Citral reduces breast tumor growth by inhibiting the cancer stem cell marker ALDH1A3.
ABSTRACT: Breast cancer stem cells (CSCs) can be identified by increased Aldefluor fluorescence caused by increased expression of aldehyde dehydrogenase 1A3 (ALDH1A3), as well as ALDH1A1 and ALDH2. In addition to being a CSC marker, ALDH1A3 regulates gene expression via retinoic acid (RA) signaling and plays a key role in the progression and chemotherapy resistance of cancer. Therefore, ALDH1A3 represents a druggable anti-cancer target of interest. Since to date, there are no characterized ALDH1A3 isoform inhibitors, drugs that were previously described as inhibiting the activity of other ALDH isoforms were tested for anti-ALDH1A3 activity. Twelve drugs (3-hydroxy-dl-kynurenine, benomyl, citral, chloral hydrate, cyanamide, daidzin, DEAB, disulfiram, gossypol, kynurenic acid, molinate, and pargyline) were compared for their efficacy in inducing apoptosis and reducing ALDH1A3, ALDH1A1 and ALDH2-associated Aldefluor fluorescence in breast cancer cells. Citral was identified as the best inhibitor of ALDH1A3, reducing the Aldefluor fluorescence in breast cancer cell lines and in a patient-derived tumor xenograft. Nanoparticle encapsulated citral specifically reduced the enhanced tumor growth of MDA-MB-231 cells overexpressing ALDH1A3. To determine the potential mechanisms of citral-mediated tumor growth inhibition, we performed cell proliferation, clonogenic, and gene expression assays. Citral reduced ALDH1A3-mediated colony formation and expression of ALDH1A3-inducible genes. In conclusion, citral is an effective ALDH1A3 inhibitor and is able to block ALDH1A3-mediated breast tumor growth, potentially via blocking its colony forming and gene expression regulation activity. The promise of ALDH1A3 inhibitors as adjuvant therapies for patients with tumors that have a large population of high-ALDH1A3 CSCs is discussed.
Project description:N,N-diethylaminobenzaldehyde (DEAB) is a commonly used "selective" inhibitor of aldehyde dehydrogenase isoenzymes in cancer stem cell biology due to its inclusion as a negative control compound in the widely utilized Aldefluor assay. Recent evidence has accumulated that DEAB is not a selective inhibitory agent when assayed in vitro versus ALDH1, ALDH2 and ALDH3 family members. We sought to determine the selectivity of DEAB toward ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, ALDH1L1, ALDH2, ALDH3A1, ALDH4A1 and ALDH5A1 isoenzymes and determine the mechanism by which DEAB exerts its inhibitory action. We found that DEAB is an excellent substrate for ALDH3A1, exhibiting a Vmax/KM that exceeds that of its commonly used substrate, benzaldehyde. DEAB is also a substrate for ALDH1A1, albeit an exceptionally slow one (turnover rate ?0.03 min(-1)). In contrast, little if any turnover of DEAB was observed when incubated with ALDH1A2, ALDH1A3, ALDH1B1, ALDH2 or ALDH5A1. DEAB was neither a substrate nor an inhibitor for ALDH1L1 or ALDH4A1. Analysis by enzyme kinetics and QTOF mass spectrometry demonstrates that DEAB is an irreversible inhibitor of ALDH1A2 and ALDH2 with apparent bimolecular rate constants of 2900 and 86,000 M(-1) s(-1), respectively. The mechanism of inactivation is consistent with the formation of quinoid-like resonance state following hydride transfer that is stabilized by local structural features that exist in several of the ALDH isoenzymes.
Project description:To discover novel therapeutic targets for triple-negative breast cancer (TNBC) and cancer stem cells (CSCs), we screened long non-coding RNAs (lncRNAs) most enriched in TNBCs for high expression in CSCs defined by high Aldefluor activity and associated with worse patient outcomes. This led to the identification of non-coding RNA in the aldehyde dehydrogenase 1?A pathway (NRAD1), also known as LINC00284. Targeting NRAD1 in TNBC tumors using antisense oligonucleotides reduced cell survival, tumor growth, and the number of cells with CSC characteristics. Expression of NRAD1 is regulated by an enzyme that causes Aldefluor activity in CSCs, aldehyde dehydrogenase 1A3 (ALDH1A3) and its product retinoic acid. Cellular fractionation revealed that NRAD1 is primarily nuclear localized, which suggested a potential function in gene regulation. This was confirmed by transcriptome profiling and chromatin isolation by RNA purification, followed by sequencing (ChIRP-seq), which demonstrated that NRAD1 has enriched chromatin interactions among the genes it regulates. Gene Ontology enrichment analysis revealed that NRAD1 regulates expression of genes involved in differentiation and catabolic processes. NRAD1 also contributes to gene expression changes induced by ALDH1A3; thereby, the induction of NRAD1 is a novel mechanism through which ALDH1A3 regulates gene expression. Together, these data identify lncRNA NRAD1 as a downstream effector of ALDH1A3, and a target for TNBCs and CSCs, with functions in cell survival and regulation of gene expression.
Project description:Previous studies indicate that breast cancer cells with high aldehyde dehydrogenase (ALDH) activity and CD44 expression (ALDHhiCD44?) contribute to metastasis and therapy resistance, and that ALDH1 correlates with poor outcome in breast cancer patients. The current study hypothesized that ALDH1 functionally contributes to breast cancer metastatic behavior and therapy resistance. Expression of ALDH1A1 or ALDH1A3 was knocked down in MDA-MB-468 and SUM159 human breast cancer cells using siRNA. Resulting impacts on ALDH activity (Aldefluor® assay); metastatic behavior and therapy response in vitro (proliferation/adhesion/migration/colony formation/chemotherapy and radiation) and extravasation/metastasis in vivo (chick choroiallantoic membrane assay) was assessed. Knockdown of ALDH1A3 but not ALDH1A1 in breast cancer cells decreased ALDH activity, and knockdown of ALDH1A1 reduced breast cancer cell metastatic behavior and therapy resistance relative to control (p < 0.05). In contrast, knockdown of ALDH1A3 did not alter proliferation, extravasation, or therapy resistance, but increased adhesion/migration and decreased colony formation/metastasis relative to control (p < 0.05). This is the first study to systematically examine the function of ALDH1 isozymes in individual breast cancer cell behaviors that contribute to metastasis. Our novel results indicate that ALDH1 mediates breast cancer metastatic behavior and therapy resistance, and that different enzyme isoforms within the ALDH1 family differentially impact these cell behaviors.
Project description:Vasculogenic mimicry (VM), referring to vasculogenic structures lined by tumor cells, can be distinguished from angiogenesis, and is responsible for the aggressiveness and metastatic potential of tumors. HCC1937/p53 cells were derived from triple-negative breast cancer (TNBC), and used to investigate the roles of breast cancer stem cells (CSCs) in the formation of VM. HCC1937/p53 cells formed mesh-like structures on matrigel culture in which expression of VM-related genes, vascular endothelial (VE)-cadherin, matrix metalloproteinase (MMP)-2 and MMP-9 was confirmed by droplet digital polymerase chain reaction (PCR). In immunofluorescence microscopy, aldehyde dehydrogenase (ALDH)1A3+ cells with properties of CSCs or progenitors and GATA binding protein 3 (GATA3)+ cells with more differentiated characteristics were localized in the bridging region and aggregated region of VM structures, respectively. In fluorescence-activated cell sorting analysis, ALDH+ cells, considered to be a subpopulation of CSCs sorted by the aldefluor assay, exhibited marked VM formation on matrigel in 24 hr, whereas ALDH- cells did not form VM, indicating possible roles of CSCs in VM formation. The stem-like cancer cells resistant to p53-induced apoptosis, which expressed a high rate of ALDH1A3 and Sex-determining region Y (SRY)-box binding protein-2 (Sox-2), completed VM formation much faster than the control. These findings may provide clues to elucidate the significance of VM formed by treatment-resistant CSCs in the metastatic potential and poor prognosis associated with TNBC.
Project description:BACKGROUND:Aldehyde dehydrogenase (ALDH) has been widely used as a marker of cancer stem cells (CSCs). However, the ALDH family includes 19 members, and the most relevant isoforms and their biological functions in cancer biology are still controversial. METHODS:We examined ALDH enzyme activity and the mRNA expression of 19 ALDH members in 58 human cell lines. The biological effect and mechanism of knocking down ALDH1A3 with siRNA and shRNA in cell lines were explored. Finally, the relationship between ALDH1A3 and CXCR4 was analysed in a large panel of cell lines. RESULTS:ALDH1A3 is the key isoform that contributed to Aldefluor positivity in cell lines. Knocking down ALDH1A3 in different cancer cells conferred opposite phenotypes due to differential effects on CXCR4 expression. There was a significant negative correlation between ALDH1A3 and CXCR4 in 58 human cell lines. CONCLUSIONS:ALDH1A3 was the main contributor to Aldefluor positivity in human cell lines, and its contrasting effects might arise from differences in CXCR4 expression.
Project description:High aldehyde dehydrogenase (ALDHhi) activity has been reported in normal and cancer stem cells. We and others have shown previously that human ALDHhi cardiac atrial appendage cells are enriched with stem/progenitor cells. The role of ALDH in these cells is poorly understood but it may come down to the specific ALDH isoform(s) expressed. This study aimed to compare ALDHhi and ALDHlo atrial cells and to identify the isoform(s) that contribute to ALDH activity, and their functional role. Methods and Results: Cells were isolated from atrial appendage specimens from patients with ischemic and/or valvular heart disease undergoing heart surgery. ALDHhi activity assessed with the Aldefluor reagent coincided with primitive surface marker expression (CD34+). Depending on their ALDH activity, RT-PCR analysis of ALDHhi and ALDHlo cells demonstrated a differential pattern of pluripotency genes (Oct 4, Nanog) and genes for more established cardiac lineages (Nkx2.5, Tbx5, Mef2c, GATA4). ALDHhi cells, but not ALDHlo cells, formed clones and were culture-expanded. When cultured under cardiac differentiation conditions, ALDHhi cells gave rise to a higher number of cardiomyocytes compared with ALDHlo cells. Among 19 ALDH isoforms known in human, ALDH1A3 was most highly expressed in ALDHhi atrial cells. Knocking down ALDH1A3, but not ALDH1A1, ALDH1A2, ALDH2, ALDH4A1, or ALDH8A1 using siRNA decreased ALDH activity and cell proliferation in ALDHhi cells. Conversely, overexpressing ALDH1A3 with a retroviral vector increased proliferation in ALDHlo cells. Conclusions: ALDH1A3 is the key isoform responsible for ALDH activity in ALDHhi atrial appendage cells, which have a propensity to differentiate into cardiomyocytes. ALDH1A3 affects in vitro proliferation of these cells.
Project description:There has been a new interest in using aldehyde dehydrogenase (ALDH) activity as one marker for stem cells since the Aldefluor flow cytometry-based assay has become available. Diethylaminobenzaldehyde (DEAB), used in the Aldeflour assay, has been considered a specific inhibitor for ALDH1A1 isoform. In this study, we explore the effects of human ALDH isoenzymes, ALDH1A2 and ALDH2, on drug resistance and proliferation, and the specificity of DEAB as an inhibitor. We also screened for the expression of 19 ALDH isoenzymes in K562 cells using TaqMan Low Density Array (TLDA). We used lentiviral vectors containing the full cDNA length of either ALDH2 or ALDH1A2 to over express the enzymes in K562 leukemia and H1299 lung cancer cell lines. Successful expression was measured by activity assay, Western blot, RT-PCR, and Aldefluor assay. Both cell lines, with either ALDH1A2 or ALDH2, exhibited higher cell proliferation rates, higher clonal efficiency, and increased drug resistance to 4-hydroperoxycyclophosphamide and doxorubicin. In order to study the specificity of known ALDH activity inhibitors, DEAB and disulfiram, we incubated each cell line with either inhibitor and measured the remaining ALDH enzymatic activity. Both inhibitors reduced ALDH activity of both isoenzymes by 65-90%. Furthermore, our TLDA results revealed that ALDH1, ALDH7, ALDH3 and ALDH8 are expressed in K562 cells. We conclude that DEAB is not a specific inhibitor for ALDH1A1 and that Aldefluor assay is not specific for ALDH1A1 activity. In addition, other ALDH isoenzymes seem to play a major role in the biology and drug resistance of various malignant cells.
Project description:BACKGROUND: Aldehyde dehydrogenases belong to a superfamily of detoxifying enzymes that protect cells from carcinogenic aldehydes. Of the superfamily, ALDH1A1 has gained most attention because current studies have shown that its expression is associated with human cancer stem cells. However, ALDH1A1 is only one of the 19 human ALDH subfamilies currently known. The purpose of the present study was to determine if the expression and activities of other major ALDH isozymes are associated with human ovarian cancer and ovarian cancer sphere cultures. METHODS: Immunohistochemistry was used to delineate ALDH isozyme localization in clinical ovarian tissues. Western Blot analyses were performed on lysates prepared from cancer cell lines and ovarian cancer spheres to confirm the immunohistochemistry findings. Quantitative reverse transcription-polymerase chain reactions were used to measure the mRNA expression levels. The Aldefluor® assay was used to measure ALDH activity in cancer cells from the four tumor subtypes. RESULTS: Immunohistochemical staining showed significant overexpression of ALDH1A3, ALDH3A2, and ALDH7A1 isozymes in ovarian tumors relative to normal ovarian tissues. The expression and activity of ALDH1A1 is tumor type-dependent, as seen from immunohistochemisty, Western blot analysis, and the Aldefluor® assay. The expression was elevated in the mucinous and endometrioid ovarian epithelial tumors than in serous and clear cell tumors. In some serous and most clear cell tumors, ALDH1A1 expression was found in the stromal fibroblasts. RNA expression of all studied ALDH isozymes also showed higher expression in endometrioid and mucinous tumors than in the serous and clear cell subtypes. The expression of ALDH enzymes showed tumor type-dependent induction in ovarian cancer cells growing as sphere suspensions in serum-free medium. CONCLUSIONS: The results of our study indicate that ALDH enzyme expression and activity may be associated with specific cell types in ovarian tumor tissues and vary according to cell states. Elucidating the function of the ALDH isozymes in lineage differentiation and pathogenesis may have significant implications for ovarian cancer pathophysiology.
Project description:High aldehyde dehydrogenase (ALDH) activity is a marker commonly used to isolate stem cells, particularly breast cancer stem cells (CSCs). Here, we determined that ALDH1A1 activity is inhibited by acetylation of lysine 353 (K353) and that acetyltransferase P300/CBP-associated factor (PCAF) and deacetylase sirtuin 2 (SIRT2) are responsible for regulating the acetylation state of ALDH1A1 K353. Evaluation of breast carcinoma tissues from patients revealed that cells with high ALDH1 activity have low ALDH1A1 acetylation and are capable of self-renewal. Acetylation of ALDH1A1 inhibited both the stem cell population and self-renewal properties in breast cancer. Moreover, NOTCH signaling activated ALDH1A1 through the induction of SIRT2, leading to ALDH1A1 deacetylation and enzymatic activation to promote breast CSCs. In breast cancer xenograft models, replacement of endogenous ALDH1A1 with an acetylation mimetic mutant inhibited tumorigenesis and tumor growth. Together, the results from our study reveal a function and mechanism of ALDH1A1 acetylation in regulating breast CSCs.
Project description:PURPOSE:Lung cancer stem cells (CSC) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic, and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated. EXPERIMENTAL DESIGN:A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH(+) versus ALDH -: cells. RT-PCR, Western blot analysis, and Aldefluor assay were used to validate identified genes. To explore the function in CSCs, we manipulated their expression followed by colony and tumor formation assays. RESULTS:We identified a subset of genes that were differentially expressed in common in ALDH(+) cells, among which ALDH1A3 was the most upregulated gene in ALDH(+) versus ALDH -: cells. shRNA-mediated knockdown of ALDH1A3 in non-small cell lung cancer (NSCLC) resulted in a dramatic reduction in ALDH activity, clonogenicity, and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. In contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH(+) cells also expressed more activated STAT3 than ALDH -: cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH(+) cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well-differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival. CONCLUSIONS:Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH(+) subpopulation in NSCLCs.