PI3K signaling specifies proximal-distal fate by driving a developmental gene regulatory network in SOX9+ mouse lung progenitors.

The tips of the developing respiratory buds are home to important progenitor cells marked by the expression of SOX9 and ID2. Early in embryonic development (prior to E13.5), SOX9+progenitors are multipotent, generating both airway and alveolar epithelium, but are selective progenitors of alveolar epithelial cells later in development. Transcription factors, including Sox9, Etv5, Irx, Mycn, and Foxp1/2 interact in complex gene regulatory networks to control proliferation and differentiation of SOX9+progenitors. Molecular mechanisms by which these transcription factors and other signaling pathways control chromatin state to establish and maintain cell-type identity are not well-defined. Herein, we analyze paired gene expression (RNA-Seq) and chromatin accessibility (ATAC-Seq) data from SOX9+ epithelial progenitor cells (EPCs) during embryonic development in Mus musculus. Widespread changes in chromatin accessibility were observed between E11.5 and E16.5, particularly at distal cis-regulatory elements (e.g. enhancers). Gene regulatory network (GRN) inference identified a common SOX9+ progenitor GRN, implicating phosphoinositide 3-kinase (PI3K) signaling in the developmental regulation of SOX9+ progenitor cells. Consistent with this model, conditional ablation of PI3K signaling in the developing lung epithelium in mouse resulted in an expansion of the SOX9+ EPC population and impaired airway epithelial cell differentiation. These data demonstrate that PI3K signaling is required for epithelial patterning during lung organogenesis, and emphasize the combinatorial power of paired RNA and ATAC seq in defining regulatory networks in development.

Studying how lungs develop has helped us understand and treat often-devastating lung diseases. This includes diseases like cystic fibrosis which result from spelling mistakes known as mutations in a person's genetic code. However, not all lung diseases involve mutations. Many other diseases, in both adults and children, are caused by genes failing to switch on or off at some point during lung development. DNA is surrounded by various proteins which package it into a compressed structure known as chromatin. Cells can control which genes are turned on or off by modifying how tightly packed parts of the genetic code are within chromatin. Changes in chromatin accessibility, also known as 'epigenetic' changes, are a normal part of development, and guide cells towards specific jobs or identities as an organ matures. However, how this happens in the developing lung is poorly understood. Here, Khattar, Fernandes et al. set out to determine how chromatin accessibility shapes development of the tissue lining the lungs, focusing on a group of progenitor cells which produce the protein SOX9. These cells are initially found at the tips of the early lung, where they go on to develop into the cells that line the whole of the mature organ. Initial experiments used large-scale genetic techniques to measure gene activity and chromatin accessibility simultaneously in progenitor cells extracted from the lungs of mice. Khattar, Fernandes et al. were then able to predict the signaling pathways that shape the lung lining based on which genes were surrounded by unpacked chromatin, and determine the proteins responsible for these epigenetic changes. This included the signaling pathway Phosphatidylinositol 3 kinase (PI3K) which is involved in a number of cellular processes. Additional experiments in mice confirmed that the PI3K pathway became active very early in lung development and remained so until adulthood. In contrast, mice lacking a gene that codes for a key part of the PI3K pathway had defective lungs which failed to develop a proper lining. The data generated in this study will provide an important resource for future studies investigating how epigenetic changes drive normal lung development. Khattar, Fernandes et al. hope that this knowledge will help researchers to better understand the cause of human lung diseases, and identify already available 'epigenetic drugs' which could be repurposed to treat them.

Sequential transcriptome analysis of human liver cancer indicates late stage acquisition of malignant traits.

BACKGROUND & AIMS: Human hepatocarcinogenesis is as a multi-step process starting from dysplastic lesions to early carcinomas (eHCC) that ultimately progress to HCC (pHCC). However, the sequential molecular alterations driving malignant transformation of the pre-neoplastic lesions are not clearly defined. This lack of information represents a major challenge in the clinical management of patients at risk. METHODS: We applied next-generation transcriptome sequencing to tumor-free surrounding liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, eHCC (n = 5) and pHCC (n = 3) from 8 HCC patients with hepatitis B infection. Integrative analyses of genetic and transcriptomic changes were performed to characterize the genomic alterations during hepatocarcinogenesis. RESULTS: We report that changes in transcriptomes of early lesions including eHCC were modest and surprisingly homogenous. Extensive genetic alterations and subsequent activation of prognostic adverse signaling pathways occurred only late during hepatocarcinogenesis and were centered on TGFß, WNT, NOTCH, and EMT-related genes highlighting the molecular diversity of pHCC. We further identify IGFALS as a key genetic determinant preferentially down-regulated in pHCC. CONCLUSIONS: Our results define new hallmarks in molecular stratification and therapy options for patients at risk for HCC, and merit larger prospective investigations to develop a modified clinical-decision making algorithm based on the individualized next-generation sequencing analyses.

Epigenetic reprogramming modulates malignant properties of human liver cancer.

UNLABELLED: Reversal of DNA hypermethylation and associated gene silencing is an emerging cancer therapy approach. Here we addressed the impact of epigenetic alterations and cellular context on functional and transcriptional reprogramming of hepatocellular carcinoma (HCC) cells. Our strategy employed a 3-day treatment of established and primary human HCC-derived cell lines grown as a monolayer at various cell densities with the DNMT1 inhibitor zebularine (ZEB) followed by a 3D culture to identify cells endowed with self-renewal potential. Differences in self-renewal, gene expression, tumorigenicity, and metastatic potential of spheres at generations G1-G5 were examined. Transient ZEB exposure produced differential cell density-dependent responses. In cells grown at low density, ZEB caused a remarkable increase in self-renewal and tumorigenicity associated with long-lasting gene expression changes characterized by a stable overexpression of cancer stem cell-related and key epithelial-mesenchymal transition genes. These effects persisted after restoration of DNMT1 expression. In contrast, at high cell density, ZEB caused a gradual decrease in self-renewal and tumorigenicty, and up-regulation of apoptosis- and differentiation-related genes. A permanent reduction of DNMT1 protein using short hairpin RNA (shRNA)-mediated DNMT1 silencing rendered HCC cells insensitive both to cell density and ZEB effects. Similarly, WRL68 and HepG2 hepatoblastoma cells expressing low DNMT1 basal levels also possessed a high self-renewal, irrespective of cell density or ZEB exposure. Spheres formed by low-density cells treated with ZEB or shDNMT1 displayed a high molecular similarity which was sustained through consecutive generations, confirming the essential role of DNMT1 depletion in the enhancement of cancer stem cell properties. CONCLUSION: These results identify DNA methylation as a key epigenetic regulatory mechanism determining the pool of cancer stem cells in liver cancer and possibly other solid tumors.

Loss of c-Met accelerates development of liver fibrosis in response to CCl(4) exposure through deregulation of multiple molecular pathways.

HGF/c-Met signaling plays a pivotal role in hepatocyte survival and tissue remodeling during liver regeneration. HGF treatment accelerates resolution of fibrosis in experimental animal models. Here, we utilized Met(fl/fl);Alb-Cre(+/-) conditional knockout mice and a carbon tetrachloride(CCl(4))-induced liver fibrosis model to formally address the role of c-Met signaling in hepatocytes in the context of chronic tissue injury. Histological changes during injury (4weeks) and healing phase (4weeks) were monitored by immunohistochemistry; expression levels of selected key fibrotic molecules were evaluated by western blotting, and time-dependent global transcriptomic changes were examined using a microarray platform. Loss of hepatocyte c-Met signaling altered hepatic microenvironment and aggravated hepatic fibrogenesis. Greater liver damage was associated with decreased hepatocyte proliferation, excessive stellate cell activation and rapid dystrophic calcification of necrotic areas. Global transcriptome analysis revealed a broad impact of c-Met on critical signaling pathways associated with fibrosis. Loss of hepatocyte c-Met caused a strong deregulation of chemotactic and inflammatory signaling (MCP-1, RANTES, Cxcl10) in addition to modulation of genes involved in reorganization of the cytoskeletal network (Actb, Tuba1a, Tuba8), intercellular communications and adhesion (Adam8, Icam1, Itgb2), control of cell proliferation (Ccng2, Csnk2a, Cdc6, cdk10), DNA damage and stress response (Rad9, Rad52, Ercc4, Gsta1 and 2, Jun). Our study demonstrates that deletion of c-Met receptor in hepatocytes results in pronounced changes in hepatic metabolism and microenvironment, and establishes an essential role for c-Met in maintaining the structural integrity and adaptive plasticity of the liver under adverse conditions.

Genomic and genetic characterization of cholangiocarcinoma identifies therapeutic targets for tyrosine kinase inhibitors.

BACKGROUND & AIMS: Cholangiocarcinoma is a heterogeneous disease with a poor outcome that accounts for 5%-10% of primary liver cancers. We characterized its genomic and genetic features and associated these with patient responses to therapy. METHODS: We profiled the transcriptomes from 104 surgically resected cholangiocarcinoma samples collected from patients in Australia, Europe, and the United States; epithelial and stromal compartments from 23 tumors were laser capture microdissected. We analyzed mutations in KRAS, epidermal growth factor receptor (EGFR), and BRAF in samples from 69 tumors. Changes in gene expression were validated by immunoblotting and immunohistochemistry; integrative genomics combined data from the patients with data from 7 human cholangiocarcinoma cell lines, which were then exposed to trastuzumab and lapatinib. RESULTS: Patients were classified into 2 subclasses, based on 5-year survival rate (72% vs 30%; χ(2) = 11.61; P < .0007), time to recurrence (13.7 vs 22.7 months; P < .001), and the absence or presence of KRAS mutations (24.6%), respectively. Class comparison identified 4 survival subgroups (SGI-IV; χ(2) = 8.34; P < .03); SGIII was characterized by genes associated with proteasomal activity and the worst prognosis. The tumor epithelium was defined by deregulation of the HER2 network and frequent overexpression of EGFR, the hepatocyte growth factor receptor (MET), pRPS6, and Ki67, whereas stroma was enriched in inflammatory cytokines. Lapatinib, an inhibitor of HER2 and EGFR, was more effective in inhibiting growth of cholangiocarcinoma cell lines than trastuzumab. CONCLUSIONS: We provide insight into the pathogenesis of cholangiocarcinoma and identify previously unrecognized subclasses of patients, based on KRAS mutations and increased levels of EGFR and HER2 signaling, who might benefit from dual-target tyrosine kinase inhibitors. The group of patients with the worst prognosis was characterized by transcriptional enrichment of genes that regulate proteasome activity, indicating new therapeutic targets.

Human hepatic cancer stem cells are characterized by common stemness traits and diverse oncogenic pathways.

UNLABELLED: Epigenetic mechanisms play critical roles in stem cell biology by maintaining pluripotency of stem cells and promoting differentiation of more mature derivatives. If similar mechanisms are relevant for the cancer stem cell (CSC) model, then epigenetic modulation might enrich the CSC population, thereby facilitating CSC isolation and rigorous evaluation. To test this hypothesis, primary human cancer cells and liver cancer cell lines were treated with zebularine (ZEB), a potent DNA methyltransferase-1 inhibitor, and putative CSCs were isolated using the side population (SP) approach. The CSC properties of ZEB-treated and untreated subpopulations were tested using standard in vitro and in vivo assays. Whole transcriptome profiling of isolated CSCs was performed to generate CSC signatures. Clinical relevance of the CSC signatures was evaluated in diverse primary human cancers. Epigenetic modulation increased frequency of cells with CSC properties in the SP fraction isolated from human cancer cells as judged by self-renewal, superior tumor-initiating capacity in serial transplantations, and direct cell tracking experiments. Integrative transcriptome analysis revealed common traits enriched for stemness-associated genes, although each individual CSC gene expression signature exhibited activation of different oncogenic pathways (e.g., EGFR, SRC, and MYC). The common CSC signature was associated with malignant progression, which is enriched in poorly differentiated tumors, and was highly predictive of prognosis in liver and other cancers. CONCLUSION: Epigenetic modulation may provide a tool for prospective isolation and in-depth analysis of CSC. The liver CSC gene signatures are defined by a pernicious interaction of unique oncogene-specific and common stemness traits. These data should facilitate the identifications of therapeutic tools targeting both unique and common features of CSCs.