BRD9-SMAD2/3 Orchestrates Stemness and Tumorigenesis in Pancreatic Ductal Adenocarcinoma.

BACKGROUND & AIMS: The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. The epigenetic mechanisms regulating CSCs are currently insufficiently understood, which hampers the development of novel strategies for eliminating CSCs. METHODS: By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodeling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signaling pathway. RESULTS: Inhibition and genetic ablation of BRD9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumors from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. CONCLUSIONS: Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

Inside the stemness engine: Mechanistic links between deregulated transcription factors and stemness in cancer.

Cell identity is largely determined by its transcriptional profile. In tumour, deregulation of transcription factor expression and/or activity enables cancer cell to acquire a stem-like state characterised by capacity to self-renew, differentiate and form tumours in vivo. These stem-like cancer cells are highly metastatic and therapy resistant, thus warranting a more complete understanding of the molecular mechanisms downstream of the transcription factors that mediate the establishment of stemness state. Here, we review recent research findings that provide a mechanistic link between the commonly deregulated transcription factors and stemness in cancer. In particular, we describe the role of master transcription factors (SOX, OCT4, NANOG, KLF, BRACHYURY, SALL, HOX, FOX and RUNX), signalling-regulated transcription factors (SMAD, ß-catenin, YAP, TAZ, AP-1, NOTCH, STAT, GLI, ETS and NF-κB) and unclassified transcription factors (c-MYC, HIF, EMT transcription factors and P53) across diverse tumour types, thereby yielding a comprehensive overview identifying shared downstream targets, highlighting unique mechanisms and discussing complexities.

Junctional Adhesion Molecule-A Is Highly Expressed on Human Hematopoietic Repopulating Cells and Associates with the Key Hematopoietic Chemokine Receptor CXCR4.

Hematopoietic stem/progenitor cells (HSPCs) reside in specialized bone marrow microenvironmental niches, with vascular elements (endothelial/mesenchymal stromal cells) and CXCR4-CXCL12 interactions playing particularly important roles for HSPC entry, retention, and maintenance. The functional effects of CXCL12 are dependent on its local concentration and rely on complex HSPC-niche interactions. Two Junctional Adhesion Molecule family proteins, Junctional Adhesion Molecule-B (JAM)-B and JAM-C, are reported to mediate HSPC-stromal cell interactions, which in turn regulate CXCL12 production by mesenchymal stromal cells (MSCs). Here, we demonstrate that another JAM family member, JAM-A, is most highly expressed on human hematopoietic stem cells with in vivo repopulating activity (p < .01 for JAM-A(high) compared to JAM-A(Int or Low) cord blood CD34(+) cells). JAM-A blockade, silencing, and overexpression show that JAM-A contributes significantly (p < .05) to the adhesion of human HSPCs to IL-1ß activated human bone marrow sinusoidal endothelium. Further studies highlight a novel association of JAM-A with CXCR4, with these molecules moving to the leading edge of the cell upon presentation with CXCL12 (p < .05 compared to no CXCL12). Therefore, we hypothesize that JAM family members differentially regulate CXCR4 function and CXCL12 secretion in the bone marrow niche. Stem Cells 2016;34:1664-1678.

The pRb/RBL2-E2F1/4-GCN5 axis regulates cancer stem cell formation and G0 phase entry/exit by paracrine mechanisms.

The lethality, chemoresistance and metastatic characteristics of cancers are associated with phenotypically plastic cancer stem cells (CSCs). How the non-cell autonomous signalling pathways and cell-autonomous transcriptional machinery orchestrate the stem cell-like characteristics of CSCs is still poorly understood. Here we use a quantitative proteomic approach for identifying secreted proteins of CSCs in pancreatic cancer. We uncover that the cell-autonomous E2F1/4-pRb/RBL2 axis balances non-cell-autonomous signalling in healthy ductal cells but becomes deregulated upon KRAS mutation. E2F1 and E2F4 induce whereas pRb/RBL2 reduce WNT ligand expression (e.g. WNT7A, WNT7B, WNT10A, WNT4) thereby regulating self-renewal, chemoresistance and invasiveness of CSCs in both PDAC and breast cancer, and fibroblast proliferation. Screening for epigenetic enzymes identifies GCN5 as a regulator of CSCs that deposits H3K9ac onto WNT promoters and enhancers. Collectively, paracrine signalling pathways are controlled by the E2F-GCN5-RB axis in diverse cancers and this could be a therapeutic target for eliminating CSCs.

BRD9-SMAD2/3 orchestrates stemness and tumorigenesis in pancreatic ductal adenocarcinoma.

The dismal prognosis of pancreatic ductal adenocarcinoma (PDAC) is linked to the presence of pancreatic cancer stem-like cells (CSCs) that respond poorly to current chemotherapy regimens. By small molecule compound screening targeting 142 epigenetic enzymes, we identified that bromodomain-containing protein BRD9, a component of the BAF histone remodelling complex, is a key chromatin regulator to orchestrate the stemness of pancreatic CSCs via cooperating with the TGFß/Activin-SMAD2/3 signalling pathway. Inhibition and genetic ablation of BDR9 block the self-renewal, cell cycle entry into G0 phase and invasiveness of CSCs, and improve the sensitivity of CSCs to gemcitabine treatment. In addition, pharmacological inhibition of BRD9 significantly reduced the tumorigenesis in patient-derived xenografts mouse models and eliminated CSCs in tumours from pancreatic cancer patients. Mechanistically, inhibition of BRD9 disrupts enhancer-promoter looping and transcription of stemness genes in CSCs. Collectively, the data suggest BRD9 as a novel therapeutic target for PDAC treatment via modulation of CSC stemness.

A novel function for the haemopoietic supportive murine bone marrow MS-5 mesenchymal stromal cell line in promoting human vasculogenesis and angiogenesis.

The bone marrow contains specific microenvironmental stem cell niches that maintain haemopoiesis. CXCL12-expressing mesenchymal stromal cells are closely associated with the bone marrow sinusoidal endothelia, forming key elements of the haemopoietic stem cell niche, yet their ability to regulate endothelial function is not clearly defined. Given that the murine nestin(+) cell line, MS-5, provides a clonal surrogate bone marrow stromal niche capable of regulating both murine and human primitive haemopoietic stem/progenitor cell (HSC/HPC) fate in vitro, we hypothesized that MS-5 cells might also support new blood vessel formation and function. Here, for the first time, we demonstrate that this is indeed the case. Using proteome arrays, we identified HSC/HPC active angiogenic factors that are preferentially secreted by haemopoietic supportive nestin(+) MS-5 cells, including CXCL12 (SDF-1), NOV (CCN3), HGF, Angiopoietin-1 and CCL2 (MCP-1). Concentrating on CXCL12, we confirmed its presence in MS-5 conditioned media and demonstrated that its antagonist in receptor binding, AMD-3100, which mobilizes HSC/HPCs and endothelial progenitors from bone marrow, could significantly reduce MS-5 mediated human vasculogenesis in vitro, principally by regulating human endothelial cell migration. Thus, the clonal nestin(+) MS-5 murine bone marrow stromal cell line not only promotes human haemopoiesis but also induces human vasculogenesis, with CXCL12 playing important roles in both processes.

ROS and TGFß: from pancreatic tumour growth to metastasis.

Transforming growth factor ß (TGFß) signalling pathway switches between anti-tumorigenic function at early stages of cancer formation and pro-tumorigenic effects at later stages promoting cancer metastasis. A similar contrasting role has been uncovered for reactive oxygen species (ROS) in pancreatic tumorigenesis. Down-regulation of ROS favours premalignant tumour development, while increasing ROS level in pancreatic ductal adenocarcinoma (PDAC) enhances metastasis. Given the functional resemblance, we propose that ROS-mediated processes converge with the spatial and temporal activation of TGFß signalling and thereby differentially impact early tumour growth versus metastatic dissemination. TGFß signalling and ROS could extensively orchestrate cellular processes and this concerted function can be utilized by cancer cells to facilitate their malignancy. In this article, we revisit the interplay of canonical and non-canonical TGFß signalling with ROS throughout pancreatic tumorigenesis and metastasis. We also discuss recent insight that helps to understand their conflicting effects on different stages of tumour development. These considerations open new strategies in cancer therapeutics.

Extracellular vesicles in pancreatic cancer progression and therapies.

Pancreatic cancer (PC) is one of the leading causes of cancer-related death worldwide due to delayed diagnosis and limited treatments. More than 90% of all pancreatic cancers are pancreatic ductal adenocarcinoma (PDAC). Extensive communication between tumour cells and other cell types in the tumour microenvironment have been identified which regulate cancer hallmarks during pancreatic tumorigenesis via secretory factors and extracellular vesicles (EVs). The EV-capsuled factors not only facilitate tumour growth locally, but also enter circulation and reach distant organs to construct a pre-metastatic niche. In this review, we delineate the key factors in pancreatic ductal adenocarcinoma derived EVs that mediate different tumour processes. Also, we highlight the factors that are related to the crosstalk with cancer stem cells/cancer-initiating cells (CSC/CIC), the subpopulation of cancer cells that can efficiently metastasize and resist currently used chemotherapies. Lastly, we discuss the potential of EV-capsuled factors in early diagnosis and antitumour therapeutic strategies.

Molecular Therapeutics of Pancreatic Ductal Adenocarcinoma: Targeted Pathways and the Role of Cancer Stem Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers in humans due to late detection and highly metastatic characteristics. PDAC cells vary in their tumorigenic capabilities with the presence of a subset of PDAC cells known as pancreatic cancer stem cells (CSCs), which are more resistant to currently used therapeutics. Here, we describe the role of CSCs and tumour stroma in developing therapeutic strategies for PDAC and suggest that developmental plasticity could be considered a hallmark of cancers. We provide an overview of the molecular targets in PDAC treatments, including targeted therapies of cellular processes such as proliferation, evasion of growth suppressors, activating metastasis, and metabolic effects. Since PDAC is an inflammation-driven cancer, we also revisit therapeutic strategies targeting inflammation and immunotherapy. Lastly, we suggest that targeting epigenetic mechanisms opens therapeutic routes for heterogeneous cancer cell populations, including CSCs.

PhosphoPOINT: a comprehensive human kinase interactome and phospho-protein database.

MOTIVATION: To fully understand how a protein kinase regulates biological processes, it is imperative to first identify its substrate(s) and interacting protein(s). However, of the 518 known human serine/threonine/tyrosine kinases, 35% of these have known substrates, while 14% of the kinases have identified substrate recognition motifs. In contrast, 85% of the kinases have protein-protein interaction (PPI) datasets, raising the possibility that we might reveal potential kinase-substrate pairs from these PPIs. RESULTS: PhosphoPOINT, a comprehensive human kinase interactome and phospho-protein database, is a collection of 4195 phospho-proteins with a total of 15 738 phosphorylation sites. PhosphoPOINT annotates the interactions among kinases, with their down-stream substrates and with interacting (phospho)-proteins to modulate the kinase-substrate pairs. PhosphoPOINT implements various gene expression profiles and Gene Ontology cellular component information to evaluate each kinase and their interacting (phospho)-proteins/substrates. Integration of cSNPs that cause amino acids change with the proteins with the phosphoprotein dataset reveals that 64 phosphorylation sites result in a disease phenotypes when changed; the linked phenotypes include schizophrenia and hypertension. PhosphoPOINT also provides a search function for all phospho-peptides using about 300 known kinase/phosphatase substrate/binding motifs. Altogether, PhosphoPOINT provides robust annotation for kinases, their downstream substrates and their interaction (phospho)-proteins and this should accelerate the functional characterization of kinomemediated signaling. AVAILABILITY: PhosphoPOINT can be freely accessed in http://kinase. bioinformatics.tw/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Detection of the inferred interaction network in hepatocellular carcinoma from EHCO (Encyclopedia of Hepatocellular Carcinoma genes Online).

BACKGROUND: The significant advances in microarray and proteomics analyses have resulted in an exponential increase in potential new targets and have promised to shed light on the identification of disease markers and cellular pathways. We aim to collect and decipher the HCC-related genes at the systems level. RESULTS: Here, we build an integrative platform, the Encyclopedia of Hepatocellular Carcinoma genes Online, dubbed EHCO http://ehco.iis.sinica.edu.tw, to systematically collect, organize and compare the pileup of unsorted HCC-related studies by using natural language processing and softbots. Among the eight gene set collections, ranging across PubMed, SAGE, microarray, and proteomics data, there are 2,906 genes in total; however, more than 77% genes are only included once, suggesting that tremendous efforts need to be exerted to characterize the relationship between HCC and these genes. Of these HCC inventories, protein binding represents the largest proportion (~25%) from Gene Ontology analysis. In fact, many differentially expressed gene sets in EHCO could form interaction networks (e.g. HBV-associated HCC network) by using available human protein-protein interaction datasets. To further highlight the potential new targets in the inferred network from EHCO, we combine comparative genomics and interactomics approaches to analyze 120 evolutionary conserved and overexpressed genes in HCC. 47 out of 120 queries can form a highly interactive network with 18 queries serving as hubs. CONCLUSION: This architectural map may represent the first step toward the attempt to decipher the hepatocarcinogenesis at the systems level. Targeting hubs and/or disruption of the network formation might reveal novel strategy for HCC treatment.