FOXO1 Couples KGF and PI-3K/AKT Signaling to NKX2.1-Regulated Differentiation of Alveolar Epithelial Cells.

NKX2.1 is a master regulator of lung morphogenesis and cell specification; however, interactions of NKX2.1 with various transcription factors to regulate cell-specific gene expression and cell fate in the distal lung remain incompletely understood. FOXO1 is a key regulator of stem/progenitor cell maintenance/differentiation in several tissues but its role in the regulation of lung alveolar epithelial progenitor homeostasis has not been evaluated. We identified a novel role for FOXO1 in alveolar epithelial cell (AEC) differentiation that results in the removal of NKX2.1 from surfactant gene promoters and the subsequent loss of surfactant expression in alveolar epithelial type I-like (AT1-like) cells. We found that the FOXO1 forkhead domain potentiates a loss of surfactant gene expression through an interaction with the NKX2.1 homeodomain, disrupting NKX2.1 binding to the SFTPC promoter. In addition, blocking PI-3K/AKT signaling reduces phosphorylated FOXO-1 (p-FOXO1), allowing accumulated nuclear FOXO1 to interact with NKX2.1 in differentiating AEC. Inhibiting AEC differentiation in vitro with keratinocyte growth factor (KGF) maintained an AT2 cell phenotype through increased PI3K/AKT-mediated FOXO1 phosphorylation, resulting in higher levels of surfactant expression. Together these results indicate that FOXO1 plays a central role in AEC differentiation by directly binding NKX2.1 and suggests an essential role for FOXO1 in mediating AEC homeostasis.

High-Throughput CRISPR Screening Identifies Genes Involved in Macrophage Viability and Inflammatory Pathways.

Macrophages are critical effector cells of the immune system, and understanding genes involved in their viability and function is essential for gaining insights into immune system dysregulation during disease. We use a high-throughput, pooled-based CRISPR-Cas screening approach to identify essential genes required for macrophage viability. In addition, we target 3' UTRs to gain insights into previously unidentified cis-regulatory regions that control these essential genes. Next, using our recently generated nuclear factor κB (NF-κB) reporter line, we perform a fluorescence-activated cell sorting (FACS)-based high-throughput genetic screen and discover a number of previously unidentified positive and negative regulators of the NF-κB pathway. We unravel complexities of the TNF signaling cascade, showing that it can function in an autocrine manner in macrophages to negatively regulate the pathway. Utilizing a single complex library design, we are capable of interrogating various aspects of macrophage biology, thus generating a resource for future studies.

LINC00261 Is an Epigenetically Regulated Tumor Suppressor Essential for Activation of the DNA Damage Response.

Lung cancer is the leading cause of cancer-related death in the United States. Long noncoding RNAs (lncRNA) are a class of regulatory molecules whose role in lung carcinogenesis is poorly understood. In this study, we profiled lncRNA expression in lung adenocarcinoma (LUAD) cell lines, compared their expression with that of purified alveolar epithelial type II cells (the purported cell of origin for LUAD), cross-referenced these with lncRNAs altered in the primary human tumors, and interrogated for lncRNAs whose expression correlated with patient survival. We identified LINC00261, a lncRNA with unknown function in LUAD, adjacent to the pioneering transcription factor FOXA2. Loss of LINC00261 was observed in multiple tumor types, including liver, breast, and gastric cancer. Reintroduction of LINC00261 into human LUAD cell lines inhibited cell migration and slowed proliferation by inducing G2-M cell-cycle arrest, while upregulating DNA damage pathway genes and inducing phosphorylation-mediated activation of components of the DNA damage pathway. FOXA2 was able to induce LINC00261 expression, and the entire locus underwent hypermethylation in LUAD, leading to loss of expression. We have thus identified an epigenetically deregulated lncRNA, whose loss of expression in LUAD promotes the malignant phenotype and blocks activation of the DNA damage machinery, predisposing lung cells to cancer development. SIGNIFICANCE: These findings identify LINC00261 as a tumor suppressor that blocks cellular proliferation by activating the DNA damage response and suggest that epigenetic therapy to inhibit DNA methylation may enhance treatment of LUAD. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/12/3050/F1.large.jpg.See related commentary by Davalos and Esteller, p. 3028.