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Sulfasalazine, an xCT inhibitor, is being used as a repurposed antineoplastic drug to induce ferroptosis. Ferroptosis is a regulated necrotic cell death pathway that is dependent on iron reserves. Interestingly, cancer stem cells (CSCs) that are regarded as major drivers of resistance to conventional therapies accompanied with tumor relapse and recurrence have bulk amount of iron reserves in the form of ferritin. This suggests that inducing ferroptosis might disrupt stemness and drug-resistant mechanisms in cancer stem cells, thereby reducing the risk of drug-resistance, cancer recurrence, and relapse. In the present study, ALDH1A1 expressing oral (OCSCs) and breast (BCSCs) cancer stem cells were sorted and used to investigate the role of sulfasalazine to induce ferroptosis. To check the self-renewability of CSCs spheroid formation, assay was performed and the resultant CSCs were treated with sulfasalazine (SAS) and subjected to gene expression analysis RT-PCR and flow cytometry. FACS was performed to check stem cell marker expression, cell cycle arrest, and apoptosis. Our results suggest that the cells showed a gradual increase in sphere formation till S3 in the case of OCSCs and S2 in the case of BCSCs, with a gradual decrease in sphere-forming efficiency from the respective generations. When treated with 0.6mM SAS, these cells induced ferroptosis by downregulating stem cell markers like ALDH1A1, SLC7A11, ferritin, and GPx-4 with a concomitant increase in transferrin and STEAP-3. Flow cytometry studies revealed that the cells have undergone mitochondrial dysfunction characterized by loss of membrane potential and the cell cycle progression was halted in the G2/M phase. In the present study, we demonstrate that SAS potentially induced ferroptosis accompanied with oxidative stress in both OCSCs as well as BCSCs by lowering GPx-4 activity, a key enzyme that scavenges the products produced as a result of oxidative stress.

Ovarian cancer is a deadly disease attributed to late-stage detection as well as recurrence and the development of chemoresistance. Ovarian cancer stem cells (OCSCs) are hypothesized to be largely responsible for the emergence of chemoresistant tumors. Although chemotherapy may initially succeed at decreasing the size and number of tumors, it leaves behind residual malignant OCSCs. In this study, we demonstrate that aldehyde dehydrogenase 1A1 (ALDH1A1) is essential for the survival of OCSCs. We identified a first-in-class ALDH1A1 inhibitor, compound 974, and used 974 as a tool to decipher the mechanism of stemness regulation by ALDH1A1. The treatment of OCSCs with 974 significantly inhibited ALDH activity, the expression of stemness genes, and spheroid and colony formation. An in vivo limiting dilution assay demonstrated that 974 significantly inhibited CSC frequency. A transcriptomic sequencing of cells treated with 974 revealed a significant downregulation of genes related to stemness and chemoresistance as well as senescence and the senescence-associated secretory phenotype (SASP). We confirmed that 974 inhibited the senescence and stemness induced by platinum-based chemotherapy in functional assays. Overall, these data establish that ALDH1A1 is essential for OCSC survival and that ALDH1A1 inhibition suppresses chemotherapy-induced senescence and stemness. Targeting ALDH1A1 using small-molecule inhibitors in combination with chemotherapy therefore presents a promising strategy to prevent ovarian cancer recurrence and has the potential for clinical translation.

ALDH1A1 and ALDH1A3 paralogues of aldehyde dehydrogenase 1 (ALDH1) control myogenic differentiation of skeletal muscle satellite cells (SC) by formation of retinoic acid (RA) and subsequent cell cycle adjustments. The respective relevance of each paralogue for myogenic differentiation and the mechanistic interaction of each paralogue within RA-dependent and RA-independent pathways remain elusive. We analysed the impact of ALDH1A1 and ALDH1A3 activity on myogenesis of murine C2C12 myoblasts. Both paralogues are pivotal factors in myogenic differentiation, since CRISPR/Cas9-edited single paralogue knock-out impaired serum withdrawal-induced myogenic differentiation, while successive recombinant re-expression of ALDH1A1 or ALDH1A3, respectively, in the corresponding ALDH1 paralogue single knock-out cell lines, recovered the differentiation potential. Loss of differentiation in single knock-out cell lines was restored by treatment with RA-analogue TTNPB, while RA-receptor antagonization by AGN 193109 inhibited differentiation of wildtype cell lines, supporting the idea that RA-dependent pathway is pivotal for myogenic differentiation which is accomplished by both paralogues. However, overexpression of ALDH1-paralogues or disulfiram-mediated inhibition of ALDH1 enzymatic activity not only increased ALDH1A1 and ALDH1A3 protein levels but also induced subsequent differentiation of C2C12 myoblasts independently from serum withdrawal, indicating that ALDH1-dependent myogenic differentiation relies on different cellular conditions. Remarkably, ALDH1-paralogue knock-out impaired the autophagic flux, namely autophagosome cargo protein p62 formation and LC3B-I to LC3B-II conversion, demonstrating that ALDH1-paralogues interact with autophagy in myogenesis. Together, ALDH1 paralogues play a crucial role in myogenesis by orchestration of complex RA-dependent and RA-independent pathways.

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.

A small of population of slow cycling and chemo-resistant cells referred to as cancer stem cells (CSC) have been implicated in cancer recurrence. There is emerging interest in developing targeted therapeutics to eradicate CSCs. Aldehyde-dehydrogenase (ALDH) activity was shown to be a functional marker of CSCs in ovarian cancer (OC). ALDH activity is increased in cells grown as spheres versus monolayer cultures under differentiating conditions and in OC cells after treatment with platinum. Here, we describe the activity of CM37, a newly identified small molecule with inhibitory activity against ALDH1A1, in OC models enriched in CSCs. Treatment with CM37 reduced OC cells’ proliferation as spheroids under low attachment growth conditions and the expression of stemness-associated markers (OCT4 and SOX2) in ALDH+ cells fluorescence-activated cell sorting (FACS)-sorted from cell lines and malignant OC ascites. Likewise, siRNA-mediated ALDH1A1 knockdown reduced OC cells’ proliferation as spheres, expression of stemness markers, and delayed tumor initiation capacity in vivo. Treatment with CM37 promoted DNA damage in OC cells, as evidenced by induction of γH2AX. This corresponded to increased expression of genes involved in DNA damage response, such as NEIL3, as measured in ALDH+ cells treated with CM37 or in cells where ALDH1A1 was knocked down. By inhibiting ALDH1A1, CM37 augmented intracellular ROS accumulation, which in turn led to increased DNA damage and reduced OC cell viability. Cumulatively, our findings demonstrate that a novel ALDH1A1 small molecule inhibitor is active in OC models enriched in CSCs. Further optimization of this new class of small molecules could provide a novel strategy for targeting treatment-resistant OC.

A major contributor leading to treatment failure of ovarian cancer patients is the drug resistance of cancer cell. CSCs- (cancer stem cells) and ECM (extracellular matrix)-related models of drug resistance are described as independently occurring in cancer cells. Lysyl oxidase (LOX) is another extracellular protein involved in collagen cross-linking and remodeling of extracellular matrix and has been correlated with tumor progression. The expression of LOX, COL1A2, COL3A1, and ALDH1A1 was performed in sensitive (A2780, W1) and resistant to paclitaxel (PAC) (A2780PR1 and W1PR2) and topotecan (TOP) (W1TR) cell lines at the mRNA (real-time PCR analysis) and protein level (Western blot and immunofluorescence analysis). The ALDH1A1 activity was measured with the ALDEFLUOR test and flow cytometry analysis. The protein expression in ovarian cancer tissues was determined by immunohistochemistry. We observed an increased expression of LOX and collagens in PAC and TOP resistant cell lines. Subpopulations of ALDH1A1 positive and negative cells were also noted for examined cell lines. Additionally, the coexpression of LOX with ALDH1A1 and COL1A2 with ALDH1A1 was observed. The expression of LOX, collagens, and ALDH1A1 was also detected in ovarian cancer lesions. In our study LOX, ALDH1A1 and collagens were found to be coordinately expressed by cells resistant to PAC (LOX, ALDH1A1, and COL1A2) or to TOP (LOX and ALDH1A1). This represents the study where molecules related with CSCs (ALDH1A1) and ECM (LOX, collagens) models of drug resistance are described as occurring simultaneously in ovarian cancer cells treated with PAC and TOP.

Prostate cancer (PCa) contains a small population of cancer stem cells (CSCs) that contribute to its initiation and progression. The development of specific markers for identification of the CSCs may lead to new diagnostic strategies of PCa. Increased aldehyde dehydrogenase 1A1 (ALDH1A1) activity has been found in the stem cell populations of leukemia and some solid tumors. The aim of the study was to investigate the stem-cell-related function and clinical significance of the ALDH1A1 in human PCa. ALDEFLUOR assay was used to isolate ALDH1A1+ cells from PCa cell lines. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. The ALDH1A1 expression was also analyzed by immunohistochemistry in 18 normal prostate and 163 PCa tissues. The ALDH1A1+ PCa cells showed high clonogenic and tumorigenic capacities, and serially reinitiated transplantable tumors that resembled histopathologic characteristics and heterogeneity of the parental PCa cells in mice. Immunohistochemical analysis of human prostate tissues showed that ALDH1A1+ cells were sparse and limited to the basal component in normal prostates. However, in tumor specimens, increased ALDH1A1 immunopositivity was found not only in secretory type cancer epithelial cells but also in neuroendocrine tumor populations. Furthermore, the high ALDH1A1 expression in PCa was positively correlated with Gleason score (P=0.01) and pathologic stage (P=0.01), and inversely associated with overall survival and cancer-specific survival of the patients (P=0.00093 and 0.00017, respectively). ALDH1A1 could be a prostate CSC-related marker. Measuring its expression might provide a potential approach to study tumorigenesis of PCa and predict outcome of the disease.

The transforming growth factor‐β (TGF‐β)/Smad signaling pathway promotes malignant transformation through various mechanisms, and its effect on enhancing drug resistance can limit the efficacy of treatment. Here, we showed that pre‐stimulation of human lung cancer A549 cells with TGF‐β increases resistance to doxorubicin‐induced growth inhibition in a Smad3‐ and Smad4‐dependent manner. This effect was suppressed by the aldehyde dehydrogenase (ALDH) inhibitor oxyfedrine, suggesting that ALDH family members are involved in drug resistance. TGF‐β upregulated the mRNA and protein expression of ALDH1A1. The TGF‐β/Smad3 transcriptional enhancer region on ALDH1A1 was identified by Smad3 ChIP‐seq analysis using an open database and by reporter assays. Knockdown of ALDH1A1 in A549 cells suppressed TGF‐β–induced doxorubicin resistance, and lentivirus‐mediated introduction of ALDH1A1 into A549 SMAD3‐KO cells restored drug resistance. We also demonstrated that ALDH1A1 is required and sufficient for TGF‐β/Smad3 signaling–induced anchorage‐independent growth. The results suggest that the TGF‐β/Smad3/4 axis promotes resistance to doxorubicin and anchorage‐independent growth by inducing the transcription of ALDH1A1. We have demonstrated that prestimulation of human lung cancer cells with TGF‐β enhances resistance to doxorubicin‐induced growth inhibition, depending on Smad3 and Smad4 through induction of ALDH1A1. Knockdown of ALDH1A1 suppressed TGF‐β‐induced resistance to doxorubicin, and exogenous expression of ALDH1A1 in SMAD3‐KO cells restored the drug resistance function, indicating ALDH1A1 is required and sufficient for the TGF‐β/Smad3‐induced doxorubicin resistance. We also revealed that TGF‐β/Smad3 enhances anchorage‐independent growth through induction of ALDH1A1. Therefore, the TGF‐β/Smad3/4 axis leading to induction of ALDH1A1 expression would be a promising therapeutic target.

Background Motor symptoms of Parkinson’s disease (PD) primarily result from the degeneration of nigrostriatal dopaminergic neurons (DANs), particularly the Aldehyde Dehydrogenase 1A1-positive (ALDH1A1⁺) subpopulation. Pitx3 -deficient mice exhibit selective developmental loss of ALDH1A1⁺ DANs but paradoxically display hyperlocomotion, suggesting compensatory changes in striatal circuitry. The dorsal striatum contains four main types of spiny projection neurons (SPNs): patch (or striosome) and matrix subtypes of both direct-pathway (dSPNs) and indirect-pathway (iSPNs). Activation of patch dSPNs suppresses locomotion by inhibiting ALDH1A1⁺ DANs. Methods We combined RNAscope in situ hybridization with SPN subtype-specific reporter mice to quantify patch and total dSPNs and iSPNs in Pitx3 -deficient and control mice. Three patch SPN reporter lines ( Kremen1 2A − Cre , Nr4a1 - GFP , and Pdyn IRES−Cre ) were used to map projections. Optogenetic stimulation was performed in freely moving mice to assess the behavioral effects of activating patch dSPNs and iSPNs. Results Pitx3- deficient mice showed no change in the overall dSPN: iSPN ratio but exhibited a marked shift in the patch dSPN: patch iSPN ratio, which decreased from 1.7 in control mice to 0.7 in the Pitx3- deficient group. Accordingly, patch dSPN projections to the substantia nigra pars reticulata (SNr) were reduced, whereas patch iSPN projections to the globus pallidus externus (GPe) were enhanced. Notably, while optogenetic stimulation of patch dSPNs and iSPNs suppressed locomotion in control mice, the same stimulation promoted locomotion in Pitx3 -deficient mice. Conclusions Our findings reveal a selective reorganization of patch SPNs in response to developmental loss of ALDH1A1⁺ DANs, characterized by reduced patch dSPN and enhanced patch iSPN influence. This shift may underlie the paradoxical hyperlocomotion observed in Pitx3 -deficient mice and provides insight into circuit-level adaptations with potential therapeutic relevance for PD.

Aldehyde dehydrogenase 1A3 (ALDH1A3) has recently gained attention from researchers in the cancer field. Several studies have reported ALDH1A3 overexpression in different cancer types, which has been found to correlate with poor treatment recovery. Therefore, finding selective inhibitors against ALDH1A3 could result in new treatment options for cancer treatment. In this study, ALDH1A3-selective candidates were designed based on the physiological substrate resemblance, synthesized and investigated for ALDH1A1, ALDH1A3 and ALDH3A1 selectivity and cytotoxicity using ALDH-positive A549 and ALDH-negative H1299 cells. Two compounds (ABMM-15 and ABMM-16), with a benzyloxybenzaldehyde scaffold, were found to be the most potent and selective inhibitors for ALDH1A3, with IC50 values of 0.23 and 1.29 µM, respectively. The results also show no significant cytotoxicity for ABMM-15 and ABMM-16 on either cell line. However, a few other candidates (ABMM-6, ABMM-24, ABMM-32) showed considerable cytotoxicity on H1299 cells, when compared to A549 cells, with IC50 values of 14.0, 13.7 and 13.0 µM, respectively. The computational study supported the experimental results and suggested a good binding for ABMM-15 and ABMM-16 to the ALDH1A3 isoform. From the obtained results, it can be concluded that benzyloxybenzaldehyde might be considered a promising scaffold for further drug discovery aimed at exploiting ALDH1A3 for therapeutic intervention.