Aldehyde Dehydrogenase 1 Isoforms ALDH1A1 and ALDH1A3 are Essential for Myogenic Differentiation

Background Satellite cells (SC) constitute the stem cell population of skeletal muscle tissue and are determinants for myogenesis. Aldehyde Dehydrogenase 1 (ALDH1) enzymatic activity correlates with myogenic properties of SCs and, recently, we could show co-localization of its isoforms ALDH1A1 and ALDH1A3 in SCs of human skeletal muscle. ALDH1 is not only the pacemaker enzyme in retinoic acid signaling and differentiation, but also protecting cell maintenance against oxidative stress products. However, the molecular mechanism of ALDH1 in SC activation and regulation of myogenesis has not yet been characterized. In regard of ALDH1A1 and ALDH1A1 expression in myogenesis human RH30 and murine C2C12 myoblast cell lines were investigated using Western Blot, Immunofluorescence and Aldefluor Assay. Results Here, we show, that isoforms ALDH1A1 and ALDH1A3 are pivotal factors in the process of myogenic differentiation, since ALDH1A1 knock-out and ALDH1A3 knock-out, respectively, impaired differentiation potential. Recombinant re-expression of ALDH1A1 and ALDH1A3, respectively, in corresponding ALDH1-isoform knock-out cells recovered their differentiation potential. Most interestingly, the chemical inhibition of enzymatic activity by disulfiram leads to ALDH1A1 and ALDH1A3 protein upregulation and subsequent myogenic differentiation. Conclusion Our findings indicate that ALDH1A1 and ALDH1A3 proteins are important for myogenic differentiation and, therefore, seem to be essential activators and regulators of SCs.

Aldehyde Dehydrogenase 1 isoforms ALDH1A1 and ALDH1A3 are essential for myogenic differentiation

Background Satellite cells (SC) constitute the stem cell population of skeletal muscle tissue and are determinants for myogenesis. Aldehyde Dehydrogenase 1 (ALDH1) enzymatic activity correlates with myogenic properties of SCs and, recently, we could show co-localization of its isoforms ALDH1A1 and ALDH1A3 in SCs of human skeletal muscle. ALDH1 is not only the pacemaker enzyme in retinoic acid signaling and differentiation, but also protecting cell maintenance against oxidative stress products. However, the molecular mechanism of ALDH1 in SC activation and regulation of myogenesis has not yet been characterized. Method Human RH30 and murine C2C12 myoblast cell lines were investigated in regard of ALDH1A1 and ALDH1A1 expression in myogenesis using Western Blot, Immunofluorescence and Aldefluor Assay. Results Here, we show, that isoforms ALDH1A1 and ALDH1A3 are pivotal factors in the process of myogenic differentiation, since ALDH1A1 knock-out and ALDH1A3 knock-out, respectively, impaired differentiation potential. Recombinant re-expression of ALDH1A1 and ALDH1A3, respectively, in corresponding ALDH1-isoform knock-out cells recovered their differentiation potential. Most interestingly, the chemical inhibition of enzymatic activity by disulfiram leads to ALDH1A1 and ALDH1A3 protein upregulation and subsequent myogenic differentiation. Conclusion Our findings indicate that ALDH1A1 and ALDH1A3 proteins are important for myogenic differentiation and, therefore, seem to be essential activators and regulators of SCs.

The Antiasthma Medication Ciclesonide Suppresses Breast Cancer Stem Cells through Inhibition of the Glucocorticoid Receptor Signaling-dependent YAP Pathway

Background Ciclesonide is an inhaled corticosteroid used to treat mild-to-severe asthma. However, whether it has anticancer and anti-cancer stem cell (CSC) effects is unknown. This study focused on studying the effect on breast cancer and breast CSCs by ciclesonide and determining its molecular mechanism. Methods The sensitivity of breast cancer by ciclesonide was determined by cell apoptosis, migration, colony formation, and xenograft. Effect of ciclesonide on CSC formation through GR/YAP pathway was determined by SiRNA of GR and YAP, GR degradation assay, GR antagonist, nuclear localization of GR and YAP, and YAP inhibitor. CSC subpopulation was determined by mammosphere culture, CD44+/CD24-, and aldefluor assay. Results Here, we showed that ciclesonide inhibits breast cancer and CSC growth. Similar glucocorticoids (GC), dexamethasone (DEX) and prednisone, did not suppress CSC formation. Ciclesonide-induced glucocorticoid receptor (GR) degradation was dependent on ubiquitination. We showed via GR small interfering RNA (siRNA) that GR plays a crucial role in CSC formation. We showed via western blot and immunofluorescence assays that ciclesonide reduces the nuclear level of GR. The GR antagonist RU-486 also inhibited CSC formation. Ciclesonide reduced the protein level of the Hippo transducer Yes-associated protein (YAP). GR siRNA induced a decrease in YAP protein expression and inhibited mammosphere formation. The YAP inhibitor verteporfin inhibited CSC formation and transcription of the connective tissue growth factor and cysteine-rich protein 61 genes. The GR/YAP1 pathway regulated breast CSC formation. Conclusions We describe that ciclesonide inhibition CSCs through GR and YAP signaling pathway. These findings suggest that the GR/YAP signaling pathway regulates breast CSC formation and revealed a new approach for targeting GR and YAP to inhibit CSC formation.

Alternatively Activated Macrophages are the Primary Retinoic Acid Producing Cells in Human Decidua

In situ production and metabolism of all-trans retinoic acid (RA) in decidual tissue are critically important for endometrial stromal differentiation, embryo implantation, and healthy placentation. However, the cellular source(s) of RA in this tissue has yet to be determined. To identify the primary RA-producing cells in human term decidua, we isolated cells from decidua basalis of delivered placenta and quantified cellular retinal dehydrogenase (RALDH) activity, a major biosynthetic enzyme whose activity determines the synthesis of RA from retinol, using an Aldefluor assay and flow cytometry. RA production in decidual tissue and sorted cell subpopulations was evaluated by liquid chromatography-tandem mass spectrometry. CD14+ cells (macrophages/monocytes) showed >4-fold higher RALDH activity than stromal cells (CD10+), T-cells (CD3+), or non-T lymphocytes (CD3-negative). CD11c+ cells that did not co-express CD14 showed about one-third the RALDH activity of their CD14 co-expressing counterparts. The highest RALDH activity was found in “alternatively activated” M2 macrophages delineated by the simultaneous expression of CD14 and CD163. The greater RA synthesizing capacity of M2 versus CD14+CD163-ve (M1) cells was confirmed by direct quantitation of RA biosynthesis from retinol. RA levels in whole decidua correlated with M2 cell density but not with stromal cell (CD10+) number, the major cell type comprising the decidua. These results identified M2 monocyte/macrophages as the primary source of RA in human term decidua. This finding may have implications for certain pregnancy complications that are known to be associated with reduced numbers of decidual M2 cells. Author note: This manuscript has been accepted for publication in the journal Reproductive Sciences

Increased Activity of Aldehyde Dehydrogenase, a Stem/Progenitor Cell Marker, in Chronic Lymphocytic Leukemia,

Abstract 3867 Introduction: Despite their mature appearance, the B cells from chronic lymphocytic leukemia (CLL) possess immature characteristics both functionally and biochemically. CLL B cells display known biochemical markers characteristic of cells early in the blood lineage, including ROR1, Wnt16, and LEF1. In addition, CLL B cells have higher levels of Reactive Oxygen Species (ROS) and of the oxidant-induced transcription factor Nrf2 [NFE2L2], compared to normal peripheral blood mononuclear cells (PBMC). Intracellular ROS status has been suggested to be a marker of cancer stem/progenitor cells possibly due to their high expression of oncogenes. Downstream targets of Nrf2 include the Aldehyde dehydrogenase [ALDH] enzymes, which are believed to play a crucial role in stem cell biology because they protect the cells against oxidative stress caused by accumulation of aldehydes. Here, we use ALDH activity to visualize populations of CLL B cells that may have stem/progenitor properties. Materials and Methods: Isolated PBMC from normal donors and CLL patients with aggressive and indolent disease were stained for ALDH activity with an Aldefluor assay kit (StemCell Technologies). The ALDH inhibitor, diethylaminobenzaldehyde (DEAB), was used to confirm that the fluorescent activity was due to ALDH activity. At the end of the Aldefluor assay, the cells were stained for cell surface markers, CD19, CD5, CD38 and CD34. 50,000 total events were collected for FACS analysis. Normalized Mean Fluorescence Intensity (MFI) values were calculated by dividing each MFI value to average MFI value of normal CD19+ cells for each experiment. Data analyses were performed by FlowJo software and Prizm. P-values were calculated by One-Way ANOVA analysis with Post-Bonferroni's multiple comparison test. Results: We examine the level of ALDH expression and activity in CD19+ cells of healthy donors (n = 9), CLL samples that expressed unmutated IgVH and that were ZAP-70 positive (defined as “aggressive”, n = 14) or samples that expressed mutated IgVH and were ZAP-70 negative (defined as “indolent”, n=12). CLL B cells from patients with aggressive disease had significantly higher ALDH activities compared to normal B cells (p < 0.001) and indolent CLL B cells (p < 0.05) (Figure1). Indolent CLL B cells also have higher level of ALDH activities compared to normal B cells (p < 0.01) (Figure1). Treatment with the ALDH inhibitor, DEAB, suppressed the increased fluorescence observed in CLL B cells. In addition, ALDH high CLL B cells are CD34 negative. These data show that CLL B cells express a marker known to be associated with stem/progenitor cells, but these populations are different from CD34 positive hematopoietic stem cells. In addition, our data show that a stem/progenitor cell marker is associated with the pathogenesis of CLL. Disclosures: Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.

Evaluation of aldehyde dehydrogenase 1 (ALDH1) as a marker of non-small cell lung cancer (NSCLC) stem cells (SCs) and correlation with prognosis

11105 Background: ALDH1 is a cytosolic enzyme responsible for oxidizing intracellular aldehydes, and conversion of retinol to retinoic acid in SCs. ALDH1 has been previously demonstrated to be a marker for SCs in breast cancer (Ginestier C, et al., Cell Stem Cell. 2007 Nov; 1(5):555–67). The Aldefluor assay is based on conversion of a synthetic substrate BAAA, when passively infused into cytosol, to brightly fluorescent BAA by ALDH1. Combined with fluorescence-activated cell sorting (FACS), it has been used successfully in hematopoietic and breast cancer SCs isolation. ALDH1 is also found in NSCLC. We hypothesized that ALDH1 would be a marker for NSCLC SCs and a potential prognostic marker. Methods: NSCLC SCs were isolated from human NSCLC cell lines using the Aldefluor assay and FACS. ALDH1-positive cells were analyzed extensively for SC characteristics. ALDH1 expression in 303 NSCLC biopsy specimens from three independent cohorts of NSCLC patients was then analyzed by immunohistochemisty (IHC) using an ALDH1 antibody (Santa Cruz Biotechnology) and commercially available negative controls. Results: Isolated cancer cells with high ALDH1 activity displayed cancer SCs features, including capacities for proliferation, self-renewal, differentiation; resistance to chemotherapeutic agents including cisplatin, vinorelbine, gemcitabine and docetaxel; expression of the SC surface marker CD133, and high invasiveness. ALDH1-positive cells generated tumors in vivo recapitulating heterogeneity of parental cells. Xenograft tumors derived from ALDH1-positive cells (103 each) were 36±2.9 mm3 on average in size, with some of the cells having lost ALDH1 expression. Xenograft tumors derived from ALDH1-negative cells (105 each) were 4 mm3 on average in size, without ALDH1 reactivation. Statistical analysis of quantitative IHC and clinical data showed ALDH1 expression was correlated with higher stage and grade of NSCLC (p = 0.02). Expression of ALDH1 in stage I NSCLC patients was linked to decreased 5 year cancer-specific survival (62% vs. 96%, p = 0.006) and overall survival (32% vs. 72%, p = 0.009). Conclusions: 1. ALDH1 is a NSCLC SC-associated tumor marker. 2. ALDH1 expression is a negative prognostic marker in early stage NSCLC. No significant financial relationships to disclose.