcriptional regulatory network. A detailed and thorough understanding of the AR transcriptional regulatory network is therefore critical in the strategic manipulation of AR activity for the targeted eradication of prostate cancer cells. In this mini-review, we highlight some of the novel and unexpected mechanistic and functional insights of the AR transcriptional network derived from recent targeted sequencing (ChIP–Seq) studies of AR and its coregulatory factors in prostate cancer cells. ASTAR (Agency for Sci., Tech. and Research, S’pore)

AR (androgen receptor) signaling is crucial for the development and maintenance of the prostate as well as the initiation and progression of prostate cancer. Despite the AR's central role in prostate cancer progression, it is still unclear which AR-mediated processes drive the disease. Here, we identified 4 core autophagy genes: ATG4B, ATG4D, ULK1, and ULK2, in addition to the transcription factor TFEB, a master regulator of lysosomal biogenesis and function, as transcriptional targets of AR in prostate cancer. These findings were significant in light of our recent observation that androgens promoted prostate cancer cell growth in part through the induction of autophagy. Expression of these 5 genes was essential for maximal androgen-mediated autophagy and cell proliferation. In addition, expression of each of these 5 genes alone or in combination was sufficient to increase prostate cancer cell growth independent of AR activity. Further, bioinformatic analysis demonstrated that the expression of these genes correlated with disease progression in 3 separate clinical cohorts. Collectively, these findings demonstrate a functional role for increased autophagy in prostate cancer progression, provide a mechanism for how autophagy is augmented, and highlight the potential of targeting this process for the treatment of advanced prostate cancer.

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.

Neuroendocrine prostate cancer (NEPC) has a poor prognosis, with a median survival of less than 1 year after diagnosis. Following androgen deprivation therapy, prostate adenocarcinoma cells have been observed to develop an androgen receptor-negative, terminally differentiated and indolent neuroendocrine-like phenotype. However, several molecular events, including interleukin 6 (IL-6) stimulation, in the prostate microenvironment result in the appearance of aggressive, highly proliferative castrate-resistant NEPC. In this study, we examined the mechanistic effects of a natural prenylflavonoid, icaritin (ICT), on neuroendocrine differentiation in IL-6-induced LNCaP cells and NEPC development in the male transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice received daily intraperitoneal injection of ICT or vehicle. ICT induced apoptosis in prostate tumor, suppressed NEPC development and, accordingly, improved overall survival in TRAMP mice. Expression of neuroendocrine markers (synaptophysin) and androgen receptor in TRAMP mice and neuroendocrine-like LNCaP cells were inhibited by ICT. Suppression of neuroendocrine and NEPC development by ICT was associated with dose-dependent inhibitory effects on abnormally elevated IL-6/STAT3 and Aurora kinase A in vitro and in vivo Since ICT demonstrated favorable pharmacokinetic and safety profiles with marked enrichment in prostate tissues, our study provides evidence for the development of prenylflavonoid as a multimodal therapeutic agent against NEPC.

Persistent androgen receptor (AR) signaling is the key driving force behind progression and development of castration-resistant prostate cancer (CRPC). In many patients, AR COOH-terminal truncated splice variants (ARvs) play a critical role in contributing to the resistance against androgen depletion therapy. Unfortunately, clinically used antiandrogens like bicalutamide (BIC) and enzalutamide (MDV), which target the ligand binding domain, have failed to suppress these AR variants. Here, we report for the first time that a natural prenylflavonoid, icaritin (ICT), can co-target both persistent AR and ARvs. ICT was found to inhibit transcription of key AR-regulated genes, such as KLK3 [prostate-specific antigen (PSA)] and ARvs-regulated genes, such as UBE2C and induce apoptosis in AR-positive prostate cancer (PC) cells. Mechanistically, ICT promoted the degradation of both AR and ARvs by binding to arylhydrocarbon-receptor (AhR) to mediate ubiquitin-proteasomal degradation. Therefore, ICT impaired AR transactivation in PC cells. Knockdown of AhR gene restored AR stability and partially prevented ICT-induced growth suppression. In clinically relevant murine models orthotopically implanted with androgen-sensitive and CRPC cells, ICT was able to target AR and ARvs, to inhibit AR signaling and tumor growth with no apparent toxicity. Our results provide a mechanistic framework for the development of ICT, as a novel lead compound for AR-positive PC therapeutics, especially for those bearing AR splice variants.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0391-4) contains supplementary material, which is available to authorized users.

Androgen and the androgen receptor (AR) are critical effectors of prostate cancer. Consequently, androgen deprivation therapy is typically employed as a first-line treatment for prostate cancer patients. While initial responses are generally positive, prostate tumors frequently recur and progress to a lethal form known as castration-resistant prostate cancer (CRPC). Recently, considerable effort has been directed toward elucidating the molecular mechanisms of CRPC. Results from both preclinical and clinical studies suggest that AR-mediated signaling persists and remains functionally important in CRPC despite the elimination of androgens. Understanding the role of this pathway in the development of resistance will therefore be critical to identify alternative diagnostic markers as well as more effective therapies for the treatment of CRPC. Using next-generation sequencing and other high-throughput approaches, numerous groups are beginning to identify the key differences in the transcriptional regulatory and gene expression programs between androgen-dependent and CRPC. A number of mechanisms have been proposed for the differences and these mostly involve alterations to components of the AR co-regulatory network. In this review, we summarize current knowledge on co-regulators of the AR and discuss their potential roles in CRPC. It is anticipated that a deeper understanding of these factors will undercover new targets that can assist in the diagnosis and treatment of CRPC.

Introduction: Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancers, predominantly an arginine to histidine amino acid change at codon 132. This structural alteration drives the catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate (2HG), which enhances DNA and protein hypermethylation and cellular dedifferentiation. Pharmacologic inhibitors of the mutant IDH1 protein show promise in clinical trials, yet the regulation of IDH1 has not been fully elucidated. We recently discovered in wild type IDH1 tumors that the regulatory RNA-binding and stress response protein, HuR (ELAVL1), protects cancer cells under nutrient deprivation by regulating the expression of core metabolic enzymes, including IDH1. We mapped the regulatory HuR binding site on the IDH1 transcript to the 3’-untranslated region. Since wild type and mutant IDH1 alleles both contain this binding sequence, we hypothesize that HuR is an important regulator of both isozymes and is biologically important in mutant IDH1 tumors. Methods: HuR expression was suppressed by siRNA in a fibrosarcoma cell line (HT-1080) harboring a natural heterozygous IDH1 mutation and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Sensitivity to pharmacologic inhibition of the mutant IDH1 protein was assessed. Sanger sequencing and mRNP-IP were performed to determine HuR's impact on the expression of each IDH1 allele. To further characterize the post-transcriptional regulation of IDH1 by HuR, CRISPR/CAS 9 editing of the HuR gene was performed. Results: Sanger sequencing of IDH1 after depletion of HuR in HT1080 cells, as well as after HuR mRNP-IP, revealed that HuR regulates both the mutant and wild type IDH1. Drug sensitivity assays to a mutant IDH1 inhibitor (AGI-5198) under varying glucose concentrations revealed glucose deprivation to be a novel driver of chemo-resistance. However, HuR silencing abrogated HT1080 resistance to AGI-5198 under these conditions. Targeted knockout of HuR using the CRISPR/CAS9 system resulted in potent suppression of mutant and wild type IDH1, at both the mRNA and protein levels when compared to CRISPR/Cas9 control. Conclusions: These findings reveal that harsh metabolic conditions present in the tumor microenvironment induce chemo-resistance in an IDH1 mutant cell line to a mutant IDH1 inhibitor, in an HuR dependent manner. Moreover, both mutant and wild type IDH1 alleles are potently regulated by HuR. Therapeutic strategies that target the HuR-IDH1 axis and block this resistance mechanism may enhance the efficacy of mutant IDH1 inhibitors for relevant tumors. Citation Format: Mahsa Zarei, Shruti Lal, Nicole C. Mambelli-Lisboa, Edwin Cheung, Saswati N. Chand, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. The mRNA-binding protein HuR, regulates mutant and wild type IDH1 expression in IDH1-mutated cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1847.

The NKX3-1 gene is a homeobox gene required for prostate tumor progression, but how it functions is unclear. Here, using chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP-seq) we showed that NKX3-1 colocalizes with the androgen receptor (AR) across the prostate cancer genome. We uncovered two distinct mechanisms by which NKX3-1 controls the AR transcriptional network in prostate cancer. First, NKX3-1 and AR directly regulate each other in a feed-forward regulatory loop. Second, NKX3-1 collaborates with AR and FoxA1 to mediate genes in advanced and recurrent prostate carcinoma. NKX3-1- and AR-coregulated genes include those found in the “protein trafficking” process, which integrates oncogenic signaling pathways. Moreover, we demonstrate that NKX3-1, AR, and FoxA1 promote prostate cancer cell survival by directly upregulating RAB3B, a member of the RAB GTPase family. Finally, we show that RAB3B is overexpressed in prostate cancer patients, suggesting that RAB3B together with AR, FoxA1, and NKX3-1 are important regulators of prostate cancer progression. Collectively, our work highlights a novel hierarchical transcriptional regulatory network between NKX3-1, AR, and the RAB GTPase signaling pathway that is critical for the genetic-molecular-phenotypic paradigm in androgen-dependent prostate cancer.

Prostate cancer is one of the most common types of cancer afflicting the male population. Although prostate cancer is initially slow-growing and regresses upon androgen ablation, the disease is capable of transiting into an aggressive and metastatic form that is hormone refractory. Studies have attributed alterations in the cancer genome and transcriptome as being integral to this transition process. With the aim of developing alternative therapeutic strategies for advanced prostate ­cancer, research efforts are now directed towards a more comprehensive understanding of prostate cancer genomics. Herein, we review the progress made recently in prostate cancer genomics research with a focus on the application of next generation ­sequencing technologies.

BackgroundThe growth of sequencing-based Chromatin Immuno-Precipitation studies call for a more in-depth understanding of the nature of the technology and of the resultant data to reduce false positives and false negatives. Control libraries are typically constructed to complement such studies in order to mitigate the effect of systematic biases that might be present in the data. In this study, we explored multiple control libraries to obtain better understanding of what they truly represent.MethodologyFirst, we analyzed the genome-wide profiles of various sequencing-based libraries at a low resolution of 1 Mbp, and compared them with each other as well as against aCGH data. We found that copy number plays a major influence in both ChIP-enriched as well as control libraries. Following that, we inspected the repeat regions to assess the extent of mapping bias. Next, significantly tag-rich 5 kbp regions were identified and they were associated with various genomic landmarks. For instance, we discovered that gene boundaries were surprisingly enriched with sequenced tags. Further, profiles between different cell types were noticeably distinct although the cell types were somewhat related and similar.ConclusionsWe found that control libraries bear traces of systematic biases. The biases can be attributed to genomic copy number, inherent sequencing bias, plausible mapping ambiguity, and cell-type specific chromatin structure. Our results suggest careful analysis of control libraries can reveal promising biological insights.

Although Kruppel-like factor 5 (KLF5) is a transcription factor that has been implicated in pathways critical to carcinogenesis, controversy persists as to whether it functions as a tumor suppressor or as an oncogene. Here, we describe a novel role for KLF5 in a p53-independent apoptotic pathway. Using RNA-interference technology, we show that cells deficient in KLF5 have increased sensitivity to DNA damage, regardless of p53 status. Both p53 and p53-dependent factors are unaffected by KLF5 depletion. Instead, the apoptotic phenotype consequent to damage is associated with reduced bad phosphorylation, and downregulation of Pim1. Consistently, transfection of wild-type Pim1 is sufficient to rescue this phenotype. Previous data have shown a number of putative Sp1-binding consensus sequences on the Pim1 promoter. Remarkably, chromatin immunoprecipitation studies show that KLF5 binds to the Pim1 promoter, and that binding increases soon after damage. These results identify a novel, p53-independent apoptotic pathway through which KLF5 functions in response to DNA damage. Therapeutic deregulation of this pathway could be used to modulate chemosensitivity.

Protein acetylation is important in regulating DNA-templated processes specifically and protein-protein interactions more generally in eukaryotes. The geminivirus movement protein NSP is essential for virus movement, shuttling the viral DNA genome between the nucleus and the cytoplasm. We have identified a novel Arabidopsis protein, AtNSI, that interacts with NSP. AtNSI is highly conserved among widely divergent plants. Biochemical studies show that its interaction with NSP is direct and that AtNSI acetylates histones, but not NSP, in vitro. Rather, AtNSI specifically acetylates the viral coat protein. AtNSI is a nuclear protein but does not act as a transcriptional coactivator in vitro, which distinguishes it from known eukaryotic histone acetyltransferases. Its overexpression enhances the efficiency of infection by Cabbage leaf curl virus. These findings suggest a role for protein acetylation in coordinating replication of the viral DNA genome with its export from the nucleus.

In the majority of breast cancers, estrogen and its cellular receptor, estrogen receptor (ER), are involved in regulating tumor cell proliferation. ER is both a prognostic marker and a therapeutic target in the management of hormone-dependent tumors. Current research efforts and the paradigm of ER transcriptional regulatory functions in breast cancer have focused on the role of ER in activating target gene transcription. Microarray studies and whole-genome ER binding site mapping experiments indicate, however, that ER can alternatively directly repress the expression of a significant subset of target genes. We posit that transcriptional repression by ER likely involves interactions with other co-regulatory transcription factors found in the proximity of ER binding sites in the cis-regulatory regions of repressed target genes and have identified the runt-related transcription factor 1 (RUNX1), a key regulator of hematopoiesis and leukemogenesis, as a candidate co-regulatory factor involved in transcriptional repression by ER. Here, we demonstrate that RUNX1 interacts with ER, specifically the ligand-binding domain, and is involved in the repression of ER target genes and estrogen-dependent and -independent proliferation of breast cancer cells. We also show that putative ER-RUNX1 target genes are associated with disease outcome in breast cancer patients. These findings not only provide the first evidence for the involvement of RUNX1 in hormonal carcinogenesis and additional insights into mechanisms of transcriptional regulation by ER but may potentially lead to more effective strategies for targeting ER and estrogen signaling in the prevention and treatment of breast cancer. Citation Format: Ka Liu, Richard L. Smith, Trang Nguyen-Vu, Nicholes R. Candelaria, Colin M. Rogerson, W. Evan Johnson, Edwin Cheung, Boris R. Jankovic, Wail Ba alawi, Jinsong Zhang, Vladimir B. Bajic, Chin-Yo Lin. Runt-related transcription factor 1 (RUNX1) is involved in transcriptional repression by estrogen receptor and breast cancer cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3107. doi:10.1158/1538-7445.AM2013-3107