Epigenetic Activation of WNT5A Drives Glioblastoma Stem Cell Differentiation and Invasive Growth.

Glioblastoma stem cells (GSCs) are implicated in tumor neovascularization, invasiveness, and therapeutic resistance. To illuminate mechanisms governing these hallmark features, we developed a de novo glioblastoma multiforme (GBM) model derived from immortalized human neural stem/progenitor cells (hNSCs) to enable precise system-level comparisons of pre-malignant and oncogene-induced malignant states of NSCs. Integrated transcriptomic and epigenomic analyses uncovered a PAX6/DLX5 transcriptional program driving WNT5A-mediated GSC differentiation into endothelial-like cells (GdECs). GdECs recruit existing endothelial cells to promote peritumoral satellite lesions, which serve as a niche supporting the growth of invasive glioma cells away from the primary tumor. Clinical data reveal higher WNT5A and GdECs expression in peritumoral and recurrent GBMs relative to matched intratumoral and primary GBMs, respectively, supporting WNT5A-mediated GSC differentiation and invasive growth in disease recurrence. Thus, the PAX6/DLX5-WNT5A axis governs the diffuse spread of glioma cells throughout the brain parenchyma, contributing to the lethality of GBM.

Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression.

UNLABELLED: The signaling mechanisms between prostate cancer cells and infiltrating immune cells may illuminate novel therapeutic approaches. Here, utilizing a prostate adenocarcinoma model driven by loss of Pten and Smad4, we identify polymorphonuclear myeloid-derived suppressor cells (MDSC) as the major infiltrating immune cell type, and depletion of MDSCs blocks progression. Employing a novel dual reporter prostate cancer model, epithelial and stromal transcriptomic profiling identified CXCL5 as a cancer-secreted chemokine to attract CXCR2-expressing MDSCs, and, correspondingly, pharmacologic inhibition of CXCR2 impeded tumor progression. Integrated analyses identified hyperactivated Hippo-YAP signaling in driving CXCL5 upregulation in cancer cells through the YAP-TEAD complex and promoting MDSC recruitment. Clinicopathologic studies reveal upregulation and activation of YAP1 in a subset of human prostate tumors, and the YAP1 signature is enriched in primary prostate tumor samples with stronger expression of MDSC-relevant genes. Together, YAP-driven MDSC recruitment via heterotypic CXCL5-CXCR2 signaling reveals an effective therapeutic strategy for advanced prostate cancer. SIGNIFICANCE: We demonstrate a critical role of MDSCs in prostate tumor progression and discover a cancer cell nonautonomous function of the Hippo-YAP pathway in regulation of CXCL5, a ligand for CXCR2-expressing MDSCs. Pharmacologic elimination of MDSCs or blocking the heterotypic CXCL5-CXCR2 signaling circuit elicits robust antitumor responses and prolongs survival.

Telomere dysfunction drives aberrant hematopoietic differentiation and myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) risk correlates with advancing age, therapy-induced DNA damage, and/or shorter telomeres, but whether telomere erosion directly induces MDS is unknown. Here, we provide the genetic evidence that telomere dysfunction-induced DNA damage drives classical MDS phenotypes and alters common myeloid progenitor (CMP) differentiation by repressing the expression of mRNA splicing/processing genes, including SRSF2. RNA-seq analyses of telomere dysfunctional CMP identified aberrantly spliced transcripts linked to pathways relevant to MDS pathogenesis such as genome stability, DNA repair, chromatin remodeling, and histone modification, which are also enriched in mouse CMP haploinsufficient for SRSF2 and in CD34(+) CMML patient cells harboring SRSF2 mutation. Together, our studies establish an intimate link across telomere biology, aberrant RNA splicing, and myeloid progenitor differentiation.

Yap1 activation enables bypass of oncogenic Kras addiction in pancreatic cancer.

Activating mutations in KRAS are among the most frequent events in diverse human carcinomas and are particularly prominent in human pancreatic ductal adenocarcinoma (PDAC). An inducible Kras(G12D)-driven mouse model of PDAC has established a critical role for sustained Kras(G12D) expression in tumor maintenance, providing a model to determine the potential for and the underlying mechanisms of Kras(G12D)-independent PDAC recurrence. Here, we show that some tumors undergo spontaneous relapse and are devoid of Kras(G12D) expression and downstream canonical MAPK signaling and instead acquire amplification and overexpression of the transcriptional coactivator Yap1. Functional studies established the role of Yap1 and the transcriptional factor Tead2 in driving Kras(G12D)-independent tumor maintenance. The Yap1/Tead2 complex acts cooperatively with E2F transcription factors to activate a cell cycle and DNA replication program. Our studies, along with corroborating evidence from human PDAC models, portend a novel mechanism of escape from oncogenic Kras addiction in PDAC.

From the Cover: Neutralization of terminal differentiation in gliomagenesis.

An immature state of cellular differentiation--characterized by stem cell-like tendencies and impaired differentiation--is a hallmark of cancer. Using glioblastoma multiforme (GBM) as a model system, we sought to determine whether molecular determinants that drive cells toward terminal differentiation are also genetically targeted in carcinogenesis and whether neutralizing such genes also plays an active role to reinforce the impaired differentiation state and promote malignancy. To that end, we screened 71 genes with known roles in promoting nervous system development that also sustain copy number loss in GBM through antineoplastic assay and identified A2BP1 (ataxin 2 binding protein 1, Rbfox1), an RNA-binding and splicing regulator that is deleted in 10% of GBM cases. Integrated in silico analysis of GBM profiles to elucidate the A2BP1 pathway and its role in glioma identified myelin transcription factor 1-like (Myt1L) as a direct transcriptional regulator of A2BP1. Reintroduction of A2BP1 or Myt1L in GBM cell lines and glioma stem cells profoundly inhibited tumorigenesis in multiple assays, and conversely, shRNA-mediated knockdown of A2BP1 or Myt1L in premalignant neural stem cells compromised neuronal lineage differentiation and promoted orthotopic tumor formation. On the mechanistic level, with the top-represented downstream target TPM1 as an illustrative example, we demonstrated that, among its multiple functions, A2BP1 serves to regulate TPM1's alternative splicing to promote cytoskeletal organization and terminal differentiation and suppress malignancy. Thus, in addition to the activation of self-renewal pathways, the neutralization of genetic programs that drive cells toward terminal differentiation may also promote immature and highly plastic developmental states that contribute to the aggressive malignant properties of GBM.

Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway.

Cancer cells have metabolic dependencies that distinguish them from their normal counterparts. Among these dependencies is an increased use of the amino acid glutamine to fuel anabolic processes. Indeed, the spectrum of glutamine-dependent tumours and the mechanisms whereby glutamine supports cancer metabolism remain areas of active investigation. Here we report the identification of a non-canonical pathway of glutamine use in human pancreatic ductal adenocarcinoma (PDAC) cells that is required for tumour growth. Whereas most cells use glutamate dehydrogenase (GLUD1) to convert glutamine-derived glutamate into α-ketoglutarate in the mitochondria to fuel the tricarboxylic acid cycle, PDAC relies on a distinct pathway in which glutamine-derived aspartate is transported into the cytoplasm where it can be converted into oxaloacetate by aspartate transaminase (GOT1). Subsequently, this oxaloacetate is converted into malate and then pyruvate, ostensibly increasing the NADPH/NADP(+) ratio which can potentially maintain the cellular redox state. Importantly, PDAC cells are strongly dependent on this series of reactions, as glutamine deprivation or genetic inhibition of any enzyme in this pathway leads to an increase in reactive oxygen species and a reduction in reduced glutathione. Moreover, knockdown of any component enzyme in this series of reactions also results in a pronounced suppression of PDAC growth in vitro and in vivo. Furthermore, we establish that the reprogramming of glutamine metabolism is mediated by oncogenic KRAS, the signature genetic alteration in PDAC, through the transcriptional upregulation and repression of key metabolic enzymes in this pathway. The essentiality of this pathway in PDAC and the fact that it is dispensable in normal cells may provide novel therapeutic approaches to treat these refractory tumours.

A comprehensive nuclear receptor network for breast cancer cells.

In breast cancer, nuclear receptors (NRs) play a prominent role in governing gene expression, have prognostic utility, and are therapeutic targets. We built a regulatory map for 24 NRs, six chromatin state markers, and 14 breast-cancer-associated transcription factors (TFs) that are expressed in the breast cancer cell line MCF-7. The resulting network reveals a highly interconnected regulatory matrix where extensive crosstalk occurs among NRs and other breast -cancer-associated TFs. We show that large numbers of factors are coordinately bound to highly occupied target regions throughout the genome, and these regions are associated with active chromatin state and hormone-responsive gene expression. This network also provides a framework for stratifying and predicting patient outcomes, and we use it to show that the peroxisome proliferator-activated receptor delta binds to a set of genes also regulated by the retinoic acid receptors and whose expression is associated with poor prognosis in breast cancer.

Oncogenic Kras maintains pancreatic tumors through regulation of anabolic glucose metabolism.

Tumor maintenance relies on continued activity of driver oncogenes, although their rate-limiting role is highly context dependent. Oncogenic Kras mutation is the signature event in pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible Kras(G12D)-driven PDAC mouse model establishes that advanced PDAC remains strictly dependent on Kras(G12D) expression. Transcriptome and metabolomic analyses indicate that Kras(G12D) serves a vital role in controlling tumor metabolism through stimulation of glucose uptake and channeling of glucose intermediates into the hexosamine biosynthesis and pentose phosphate pathways (PPP). These studies also reveal that oncogenic Kras promotes ribose biogenesis. Unlike canonical models, we demonstrate that Kras(G12D) drives glycolysis intermediates into the nonoxidative PPP, thereby decoupling ribose biogenesis from NADP/NADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in PDAC.

FoxOs enforce a progression checkpoint to constrain mTORC1-activated renal tumorigenesis.

mTORC1 is a validated therapeutic target for renal cell carcinoma (RCC). Here, analysis of Tsc1-deficient (mTORC1 hyperactivation) mice uncovered a FoxO-dependent negative feedback circuit constraining mTORC1-mediated renal tumorigenesis. We document robust FoxO activation in Tsc1-deficient benign polycystic kidneys and FoxO extinction on progression to murine renal tumors; murine renal tumor progression on genetic deletion of both Tsc1 and FoxOs; and downregulated FoxO expression in most human renal clear cell and papillary carcinomas, yet continued expression in less aggressive RCCs and benign renal tumor subtypes. Mechanistically, integrated analyses revealed that FoxO-mediated block operates via suppression of Myc through upregulation of the Myc antagonists, Mxi1-SRα and mir-145, establishing a FoxO-Mxi1-SRα/mir-145 axis as a major progression block in renal tumor development.

Genomic antagonism between retinoic acid and estrogen signaling in breast cancer.

Retinoic acid (RA) triggers antiproliferative effects in tumor cells, and therefore RA and its synthetic analogs have great potential as anticarcinogenic agents. Retinoic acid receptors (RARs) mediate RA effects by directly regulating gene expression. To define the genetic network regulated by RARs in breast cancer, we identified RAR genomic targets using chromatin immunoprecipitation and expression analysis. We found that RAR binding throughout the genome is highly coincident with estrogen receptor alpha (ERalpha) binding, resulting in a widespread crosstalk of RA and estrogen signaling to antagonistically regulate breast cancer-associated genes. ERalpha- and RAR-binding sites appear to be coevolved on a large scale throughout the human genome, often resulting in competitive binding activity at nearby or overlapping cis-regulatory elements. The highly coordinated intersection between these two critical nuclear hormone receptor signaling pathways provides a global mechanism for balancing gene expression output via local regulatory interactions dispersed throughout the genome.

Genomic mapping of binding regions for the Ecdysone receptor protein complex.

We determined the physical locations of the heterodimeric Ecdysone receptor/Ultraspiracle (ECR/USP) nuclear hormone receptor complex throughout the entire nonrepetitive genome of Drosophila melanogaster using a cell line (Kc167) that differentiates in response to 20-hydroxyecdysone (20-HE). 20-HE, the natural ligand of this complex, controls major aspects of insect development, including molting, metamorphosis, and reproduction. Direct gene targets of 20-HE signaling were identified by combining this physical binding-site profiling with gene expression profiling after treatment with 20-HE. We found 502 significant regions of ECR/USP binding throughout the genome. Only 42% of these regions are nearby genes that are 20-HE responsive in these cells. However, at least three quarters of the remaining ECR/USP regions are near 20-HE-regulated genes in other tissue and cell types during metamorphosis, suggesting that binding at many regulatory elements in the genome is largely noncell-type specific. The majority (21/26) of the early targets of 20-HE encode transcriptional regulatory factors. To determine whether any of these targets are required for the morphological differentiation of these cells, we used RNAi to reduce the expression of each of the 26 early genes. Accordingly, we found that three direct targets of ECR/USP--hairy, vrille, and Hr4--are required for cellular differentiation in response to the hormone. Initial mutational analysis of vrille in vivo reveals that it is required for metamorphosis.

Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer.

We constructed a large-scale functional network model in Drosophila melanogaster built around two key transcription factors involved in the process of embryonic segmentation. Analysis of the model allowed the identification of a new role for the ubiquitin E3 ligase complex factor SPOP. In Drosophila, the gene encoding SPOP is a target of segmentation transcription factors. Drosophila SPOP mediates degradation of the Jun kinase phosphatase Puckered, thereby inducing tumor necrosis factor (TNF)/Eiger-dependent apoptosis. In humans, we found that SPOP plays a conserved role in TNF-mediated JNK signaling and was highly expressed in 99% of clear cell renal cell carcinomas (RCCs), the most prevalent form of kidney cancer. SPOP expression distinguished histological subtypes of RCC and facilitated identification of clear cell RCC as the primary tumor for metastatic lesions.

Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression.

We demonstrate an integrated approach to the study of a transcriptional regulatory cascade involved in the progression of breast cancer and we identify a protein associated with disease progression. Using chromatin immunoprecipitation and genome tiling arrays, whole genome mapping of transcription factor-binding sites was combined with gene expression profiling to identify genes involved in the proliferative response to estrogen (E2). Using RNA interference, selected ERalpha and c-MYC gene targets were knocked down to identify mediators of E2-stimulated cell proliferation. Tissue microarray screening revealed that high expression of an epigenetic factor, the E2-inducible histone variant H2A.Z, is significantly associated with lymph node metastasis and decreased breast cancer survival. Detection of H2A.Z levels independently increased the prognostic power of biomarkers currently in clinical use. This integrated approach has accelerated the identification of a molecule linked to breast cancer progression, has implications for diagnostic and therapeutic interventions, and can be applied to a wide range of cancers.

BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.

The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.

Detecting transcriptionally active regions using genomic tiling arrays.

We have developed a method for interpreting genomic tiling array data, implemented as the program TranscriptionDetector. Probed loci expressed above background are identified by combining replicates in a way that makes minimal assumptions about the data. We performed medium-resolution Anopheles gambiae tiling array experiments and found extensive transcription of both coding and non-coding regions. Our method also showed improved detection of transcriptional units when applied to high-density tiling array data for ten human chromosomes.

Comparative genome sequencing of Drosophila pseudoobscura: chromosomal, gene, and cis-element evolution.

We have sequenced the genome of a second Drosophila species, Drosophila pseudoobscura, and compared this to the genome sequence of Drosophila melanogaster, a primary model organism. Throughout evolution the vast majority of Drosophila genes have remained on the same chromosome arm, but within each arm gene order has been extensively reshuffled, leading to a minimum of 921 syntenic blocks shared between the species. A repetitive sequence is found in the D. pseudoobscura genome at many junctions between adjacent syntenic blocks. Analysis of this novel repetitive element family suggests that recombination between offset elements may have given rise to many paracentric inversions, thereby contributing to the shuffling of gene order in the D. pseudoobscura lineage. Based on sequence similarity and synteny, 10,516 putative orthologs have been identified as a core gene set conserved over 25-55 million years (Myr) since the pseudoobscura/melanogaster divergence. Genes expressed in the testes had higher amino acid sequence divergence than the genome-wide average, consistent with the rapid evolution of sex-specific proteins. Cis-regulatory sequences are more conserved than random and nearby sequences between the species--but the difference is slight, suggesting that the evolution of cis-regulatory elements is flexible. Overall, a pattern of repeat-mediated chromosomal rearrangement, and high coadaptation of both male genes and cis-regulatory sequences emerges as important themes of genome divergence between these species of Drosophila.

A gene expression map for the euchromatic genome of Drosophila melanogaster.

We used a maskless photolithography method to produce DNA oligonucleotide microarrays with unique probe sequences tiled throughout the genome of Drosophila melanogaster and across predicted splice junctions. RNA expression of protein coding and nonprotein coding sequences was determined for each major stage of the life cycle, including adult males and females. We detected transcriptional activity for 93% of annotated genes and RNA expression for 41% of the probes in intronic and intergenic sequences. Comparison to genome-wide RNA interference data and to gene annotations revealed distinguishable levels of expression for different classes of genes and higher levels of expression for genes with essential cellular functions. Differential splicing was observed in about 40% of predicted genes, and 5440 previously unknown splice forms were detected. Genes within conserved regions of synteny with D. pseudoobscura had highly correlated expression; these regions ranged in length from 10 to 900 kilobase pairs. The expressed intergenic and intronic sequences are more likely to be evolutionarily conserved than nonexpressed ones, and about 15% of them appear to be developmentally regulated. Our results provide a draft expression map for the entire nonrepetitive genome, which reveals a much more extensive and diverse set of expressed sequences than was previously predicted.

An arabidopsis promoter microarray and its initial usage in the identification of HY5 binding targets in vitro.

To analyze transcription factor-promoter interactions in Arabidopsis, a general strategy for generating a promoter microarray has been established. This includes an integrated platform for promoter sequence extraction and the design of primers for the PCR amplification of the promoter regions of annotated genes in the Arabidopsis genome. A web-interfaced primer-retrieval program was used to obtain up to 10 primer pairs with a suitability ranking given to each gene. We selected primer pairs for the promoters of about 3800 genes, and greater than 95% of the promoter fragments from the total genomic DNA were successfully amplified by PCR. These PCR products were purified and used to print an Arabidopsis promoter microarray. This initial promoter microarray was used to study the in vitro binding of the transcription factor HY5 to its promoter targets. A set of promoter fragments exhibited consistent and strong interaction with the HY5 protein in vitro, and computational analysis revealed that they were enriched with the HY5 consensus binding G-box motif. Thus, a promoter microarray can be a useful tool for identifying transcription factor binding sites at the genomic scale in higher plants.

DBSubLoc: database of protein subcellular localization.

We have built a protein subcellular localization annotation database, the DBSubLoc database, which is available at http://www.bioinfo.tsinghua. edu.cn/dbsubloc.html. Annotations were taken from primary protein databases, model organism genome projects and literature texts, and then were analyzed to dig out the subcellular localization features of the proteins. The proteins are also classified into different categories. Based on sequence alignment, non-redundant subsets of the database have been built, which may provide useful information for subcellular localization prediction. The database now contains >60,000 protein sequences including approximately 30,000 protein sequences in the non-redundant data sets. Online download, search and Blast tools are also available.