Search

The alveolar epithelium is comprised of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells, the latter being capable of self-renewal and transdifferentiation into AT1 cells for normal maintenance and restoration of epithelial integrity following injury. MicroRNAs (miRNAs) are critical regulators of several biological processes, including cell differentiation; however, their role in establishment/maintenance of cellular identity in adult alveolar epithelium is not well understood. To investigate this question, we performed genome-wide analysis of sequential changes in miRNA and gene expression profiles using a well-established model in which human AT2 (hAT2) cells transdifferentiate into AT1-like cells over time in culture that recapitulates many aspects of transdifferentiation in vivo. We defined three phases of miRNA expression during the transdifferentiation process as “early,” “late,” and “consistently” changed, which were further subclassified as up- or downregulated. miRNAs with altered expression at all time points during transdifferentiation were the largest subgroup, suggesting the need for consistent regulation of signaling pathways to mediate this process. Target prediction analysis and integration with previously published gene expression data identified glucocorticoid signaling as the top pathway regulated by miRNAs. Serum/glucocorticoid–regulated kinase 1 (SGK1) emerged as a central regulatory factor, whose downregulation correlated temporally with gain of hsa-miR-424 and hsa-miR-503 expression. Functional validation demonstrated specific targeting of these miRNAs to the 3′-untranslated region of SGK1. These data demonstrate the time-related contribution of miRNAs to the alveolar transdifferentiation process and suggest that inhibition of glucocorticoid signaling is necessary to achieve the AT1-like cell phenotype.

Cancer stem cells (CSCs) initiate tumors, resist treatment, and seed lethal metastases; yet CSC-specific treatments are lacking. Aggressive, treatment-resistant triple-negative breast cancers (TNBC) exhibit WNT pathway activation and are CSC enriched. Here, we show that Limb-Bud- and-Heart (LBH), a WNT/β-catenin target required for normal mammary stem cell self-renewal, marks poor prognosis, stem-like TNBC, and is a key controller of breast cancer stemness. LBH is specifically expressed in tumor-initiating CD44+CD24-/low breast CSCs. LBH overexpression confers stem-like, metastatic traits on both TNBC and luminal origin, non-TNBC breast cancer cells by activating stem cell transcriptional programs. Importantly, silencing LBH potently suppresses tumor initiation and metastasis in vivo, and sensitizes TNBC cells to chemotherapy. LBH knockout in the MMTV-Wnt1 breast cancer mouse model, furthermore, revealed LBH is required for WNT-driven breast CSC expansion. Our findings identify LBH as an essential CSC driver downstream of WNT, and a new molecular target for anti-cancer stem cell therapy.

Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell behavior in disease.

Diseases involving the distal lung alveolar epithelium include chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and lung adenocarcinoma. Accurate labeling of specific cell types is critical for determining the contribution of each to the pathogenesis of these diseases. The distal lung alveolar epithelium is composed of two cell types, alveolar epithelial type 1 (AT1) and type 2 (AT2) cells. Although cell type-specific markers, most prominently surfactant protein C, have allowed detailed lineage tracing studies of AT2 cell differentiation and the cells' roles in disease, studies of AT1 cells have been hampered by a lack of genes with expression unique to AT1 cells. In this study, we performed genome-wide expression profiling of multiple rat organs together with purified rat AT2, AT1, and in vitro differentiated AT1-like cells, resulting in the identification of 54 candidate AT1 cell markers. Cross-referencing with genes up-regulated in human in vitro differentiated AT1-like cells narrowed the potential list to 18 candidate genes. Testing the top four candidate genes at RNA and protein levels revealed GRAM domain 2 (GRAMD2), a protein of unknown function, as highly specific to AT1 cells. RNA sequencing (RNAseq) confirmed that GRAMD2 is transcriptionally silent in human AT2 cells. Immunofluorescence verified that GRAMD2 expression is restricted to the plasma membrane of AT1 cells and is not expressed in bronchial epithelial cells, whereas reverse transcription-polymerase chain reaction confirmed that it is not expressed in endothelial cells. Using GRAMD2 as a new AT1 cell-specific gene will enhance AT1 cell isolation, the investigation of alveolar epithelial cell differentiation potential, and the contribution of AT1 cells to distal lung diseases.

Human bone marrow multipotent mesenchymal stromal cell (hMSC) number decreases with aging. Subpopulations of hMSCs can differentiate into cells found in bone, vasculature, cartilage, gut, and other tissues and participate in their repair. Maintaining throughout adult life such cell subpopulations should help prevent or delay the onset of age-related degenerative conditions. Low oxygen tension, the physiological environment in progenitor cell-rich regions of the bone marrow microarchitecture, stimulates the self-renewal of marrow-isolated adult multilineage inducible (MIAMI) cells and expression of Sox2, Nanog, Oct4a nuclear accumulation, Notch intracellular domain, notch target genes, neuronal transcriptional repressor element 1 (RE1)-silencing transcription factor (REST), and hypoxia-inducible factor-1 alpha (HIF-1α), and additionally, by decreasing the expression of (i) the proapoptotic proteins, apoptosis-inducing factor (AIF) and Bak, and (ii) senescence-associated p53 expression and β-galactosidase activity. Furthermore, low oxygen increases canonical Wnt pathway signaling coreceptor Lrp5 expression, and PI3K/Akt pathway activation. Lrp5 inhibition decreases self-renewal marker Sox2 mRNA, Oct4a nuclear accumulation, and cell numbers. Wortmannin-mediated PI3K/Akt pathway inhibition leads to increased osteoblastic differentiation at both low and high oxygen tension. We demonstrate that low oxygen stimulates a complex signaling network involving PI3K/Akt, Notch, and canonical Wnt pathways, which mediate the observed increase in nuclear Oct4a and REST, with simultaneous decrease in p53, AIF, and Bak. Collectively, these pathway activations contribute to increased self-renewal with concomitant decreased differentiation, cell cycle arrest, apoptosis, and/or senescence in MIAMI cells. Importantly, the PI3K/Akt pathway plays a central mechanistic role in the oxygen tension-regulated self-renewal versus osteoblastic differentiation of progenitor cells.

Limb-bud and heart (LBH) is a key transcriptional regulator in vertebrates with pivotal roles in embryonic development and human disease. Herein, using a diverse array of biophysical techniques, we report the first structural characterization of LBH pertinent to its biological function. Our data reveal that LBH is structurally disordered with no discernable secondary or tertiary structure and exudes rod-like properties in solution. Consistent with these observations, we also demonstrate that LBH is conformationally flexible and thus may be capable of adapting distinct conformations under specific physiological contexts. We propose that LBH is a member of the intrinsically disordered protein (IDP) family, and that conformational plasticity may play a significant role in modulating LBH-dependent transcriptional processes.

Epigenetic regulation of differentiation-related genes is poorly understood. We previously reported that transcription factors GATA6 and Sp1 interact with and activate the rat proximal 358-bp promoter/enhancer (p358P/E) of lung alveolar epithelial type I (AT1) cell-specific gene aquaporin-5 (Aqp5). In this study, we found that histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) increased AQP5 expression and Sp1-mediated transcription of p358P/E. HDAC3 overexpression inhibited Sp1-mediated Aqp5 activation, while HDAC3 knockdown augmented AQP5 protein expression. Knockdown of GATA6 or transcriptional co-activator/histone acetyltransferase p300 decreased AQP5 expression, while p300 overexpression enhanced p358P/E activation by GATA6 and Sp1. GATA6 overexpression, SAHA treatment or HDAC3 knockdown increased histone H3 (H3) but not histone H4 (H4) acetylation within the homologous p358P/E region of mouse Aqp5. HDAC3 binds to Sp1 and HDAC3 knockdown increased interaction of GATA6/Sp1, GATA6/p300 and Sp1/p300. These results indicate that GATA6 and HDAC3 control Aqp5 transcription via modulation of H3 acetylation/deacetylation, respectively, through competition for binding to Sp1, and suggest that p300 modulates acetylation and/or interacts with GATA6/Sp1 to regulate Aqp5 transcription. Cooperative interactions among transcription factors and histone modifications regulate Aqp5 expression during alveolar epithelial cell transdifferentiation, suggesting that HDAC inhibitors may enhance repair by promoting acquisition of AT1 cell phenotype.

Liver cancer is one of the most common malignant tumors. It is reported to be the third most lethal malignancy worldwide. Recent studies including our own identified CD133+ cell population as the tumor initiating cells for liver cancer. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) null mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. Concomitantly, expression of mRNA levels for Wnt ligands and receptors also increased progressively. Wnt/β-Catenin signaling pathway has various roles in regulating embryonic development and tumorigenesis. In Pten null liver progenitor cell line and tissues; we observed high levels of βcatenin, a downstream target component of the Wnt signaling pathway compared to control cells. In this study, we investigate the role of the Wnt/β-Catenin pathway in the activation of hepatic progenitor cells in the Pten deletion liver cancer model using two methods: a novel Wnt/β-Catenin inhibitor ICG-001 and β-Catenin specific RNAi. ICG-001 is a small molecule, which specifically inhibits β-Catenin/CBP interaction. Coactivator CBP/β-Catenin/T cell factor (TCF) mediated transcription has been reported as critical for stem cell/progenitor cell proliferation. ICG-001 significantly attenuates the proliferation of hepatic cancer stem cells in vitro and in vivo. Our preliminary results demonstrate inhibition of Wnt/β-Catenin pathway using sh-β-Catenin in vitro can effectively inhibit the proliferation of CD133+ hepatic cancer stem cells. These observations indicate that Wnt/β- Catenin pathway likely mediates proliferation of hepatic cancer stem cell. Targeting this pathway using ICG-001 and/or β-Catenin directed shRNA holds promise for the treatment and eradication of liver cancer. Citation Format: Anketse D. Kassa, Vivian G. Medina, Ni Zeng, Lina He, Cu Nguyen, Goar Smbatyan, Megan Rieger, Michael Kahn, Bangyan L. Stiles. The therapeutic potential of ICG-001 and β-catenin specific RNAi in liver cancer development. [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 4014. doi:10.1158/1538-7445.AM2013-4014