Establishing nomograms for predicting disease-free survival and overall survival in patients with breast cancer.

BACKGROUND: Prognostic factors-based nomograms have been utilised to detect the likelihood of the specific cancer events. We have focused on the roles of aldehyde dehydrogenase 1 (ALDH1) and p-AKT in predicting the prognosis of BC patients. This study was designed to establish nomograms based on the integration of aldehyde dehydrogenase 1 (ALDH1) and p-AKT in predicting the disease-free survival (DFS) and overall survival (OS) of breast cancer (BC) patients. METHODS: Demographic and clinical data were obtained from BC patients admitted to our hospital between September 2015 and August 2016. Univariate and multivariate Cox regression analyses were utilised to analyse the risk factors of recurrence and mortality. The nomograms for predicting the DFS and OS were established using the screened risk factors. Stratified analysis was performed with the cut-off value of exp (pi) of 4.0-fold in DFS and OS, respectively. RESULTS: Multivariate Cox regression analysis indicated that ALDH, p-AKT and pathological stage III were independent risk factors for the recurrence among BC patients. ALDH1, p-AKT, pathological stage III and ER-/PR-/HER2- were independent risk factors for the mortality among BC patients. The established nomograms based on these factors were effective for predicting the DFS and OS with good agreement to the calibration curve and acceptable area under the receiver operating characteristic (ROC) curve. Finally, stratified analyses showed patients with a low pi showed significant decrease in the DFS and OS compared with those of high risk. CONCLUSION: We established nomograms for predicting the DFS and OS of BC patients based on ALDH1, p-AKT and pathological stages. The ER-/PR-/HER2- may be utilised to predict the OS rather than DFS in the BC patients.

Many breast cancer patients show poor response after treatment due to recurrence and metastasis. Therefore, early prediction of the disease-free survival and overall survival is crucial to the treatment outcome and clinical decision-making. In this study, we established nomograms with the demographic and clinical data from breast cancer patients admitted to our hospital between September 2015 and August 2016. Univariate and multivariate Cox regression analyses showed that some important proteins and signalling pathways were risk factors for decreased disease-free survival and overall survival of breast cancer patients. On this basis, we established an effective nomogram for predicting the disease-free survival and overall survival of these patients based on these factors. This study offers new options in the predicting the treatment outcome of breast cancer patients.

RUNX3 exerts tumor-suppressive role through inhibiting EXOSC4 expression.

Breast cancer severely affects women health. 70% of breast cancer are estrogen receptor positive. Breast cancer stem cells are a group of tumor with plasticity, causing tumor relapse and metastasis. RUNX3 is a tumor suppressor frequently inactivated in estrogen receptor positive breast cancer. However, the mechanism of how RUNX3 is involved in the regualation of cancer stem cell traits in estrogen receptor positive breast cancer remains elusive. In this study, we utilized cut-tag assay to investigate the binding profile RUNX3 in BT474 and T47D cell, and confirmed EXOSC4 as the bona-fide target of RUNX3; RUNX3 could bind to the promoter are of EXOSC4 to suppress its expression. Furthermore, EXOSC4 could increase the colony formation, cell invasion and mammosphere formation ability of breast cancer cells and upregulate the the expression of SOX2 and ALDH1. Consistent with these findings, EXOSC4 was associated with poorer survival for Luminal B/Her2 breast cancer patiens. At last, we confirmed that EXOSC4 mediated the tumor suppressive role of RUNX3 in breast cancer cells. In conclusion, we demonstrate that RUNX3 directly binds to the promoter region of EXOSC4, leading to the suppression of EXOSC4 expression and exerting a tumor-suppressive effect in estrogen receptor postivive breast cancer cells.

Dual-responsive near-infrared turn-on fluorescent probe for cancer stem cell-specific visualization.

Aldehyde dehydrogenase 1A1 (ALDH1A1) stands out as one of the most reliable intracellular biomarkers for stem cells because it is expressed in both cancer stem cells (CSCs) and normal somatic stem cells (NSCs). Although several turn-on fluorescent probes for ALDH1A1 have been developed to visualize CSCs in cancer cells, the discrimination of CSCs from NSCs is difficult. We here report an AND-type dual-responsive fluorescent probe, CHO_ßgal, the near-infrared fluorescence of which can be turned on after responding to both ALDH1A1 and ß-galactosidase. The AND-type dual responsiveness enables CSCs to be clearly visualized, whereas NSCs are non-emissive in microscopy. CSC-positive metastasis model lungs were successfully discriminated from normal lungs in ex vivo staining experiments using CHO_ßgal, whereas the single-input ALDH1A1-responsive probe failed to achieve this discrimination owing to pronounced false-positive fluorescence output from lung NSCs. In tissue slice staining experiments, even in the presence of adjacent normal tissues, the peripheral region-specific localization of CSCs was clear. The versatility of CHO_ßgal holds promise not only as a fundamental in vitro research tool for visualizing CSCs but also as a valuable asset in practical tissue staining diagnosis, significantly contributing to the assessment of cancer malignancy.

Molecular characterization and expression profile of the ALDH1A1 gene and its functions in yak luteal cells.

The ALDH1A1 gene encodes a cytoplasmic member of the aldehyde dehydrogenase 1 family, which plays an important role in regulating animal reproductive performance, including estrus cycle and embryonic development. The aim of this study was to characterize ALDH1A1 activity in ovaries of 3-5 year-old yaks and to determine its effects on cell proliferation, apoptosis, and progesterone secretion in luteal cells (LCs). The coding sequence (CDS) of the ALDH1A1 gene was cloned by reverse transcription-PCR and immunohistochemical analysis was used to confirm localization of the ALDH1A1 protein in the ovary. To assess the activity of ALDH1A1 in regulating progesterone secretion, si-ALDH1A1 was transfected into LCs in vitro and progesterone levels in LC supernatants were measured by ELISA. The interference efficiency was assessed by real-time quantitative PCR (RT-qPCR) and immunofluorescence staining, and cell proliferation and apoptosis were evaluated by EdU and TUNEL staining, respectively. The cloned ALDH1A1 sequence contained 1462 bp, encoding 487 amino acids. Immunohistochemical analysis showed that ALDH1A1 protein expression, which was significantly higher in LCs, was mainly found in antral follicles and the corpus luteum (CL). The expression of ALDH1A1 mRNA in LCs was effectively inhibited by si-ALDH1A1transfection, and progesterone secretion was markedly decreased along with the significant down-regulation of progesterone pathway-related genes, STAR, CYP11A1, CYP19A1, CYP17A1, 3ß-HSD, and HSD17B1. Knockdown of ALDH1A1 mRNA expression decreased cell proliferation and increased apoptosis in LCs. The mRNA expression of the proliferation-related genes, PCNA, CCND1, CCNB1 and CDC25A, was significantly down-regulated, while expression of the apoptosis-promoting CASP3 gene was significantly increased. In summary, we characterized the yak ALDH1A1 gene and revealed that ALDH1A1 knockdown promoted apoptosis, repressed cell proliferation, and decreased progesterone secretion by yak LCs, potentially by regulating the mRNA expression of genes related to proliferation, apoptosis, and progesterone synthesis and secretion.

ALDH1A1 confers resistance to RAF/MEK inhibitors in melanoma cells by maintaining stemness phenotype and activating PI3K/AKT signaling.

The mitogen-activated protein kinase (MAPK/ERK) pathway is pivotal in controlling the proliferation and survival of melanoma cells. Several mutations, including those in BRAF, exhibit an oncogenic effect leading to increased cellular proliferation. As a result, the combination therapy of a MEK inhibitor with a BRAF inhibitor demonstrated higher efficacy and lower toxicity than BRAF inhibitor alone. This combination has become the preferred standard of care for tumors driven by BRAF mutations. Aldehyde dehydrogenase 1A1 (ALDH1A1) is a known marker of stemness involved in drug resistance in several type of tumors, including melanoma. This study demonstrates that melanoma cells overexpressing ALDH1A1 displayed resistance to vemurafenib and trametinib through the activation of PI3K/AKT signaling instead of MAPK axis. Inhibition of PI3K/AKT signaling partially rescued sensitivity to the drugs. Consistently, pharmacological inhibition of ALDH1A1 activity downregulated the activation of AKT and partially recovered responsiveness to vemurafenib and trametinib. We propose ALDH1A1 as a new potential target for treating melanoma resistant to MAPK/ERK inhibitors.

CSChighE-cadherinlow immunohistochemistry panel predicts poor prognosis in oral squamous cell carcinoma.

Identifying marker combinations for robust prognostic validation in primary tumour compartments remains challenging. We aimed to assess the prognostic significance of CSC markers (ALDH1, CD44, p75NTR, BMI-1) and E-cadherin biomarkers in OSCC. We analysed 94 primary OSCC and 67 metastatic lymph node samples, including central and invasive tumour fronts (ITF), along with clinicopathological data. We observed an increase in ALDH1+/CD44+/BMI-1- tumour cells in metastatic lesions compared to primary tumours. Multivariate analysis highlighted that elevated p75NTR levels (at ITF) and reduced E-cadherin expression (at the tumour centre) independently predicted metastasis, whilst ALDH1high exhibited independent predictive lower survival at the ITF, surpassing the efficacy of traditional tumour staging. Then, specifically at the ITF, profiles characterized by CSChighE-cadherinlow (ALDH1highp75NTRhighE-cadherinlow) and CSCintermediateE-cadherinlow (ALDH1 or p75NTRhighE-cadherinlow) were significantly associated with worsened overall survival and increased likelihood of metastasis in OSCC patients. In summary, our study revealed diverse tumour cell profiles in OSCC tissues, with varying CSC and E-cadherin marker patterns across primary tumours and metastatic sites. Given the pivotal role of reduced survival rates as an indicator of unfavourable prognosis, the immunohistochemistry profile identified as CSChighE-cadherinlow at the ITF of primary tumours, emerges as a preferred prognostic marker closely linked to adverse outcomes in OSCC.

Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies.

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .

Associations of Early-Life and Adult Anthropometric Measures with the Expression of Stem Cell Markers CD44, CD24, and ALDH1A1 in Women with Benign Breast Biopsies.

BACKGROUND: According to the stem cell hypothesis, breast carcinogenesis may be related to the breast stem cell pool size. However, little is known about associations of breast cancer risk factors, such as anthropometric measures, with the expression of stem cell markers in noncancerous breast tissue. METHODS: The analysis included 414 women with biopsy-confirmed benign breast disease in the Nurses' Health Study and Nurses' Health Study II. Birthweight, weight at age 18, current weight, and current height were reported via self-administered questionnaires. IHC staining of stem cell markers (CD44, CD24, and aldehyde dehydrogenase family 1 member A1) in histopathologically normal epithelial and stromal breast tissue was quantified using an automated computational image analysis system. Linear regression was used to examine the associations of early-life and adult anthropometric measures with log-transformed stem cell marker expression, adjusting for potential confounders. RESULTS: Birthweight [≥10.0 vs. <5.5 lbs: ß (95% confidence interval) = 4.29 (1.02, 7.56); P trend = 0.001 in the stroma] and adult height [≥67.0 vs. <63.0 inch: 0.86 (0.14, 1.58); P trend = 0.02 in the epithelium and stroma combined] were positively associated with CD44 expression. Childhood body fatness was inversely associated (P trend = 0.03) whereas adult height was positively associated with CD24 expression in combined stroma and epithelium (P trend = 0.03). CONCLUSIONS: Our data suggest that anthropometric measures, such as birthweight, adult height, and childhood body fatness, may be associated with the stem cell expression among women with benign breast disease. IMPACT: Anthropometric measures, such as birthweight, height, and childhood body fatness, may have long-term impacts on stem cell population in the breast.

Aldehyde Dehydrogenese-1 High Cancer Stem-like Cells/Cancer-initiating Cells Escape from Cytotoxic T Lymphocytes due to Lower Expression of Human Leukocyte Antigen Class 1.

BACKGROUND/AIM: Human gastric cancer stem-like cells (CSCs)/cancer-initiating cells can be identified as aldehyde dehydrogenase-high (ALDHhigh) cells. Cancer immunotherapy employing immune checkpoint blockade has been approved for advanced gastric cancer cases. However, the effectiveness of cancer immunotherapy against gastric CSCs/CICs remains unclear. This study aimed to investigate the susceptibility of gastric CSCs/CICs to immunotherapy. MATERIALS AND METHODS: Gastric CSCs/CICs were isolated as ALDHhigh cells using the human gastric cancer cell line, MKN-45. ALDHhigh clone cells and ALDHlow clone cells were isolated using the ALDEFLUOR assay. ALDH1A1 expression was assessed via qRT-PCR. Sphere-forming ability was evaluated to confirm the presence of CSCs/CICs. A model neoantigen, AP2S1, was over-expressed in ALDHhigh clone cells and ALDHlow clone cells, and susceptibility to AP2S1-specific TCR-T cells was assessed using IFNγ ELISPOT assay. RESULTS: Three ALDHhigh clone cells were isolated from MKN-45 cells. ALDHhigh clone cells exhibited a stable phenotype in in vitro culture for more than 2 months. The High-36 clone cells demonstrated the highest sphere-forming ability, whereas the Low-8 cells showed the lowest sphere-forming ability. High-36 cells exhibited lower expression of HLA-A24 compared to Low-8 cells. TCR-T cells specific for AP2S1 showed lower reactivity to High-36 cells compared to Low-8 cells. CONCLUSION: High-36 cells and Low-8 cells represent novel gastric CSCs/CICs and non-CSCs/CICs, respectively. ALDHhigh CSCs/CICs evade T cells due to lower expression of HLA class 1.

Luminal B Breast Cancer Coexpressing p62 and ALDH1A3 Is Less Susceptible to Radiotherapy.

BACKGROUND/AIM: We have reported that p62 (also known as sequestosome 1) is needed for survival/proliferation and tumor formation by aldehyde dehydrogenase 1 (ALDH1) -positive cancer stem cells (CSCs) and that p62high ALDH1A3high expression is associated with a poor prognosis in luminal B breast cancer. However, the association between p62high ALDH1A3high and the benefit from radiotherapy in patients with luminal B breast cancer remains unclear. MATERIALS AND METHODS: Datasets from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas (TCGA) were downloaded, and data from p62high ALDH1A3high luminal B patients treated without or with radiotherapy were analyzed by Kaplan-Meier and multivariate Cox regression analyses. We also performed an in vitro tumor sphere formation assay after X-ray irradiation using p62-knockdown ALDH1high luminal B BT-474 cells. RESULTS: p62high ALDH1A3high patients had poorer clinical outcomes than other luminal B breast cancer patients treated with radiotherapy. The combination of p62 DsiRNA KD and X-ray irradiation suppressed in vitro tumor sphere formation by ALDH1high BT-474 cells. These results suggest that p62 is involved in the reduced effect of X-ray irradiation on ALDH1-positive luminal B breast CSCs. CONCLUSION: p62 and ALDH1A3 may serve as prognostic biomarkers for luminal B breast cancer patients treated with radiotherapy. Additionally, the combination of p62 inhibition and radiotherapy could be useful for targeted strategies against ALDH1-positive luminal B breast CSCs.

Multi-omics profiling reveals cellular pathways and functions regulated by ALDH1B1 in colon cancer cells.

Colon cancer is the third leading cause of cancer death globally. Although early screenings and advances in treatments have reduced mortality since 1970, identification of novel targets for therapeutic intervention is needed to address tumor heterogeneity and recurrence. Previous work identified aldehyde dehydrogenase 1B1 (ALDH1B1) as a critical factor in colon tumorigenesis. To investigate further, we utilized a human colon adenocarcinoma cell line (SW480) in which the ALDH1B1 protein expression has been knocked down by 80% via shRNA. Through multi-omics (transcriptomics, proteomics, and untargeted metabolomics) analysis, we identified the impact of ALDH1B1 knocking down (KD) on molecular signatures in colon cancer cells. Suppression of ALDH1B1 expression resulted in 357 differentially expressed genes (DEGs), 191 differentially expressed proteins (DEPs) and 891 differentially altered metabolites (DAMs). Functional annotation and enrichment analyses revealed that: (1) DEGs were enriched in integrin-linked kinase (ILK) signaling and growth and development pathways; (2) DEPs were mainly involved in apoptosis signaling and cellular stress response pathways; and (3) DAMs were associated with biosynthesis, intercellular and second messenger signaling. Collectively, the present study provides new molecular information associated with the cellular functions of ALDH1B1, which helps to direct future investigation of colon cancer.

High expression of PKCλ and ALDH1A3 indicates a poor prognosis, and PKCλ is required for the asymmetric cell division of ALDH1A3-positive cancer stem cells in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is the cancer with the poorest prognosis. One of the major properties reflecting its poor prognosis is high-grade heterogeneity, which leads to insensitivity to anticancer treatments. Cancer stem cells (CSCs) acquire phenotypic heterogeneity, generating abnormally differentiated cells by asymmetric cell division. However, the detailed mechanism leading to phenotypic heterogeneity is largely unknown. Here, we showed that PDAC patients with co-upregulation of PKCλ and ALDH1A3 had the poorest clinical outcome. PKCλ knockdown by DsiRNA in the ALDH1high population of PDAC MIA-PaCa-2 cells attenuated the asymmetric distribution of the ALDH1A3 protein. To monitor asymmetric cell division of ALDH1A3-positive PDAC CSCs, we established stable Panc-1 PDAC clones expressing ALDH1A3-turboGFP (Panc-1-ALDH1A3-turboGFP cells). In addition to MIA-PaCa-2-ALDH1high cells, turboGFPhigh cells sorted from Panc-1-ALDH1A3-turboGFP cells showed asymmetric cell propagation of ALDH1A3 protein. PKCλ DsiRNA in Panc-1-ALDH1A3-turboGFP cells also attenuated the asymmetric distribution of ALDH1A3 protein. These results suggest that PKCλ regulates the asymmetric cell division of ALDH1A3-positive PDAC CSCs. Furthermore, Panc-1-ALDH1A3-turboGFP cells can be useful for the visualization and monitoring of CSC properties such as asymmetric cell division of ALDH1A3-positive PDAC CSCs in time-lapse imaging.

Clinical Significance of Cancer Stem Cell Markers in Primary and Metastatic Tissues in Patients With Breast Cancer.

BACKGROUND/AIM: This study aimed to examine the clinical significance of the protein expression of the cancer stem cell (CSC) markers ALDH1A1, CD133, CD44, and MSI-1 in primary and metastatic tissues of patients with breast cancer (BC). PATIENTS AND METHODS: ALDH1A1, CD133, CD44, and MSI-1 protein expression in pairs of primary and metastatic tissues of 55 patients with BC with metastases treated at Kanagawa Cancer Center between January 1970 and December 2016 were evaluated using immunohistochemical assay and their association with clinicopathological factors and survival was examined. RESULTS: There were no significant differences in CSC marker expression rates between primary and metastatic tissues for any CSC markers. Regarding the relationship between CSC marker expression in primary tissues and survival, patients with high CD133 expression had significantly lower recurrence-free survival (DFS) and overall survival. On multivariate analysis, they were also a poor independent predictor of DFS (hazard ratio=4.993, 95%CI=2.189-11.394, p=0.0001). In contrast, there was no significant association between the expression of any CSC marker in metastatic tissues and survival. CONCLUSION: CD133 expression in the primary BC tissue may be a useful risk factor for recurrence in patients with BC.

Application of AlDeSense to Stratify Ovarian Cancer Cells Based on Aldehyde Dehydrogenase 1A1 Activity.

Relapse after cancer treatment is often attributed to the persistence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are characterized by their remarkable tumor-initiating and self-renewal capacity. Depending on the origin of the tumor (e.g., ovaries), the CSC surface biomarker profile can vary dramatically, making the identification of such cells via immunohistochemical staining a challenging endeavor. On the contrary, aldehyde dehydrogenase 1A1 (ALDH1A1) has emerged as an excellent marker to identify CSCs, owing to its conserved expression profile in nearly all progenitor cells including CSCs. The ALDH1A1 isoform belongs to a superfamily of 19 enzymes that are responsible for the oxidation of various endogenous and xenobiotic aldehydes to the corresponding carboxylic acid products. Chan et al. recently developed AlDeSense, an isoform-selective "turn-on" probe for the detection of ALDH1A1 activity, as well as a non-reactive matching control reagent (Ctrl-AlDeSense) to account for off-target staining. This isoform-selective tool has already been demonstrated to be a versatile chemical tool through the detection of ALDH1A1 activity in K562 myelogenous leukemia cells, mammospheres, and melanoma-derived CSC xenografts. In this article, the utility of the probe was showcased through additional fluorimetry, confocal microscopy, and flow cytometry experiments where the relative ALDH1A1 activity was determined in a panel of five ovarian cancer cell lines.

The Prognostic Value of Cancer Stem Cell Markers (CSCs) Expression-ALDH1A1, CD133, CD44-For Survival and Long-Term Follow-Up of Ovarian Cancer Patients.

Recurrent disease and treatment-associated chemoresistance are the two main factors accounting for poor clinical outcomes of ovarian cancer (OC) patients. Both can be associated with cancer stem cells (CSCs), which contribute to cancer formation, progression, chemoresistance, and recurrence. Hence, this study investigated whether the expression of known CSC-associated markers ALDH1A, CD44, and CD133 may predict OC patient prognosis. We analyzed their expression in primary epithelial ovarian cancer (EOC) patients using immunohistochemistry and related them to clinicopathological data, including overall survival (OS) and progression-free survival (PFS). Expression of ALDH1A1 was detected in 32%, CD133 in 28%, and CD44 in 33% of cases. While Kaplan-Meier analysis revealed no association of the expression of CD133 and CD44 with PFS and OS, ALDH1A1-positive patients were characterized with both significantly shorter OS (p = 0.00022) and PFS (p = 0.027). Multivariate analysis demonstrated that the expression of ALDH1A1, FIGO stage III-IV, and residual disease after suboptimal debulking or neoadjuvant chemotherapy correlated with shorter OS. The results of this study identify ALDH1A1 as a potential independent prognostic factor of shorter OS and PFS in EOC patients. Therefore, targeting ALDH1A1-positive cancer cells may be a promising therapeutic strategy to influence the disease course and treatment response.

Exploratory Metabolomics Underscores the Folate Enzyme ALDH1L1 as a Regulator of Glycine and Methylation Reactions.

Folate (vitamin B9) is involved in one-carbon transfer reactions and plays a significant role in nucleic acid synthesis and control of cellular proliferation, among other key cellular processes. It is now recognized that the role of folates in different stages of carcinogenesis is complex, and more research is needed to understand how folate reactions become dysregulated in cancers and the metabolic consequences that occur as a result. ALDH1L1 (cytosolic 10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism expressed in many tissues, is ubiquitously downregulated in cancers and is not expressed in cancer cell lines. The RT4 cell line (derived from papillary bladder cancer) which expresses high levels of ALDH1L1 represents an exception, providing an opportunity to explore the metabolic consequences of the loss of this enzyme. We have downregulated this protein in RT4 cells (shRNA driven knockdown or CRISPR driven knockout) and compared metabolomes of ALDH1L1-expressing and -deficient cells to determine if metabolic changes linked to the loss of this enzyme might provide proliferative and/or survival advantages for cancer cells. In this study, cell extracts were analyzed using Ultra High Performance Liquid Chromatography High Resolution Mass Spectrometry (UHPLC-HR-MS). A total of 13,339 signals were identified or annotated using an in-house library and public databases. Supervised and unsupervised multivariate analysis revealed metabolic differences between RT4 cells and ALDH1L1-deficient clones. Glycine (8-fold decrease) and metabolites derived from S-adenosylmethionine utilizing pathways were significantly decreased in the ALDH1L1-deficient clones, compared with RT4 cells. Other changes linked to ALDH1L1 downregulation include decreased levels of amino acids, Krebs cycle intermediates, and ribose-5-phosphate, and increased nicotinic acid. While the ALDH1L1-catalyzed reaction is directly linked to glycine biosynthesis and methyl group flux, its overall effect on cellular metabolism extends beyond immediate metabolic pathways controlled by this enzyme.

Variants in ALDH1A2 reveal an anti-inflammatory role for retinoic acid and a new class of disease-modifying drugs in osteoarthritis.

More than 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease-modifying treatments for this disabling condition. Common polymorphic variants in ALDH1A2, which encodes the key enzyme for synthesis of all-trans retinoic acid (atRA), are associated with severe hand OA. Here, we sought to elucidate the biological significance of this association. We first confirmed that ALDH1A2 risk variants were associated with hand OA in the U.K. Biobank. Articular cartilage was acquired from 33 individuals with hand OA at the time of routine hand OA surgery. After stratification by genotype, RNA sequencing was performed. A reciprocal relationship between ALDH1A2 mRNA and inflammatory genes was observed. Articular cartilage injury up-regulated similar inflammatory genes by a process that we have previously termed mechanoflammation, which we believe is a primary driver of OA. Cartilage injury was also associated with a concomitant drop in atRA-inducible genes, which were used as a surrogate measure of cellular atRA concentration. Both responses to injury were reversed using talarozole, a retinoic acid metabolism blocking agent (RAMBA). Suppression of mechanoflammation by talarozole was mediated by a peroxisome proliferator-activated receptor gamma (PPARγ)-dependent mechanism. Talarozole was able to suppress mechano-inflammatory genes in articular cartilage in vivo 6 hours after mouse knee joint destabilization and reduced cartilage degradation and osteophyte formation after 26 days. These data show that boosting atRA suppresses mechanoflammation in the articular cartilage in vitro and in vivo and identifies RAMBAs as potential disease-modifying drugs for OA.

Aldehyde Dehydrogenase 1B1 Is Implicated in DNA Damage Response in Human Colorectal Adenocarcinoma.

Aldehyde dehydrogenase 1B1 (ALDH1B1) has been correlated with colorectal tumorigenesis and is considered a potential biomarker for colon cancer. Its expression has been associated with attenuation of the cell cycle in the G2/M phase and resistance to DNA damaging agents. The present study examines the role of ALDH1B1 in DNA damage response (DDR) in human colorectal adenocarcinoma. To this end, we utilized an isogenic HT29 cell line pair differing in the expression of ALDH1B1. The overexpression of ALDH1B1 was related to the translational upregulation of the total and phosphorylated (at ser15) p53. Comet and apoptosis assays revealed that the expression of ALDH1B1 protected HT29 cells from etoposide-induced DNA damage as well as apoptosis, and its overexpression led to increased constitutive phosphorylation of H2AX (at ser139). Furthermore, the expression profile of a variety of DNA damage signaling (DDS)-related genes was investigated by utilizing the RT2 profiler™ PCR array. Our results demonstrated that ALDH1B1 triggered a transcriptional activation of several DNA repair-related genes (MRE11A, PMS1, RAD18 and UNG). Finally, Spearman's rank correlation coefficient analysis in 531 publicly available colorectal adenocarcinoma clinical samples indicated the statistically significant positive correlation between ALDH1B1 and DDR and repair genes or proteins, such as APEX1, FEN1, MPG, UNG, XRCC1, DDB1, XPC, CIB1, MRE11, PRKDC, RAD50, RAD21, TP53BP1, XRCC6 and H2AX. Collectively, our results suggest that ALDH1B1 may play an essential role in the DDR and DNA repair processes. Further studies on ALDH1B1 will elucidate its precise role in DDR.

Identification of Breast Cancer Stem Cells Using a Newly Developed Long-acting Fluorescence Probe, C5S-A, Targeting ALDH1A1.

BACKGROUND/AIM: Aldehyde dehydrogenase (ALDH) 1A1 is a well-known marker for cancer stem cells (CSCs), characterized by self-renewal capacity and multidrug resistance in breast cancer. We developed a near-infrared turn-on fluorescence probe for ALDH1A1, C5S-A, which is suitable for observing and analyzing viable cells. Here, we demonstrated the utility of C5S-A in CSC research using breast cancer cell lines. MATERIALS AND METHODS: To evaluate concordance between C5S-A and conventional stem cell markers, breast cancer cells sorted for ALDEFLUOR-positive cells and for CD44+/CD24- cell populations were stained with C5S-A. Tumorigenicity of C5S-A-positive cells was examined by mammosphere formation assay and subcutaneous transplantation to immunodeficient mice. Additionally, to determine how long fluorescence from a single staining remained observable, we cultured breast cancer cells for 5 days after C5S-A staining. We then evaluated whether C5S-A-positive cells possessed resistance to cytotoxic drugs by chronological imaging. RESULTS: C5S-A staining showed good concordance with conventional breast CSC markers, and good utility for research into CSC characteristics in breast cancer cell lines, including tumorigenesis. Additionally, C5S-A was observable for more than 3 days with a single staining. Using this property, we then confirmed that C5S-A-positive cells possessed resistance to cytotoxic drugs, which is one of the characteristics of CSCs. CONCLUSION: We showed that C5S-A is suitable for CSC research using breast cancer cell lines, and confirmed its utility in observing cells over time.

Liver regeneration and ethanol detoxification: A new link in YAP regulation of ALDH1A1 during alcohol-related hepatocyte damage.

Yes-associated protein (YAP), a central effector in the Hippo pathway, is involved in the regulation of organ size, stem cell self-renewal, and tissue regeneration. In this study, we observed YAP activation in patients with alcoholic steatosis, hepatitis, and cirrhosis. Accumulation of this protein in the nucleus was also observed in murine livers that were damaged after chronic-plus-single binge or moderate ethanol ingestion combined with carbon tetrachloride intoxication (ethanol/CCl4 ). To understand the role of this transcriptional coactivator in alcohol-related liver injury, we knocked out the Yap1 gene in hepatocytes of floxed homozygotes through adeno-associated virus (AAV8)-mediated deletion utilizing Cre recombinase. Yap1 hepatocyte-specific knockouts (KO) exhibited hemorrhage, massive hepatic necrosis, enhanced oxidative stress, elevated hypoxia, and extensive infiltration of CD11b+ inflammatory cells into hepatic microenvironments rich for connective tissue growth factor (Ctgf) during ethanol/CCl4 -induced liver damage. Analysis of whole-genome transcriptomics indicated upregulation of genes involved in hypoxia and extracellular matrix (ECM) remodeling, whereas genes related to hepatocyte proliferation, progenitor cell activation, and ethanol detoxification were downregulated in the damaged livers of Yap1 KO. Acetaldehyde dehydrogenase (Aldh)1a1, a gene that encodes a detoxification enzyme for aldehyde substrates, was identified as a potential YAP target because this gene could be transcriptionally activated by a hyperactive YAP mutant. The ectopic expression of the human ALDH1A1 gene caused increase in hepatocyte proliferation and decrease in hepatic necrosis, oxidative stress, ECM remodeling, and inflammation during ethanol/CCl4 -induced liver damage. Taken together, these observations indicated that YAP was crucial for liver repair during alcohol-associated injury. Its regulation of ALDH1A1 represents a new link in liver regeneration and detoxification.