ALDEFLUOR activity, ALDH isoforms, and their clinical significance in cancers.

High aldehyde dehydrogenase (ALDH) activity is a metabolic feature of adult stem cells and various cancer stem cells (CSCs). The ALDEFLUOR system is currently the most commonly used method for evaluating ALDH enzyme activity in viable cells. This system is applied extensively in the isolation of normal stem cells and CSCs from heterogeneous cell populations. For many years, ALDH1A1 has been considered the most important subtype among the 19 ALDH family members in determining ALDEFLUOR activity. However, in recent years, studies of many types of normal and tumour tissues have demonstrated that other ALDH subtypes can also significantly influence ALDEFLUOR activity. In this article, we briefly review the relationships between various members of the ALDH family and ALDEFLUOR activity. The clinical significance of these ALDH isoforms in different cancers and possible directions for future studies are also summarised.

An aldehyde dehydrogenase 1A3 inhibitor attenuates the metastasis of human colorectal cancer.

Metastasis is the leading cause of death for patients with colorectal cancer (CRC). The development of therapeutic regimens that selectively inhibit the biological processes involved in CRC cell dissemination is important. We used multiple Affymetrix DNA microarray hybridization datasets to identify genes related to metastasis and have significant prognostic value for patients with CRC. Quantitative real-time PCR, immunofluorescent and immunohistochemical staining were used to evaluate mRNA and protein expression. The function of aldehyde dehydrogenase 1A3 (ALDH1A3) in invasion was assessed by performing transwell assays and animal experiments. Real-time PCR, luciferase reporter assays, and western blotting were used to identify the genes regulated by ALDH1A3. Molecular docking, MTS assays, cellular thermal shift assays, isothermal titration calorimetry, microscale thermophoresis, and enzymatic activity assays were used to screen and verify the efficacy of the ALDH1A3-specific inhibitor YD1701 (dibenzo-30-crown10-ether). Finally, subcutaneous or orthotopic xenograft models were established to investigate the therapeutic potential of YD1701. Human ALDH1A3 was identified to correlate with a metastatic phenotype in CRC cells and a poor patient prognosis. Moreover, ALDH1A3 upregulated the expression of ZEB1 and SNAI2 by inhibiting miR-200 family members. The ALDH1A3-specific inhibitor YD1701 was screened, attenuated the invasion of CRC cells in vitro, and prolonged the survival of mice bearing subcutaneous or orthotopic xenografts. Our results show that ALDH1A3 promotes invasion and metastasis via the miR-200-ZEB1/SANI2 axis and is thus a plausible marker for predicting CRC progression. Inhibiting ALDH1A3 with the identified compound YD1701 might represent an effective therapeutic approach to prevent the metastasis of CRC.

Triple-negative breast cancer molecular subtyping and treatment progress.

Triple-negative breast cancer (TNBC), a specific subtype of breast cancer that does not express estrogen receptor (ER), progesterone receptor (PR), or human epidermal growth factor receptor 2 (HER-2), has clinical features that include high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Because TNBC tumors lack ER, PR, and HER2 expression, they are not sensitive to endocrine therapy or HER2 treatment, and standardized TNBC treatment regimens are still lacking. Therefore, development of new TNBC treatment strategies has become an urgent clinical need. By summarizing existing treatment regimens, therapeutic drugs, and their efficacy for different TNBC subtypes and reviewing some new preclinical studies and targeted treatment regimens for TNBC, this paper aims to provide new ideas for TNBC treatment.

Inhibition of the ALDH18A1-MYCN positive feedback loop attenuates MYCN-amplified neuroblastoma growth.

MYCN-amplified neuroblastoma (NB) is characterized by poor prognosis, and directly targeting MYCN has proven challenging. Here, we showed that aldehyde dehydrogenase family 18 member A1 (ALDH18A1) exerts profound impacts on the proliferation, self-renewal, and tumorigenicity of NB cells and is a potential risk factor in patients with NB, especially those with MYCN amplification. Mechanistic studies revealed that ALDH18A1 could both transcriptionally and posttranscriptionally regulate MYCN expression, with MYCN reciprocally transactivating ALDH18A1 and thus forming a positive feedback loop. Using molecular docking and screening, we identified an ALDH18A1-specific inhibitor, YG1702, and demonstrated that pharmacological inhibition of ALDH18A1 was sufficient to induce a less proliferative phenotype and confer tumor regression and prolonged survival in NB xenograft models, providing therapeutic insights into the disruption of this reciprocal regulatory loop in MYCN-amplified NB.

Author Correction: Invasion of white matter tracts by glioma stem cells is regulated by a NOTCH1-SOX2 positive-feedback loop.

The original and corrected figures are shown in the accompanying Author Correction.

Invasion of white matter tracts by glioma stem cells is regulated by a NOTCH1-SOX2 positive-feedback loop.

Early invasive growth along specific anatomical structures, especially the white matter tract, is regarded as one of the main causes of poor therapeutic outcome of people with gliomas. We show that some glioma stem cells (GSCs) are preferentially located along white matter tracts, which exhibit a demyelinated phenotype, at the invasive frontier of glioma tissues. These GSCs are CD133+Notch1+, whereas the nerve fibers express the Notch ligand Jagged1. The Notch-induced transcription factor Sox9 promotes the transcription of SOX2 and the methylation level of the NOTCH1 promoter is attenuated by the upregulation of SOX2 to reinforce NOTCH1 expression in GSCs. This positive-feedback loop in a cohort of glioma subjects is correlated with a poor prognosis. Inhibition of Notch signaling attenuates the white-matter-tract tropism of GSCs. These findings provide evidence indicating that the NOTCH1-SOX2 positive-feedback loop controls GSC invasion along white matter tracts.

Connexin 43 C-terminus directly inhibits the hyperphosphorylation of Akt/ERK through protein-protein interactions in glioblastoma.

Although the deregulation of epidermal growth factor receptor (EGFR) is one of the most common molecular mechanisms of glioblastoma (GBM) pathogenesis, the efficacy of anti-EGFR therapy is limited. Additionally, response to anti-EGFR therapy is not solely dependent on EGFR expression and is more promising in patients with reduced activity of EGFR downstream signaling pathways. Thus, there is considerable interest in identifying the compensatory regulatory factors of the EGFR signaling pathway to improve the efficacy of anti-EGFR therapies for GBM. In this study, we confirmed the low efficacy of EGFR inhibitors in GBM patients by meta-analysis. We then identified a negative correlation between connexin 43 (Cx43) expression and Akt/ERK activation, which was caused by the direct interactions between Akt/ERK and Cx43. By comparing the interactions between Akt/ERK and Cx43 using a series of truncated and mutated Cx43 variants, we revealed that the residues T286/A305/Q308/Y313 and S272/S273 at the carboxy terminus of Cx43 are critical for its binding with Akt and ERK, respectively. In addition, Kaplan-Meier survival analysis using data from The Cancer Genome Atlas datasets indicated that the expression of Cx43 significantly improved the prognosis of GBM patients who express EGFR. Together, our results suggested that Cx43 acts as an inhibitory regulator of the activation of growth factor receptor downstream signaling pathways, indicating the potential of Cx43 as a marker for predicting the efficacy of EGFR inhibitor treatments for GBM. Targeting the interaction between the carboxy terminus of Cx43 and Akt/ERK could be an effective therapeutic strategy against GBM.

Optimized dissociation protocol for isolating human glioma stem cells from tumorspheres via fluorescence-activated cell sorting.

Fluorescence-activated cell sorting (FACS) based on the surface marker CD133 is the most common method for isolating glioma stem cells (GSCs) from heterogeneous glioma cell populations. Optimization of this method will have profound implications for the future of GSC research. Five commonly used digestion reagents, Liberase-TL, trypsin, TrypLE, Accutase, and non-enzymatic cell dissociation solution (NECDS), were used to dissociate glioma tumorspheres derived from two primary glioma specimens (091214 and 090116) and the cell lines U87 and T98G. The dissociation time, cell viability, retention of CD133, and stemness capacity were assessed. The results showed that single cells derived from the Liberase-TL (200 µg/ml) group exhibited high viability and less damage to the antigen CD133. However, the efficiency of NECDS for dissociating the tumorspheres into single cells was fairly low. Meanwhile, the use of this digestion reagent resulted in obvious cellular and antigenic impairments. Taken together, Liberase-TL (200 µg/ml) is an ideal reagent for isolating GSCs from tumorspheres. In contrast, the use of NECDS for such a protocol should be carefully considered.

ALDH1A3, a metabolic target for cancer diagnosis and therapy.

Metabolism reprogramming has been linked with the initiation, metastasis, and recurrence of cancer. The aldehyde dehydrogenase (ALDH) family is the most important enzyme system for aldehyde metabolism. The human ALDH family is composed of 19 members. ALDH1A3 participates in various physiological processes in human cells by oxidizing all-trans-retinal to retinoic acid. ALDH1A3 expression is regulated by many factors, and it is associated with the development, progression, and prognosis of cancers. In addition, ALDH1A3 influences a diverse range of biological characteristics within cancer stem cells and can act as a marker for these cells. Thus, growing evidence indicates that ALDH1A3 has the potential to be used as a target for cancer diagnosis and therapy.

Aldehyde dehydrogenase 1A1 circumscribes high invasive glioma cells and predicts poor prognosis.

Glioma is the most aggressive brain tumor with high invasiveness and poor prognosis. More reliable, sensitive and practical biomarkers to reveal glioma high invasiveness remain to be explored for the guidance of therapy. We herein evaluated the diagnostic and prognostic value of aldehyde dehydrogenase 1A1 (ALDH1A1) in the glioma specimens from 237 patients, and found that ADLH1A1 was frequently overexpressed in the high-grade glioma (WHO grade III-IV) as compared to the low-grade glioma (WHO grade I-II) patients. The tumor cells with ALDH1A1 expression were more abundant in the region between tumor and the borderline of adjacent tissue as compared to the central part of the tumor. ALDH1A1 overexpression was associated with poor differentiation and dismal prognosis. Notably, the overall and disease-free survivals of the patients who had ALDH1A1(+) tumor cells sparsely located in the adjacent tissue were much worse. Furthermore, ALDH1A1 expression was correlated with the "classical-like" (CL) subtype as we examined GBM specimens from 72 patients. Multivariate Cox regression analysis revealed that ALDH1A1 was an independent marker for glioma patients' outcome. Mechanistically, both in vitro and in vivo studies revealed that ALDH1A1(+) cells isolated from either a glioblastoma cell line U251 or primary glioblastoma cells displayed significant invasiveness, clonogenicity, and proliferation as compared to ALDH1A1(-) cells, due to increased levels of mRNA and protein for matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9). These results indicate that ALDH1A1(+) cells contribute to the progression of glioma including invasion, proliferation and poor prognosis, and suggest that targeting ALDH1A1 may have important implications for the treatment of highly invasive glioma.

Distinct patterns of ALDH1A1 expression predict metastasis and poor outcome of colorectal carcinoma.

PURPOSE: Aldehyde dehydrogenase 1A1 (ALDH1A1) has been proposed as a candidate biomarker for colorectal carcinoma (CRC). However, the heterogeneity of its expression makes it difficult to predict the outcome of CRC. The aim of this study was to evaluate the diagnostic and prognostic value of this molecule in CRC. METHODS AND RESULTS: In this study, we examined ALDH1A1 expression by immunohistochemistry including 406 cases of primary CRC with corresponding adjacent mucosa, with confirmation of real-time PCR and Western blotting. We found that the expression patterns of ALDH1A1 were heterogeneous in the CRC and corresponding adjacent tissues. We defined the ratio of ALDH1A1 level in adjacent mucosa to that in tumor tissues as RA/C and found that the capabilities of tumor invasion and metastasis in the tumors with RA/C < 1 were significantly higher than those with RA/C ≥ 1. Follow-up data showed the worse prognoses in the CRC patients with RA/C < 1. For understanding the underlying mechanism, the localization of ß-catenin was detected in the CRC tissues with different patterns of ALDH1A1 expression from 221 patients and ß-catenin was found preferentially expressed in cell nuclei of the tumors with RA/C < 1 and ALDH1A1(high) expression of HT29 cell line, indicating that nuclear translocation of ß-catenin might contribute to the increased potentials of invasion and metastasis. CONCLUSION: Our results indicate that RA/C is a novel biomarker to reflect the distinct expression patterns of ALDH1A1 for predicting metastasis and prognosis of CRC.

ALDH1A1 expression correlates with clinicopathologic features and poor prognosis of breast cancer patients: a systematic review and meta-analysis.

BACKGROUND: Aldehyde dehydrogenase 1 family member A1 (ALDH1A1) has been identified as a putative cancer stem cell (CSC) marker in breast cancer. However, the clinicopathological and prognostic significance of this protein in breast cancer patients remains controversial. METHODS: This meta-analysis was conducted to address the above issues using 15 publications covering 921 ALDH1A1(+) cases and 2353 controls. The overall and subcategory analyses were performed to detect the association between ALDH1A1 expression and clinicopathological/prognostic parameters in breast cancer patients. RESULTS: The overall analysis showed that higher expression of ALDH1A1 is associated with larger tumor size, higher histological grade, greater possibility of lymph node metastasis (LNM), higher level expression of epidermal growth factor receptor 2 (HER2), and lower level expression of estrogen receptor (ER)/progesterone receptor (PR). The prognosis of breast cancer patients with ALDH1A1(+) tumors was poorer than that of the ALDH1A1(-) patients. Although the relationships between ALDH1A1 expression and some clinicopathological parameters (tumor size, LNM, and the expression of HER2) was not definitive to some degree when we performed a subcategory analysis, the predictive values of ALDH1A1 expression for histological grade and survival of breast cancer patients were significant regardless of the different cutoff values of ALDH1A1 expression, the different districts where the patients were located, the different clinical stages of the patients, the difference in antibodies used in the studies, and the surgery status. CONCLUSIONS: Our results indicate that ALDH1A1 is a biomarker to predict tumor progression and poor survival of breast cancer patients. This marker should be taken into consideration in the development of new diagnostic and therapeutic program for breast cancer.

Strategies for isolating and enriching cancer stem cells: well begun is half done.

Cancer stem cells (CSCs) constitute a subpopulation of cancer cells that have the potential for self-renewal, multipotent differentiation, and tumorigenicity. Studies on CSC biology and CSC-targeted therapies depend on CSC isolation and/or enrichment methodologies. Scientists have conducted extensive research in this field since John Dick's group successfully isolated CSCs based on the expression of the CD34 and CD38 surface markers. Progress in CSC research has been greatly facilitated by the enrichment and isolation of these cells. In this review, we summarize the current strategies used in our and other laboratories for CSC isolation and enrichment, including methods based on stem cell surface markers, intracellular enzyme activity, the concentration of reactive oxygen species, the mitochondrial membrane potential, promoter-driven fluorescent protein expression, autofluorescence, suspension/adherent culture, cell division, the identification of side population cells, resistance to cytotoxic compounds or hypoxia, invasiveness/adhesion, immunoselection, and physical property. Although many challenges remain to be overcome, it is reasonable to believe that more reliable, efficient, and convenient methods will be developed in the near future.

TGF-ß1 enhances tumor-induced angiogenesis via JNK pathway and macrophage infiltration in an improved zebrafish embryo/xenograft glioma model.

Angiogenesis plays a crucial role at the early stage of tumorigenesis and tumor progression. A suitable model will be useful not only for the clarification of the underlying molecular mechanisms, but also for high-throughput identification of novel anti-angiogenesis compounds. Here, we established a zebrafish model for the purpose to investigate angiogenesis and screen anti-angiogenic compounds. Glioma U87 cells expressing red fluorescent protein (RFP) were transplanted in fli:GFP transgenic zebrafish embryos where significant angiogenesis was observed. TGF-ß1 enhanced glioma-induced angiogenesis, which was inhibited by JNK inhibitor SP600125 but not p38 MAPK inhibitor SB202190, ERK inhibitor PD98059, or PI3K inhibitor LY294002, indicating the important role of TGF-ß1 and JNK pathways in this process. Moreover, the glioma-induced angiogenesis was associated with macrophage infiltration that was further enhanced by TGF-ß1. Therefore, our zebrafish model provides a powerful in vivo tool for the investigation of tumor-induced angiogenesis, and a cost-effective system for high-throughput screening of anti-angiogenic compounds.

Connexin 43 reverses malignant phenotypes of glioma stem cells by modulating E-cadherin.

Malfunctioned gap junctional intercellular communication (GJIC) has been thought associated with malignant transformation of normal cells. However, the role of GJIC-related proteins such as connexins in sustaining the malignant behavior of cancer stem cells remains unclear. In this study, we obtained tumorspheres formed by glioma stem cells (GSCs) and adherent GSCs and then examined their GJIC. All GSCs showed reduced GJIC, and differentiated glioma cells had more gap junction-like structures than GSCs. GSCs expressed very low level of connexins, Cx43 in particular, which are key components of gap junction. We observed hypermethylation in the promoter of gap junction protein α1, which encodes Cx43 in GSCs. Reconstitution of Cx43 in GSCs inhibited their capacity of self-renewal, invasiveness, and tumorigenicity via influencing E-cadherin and its coding protein, which leads to changes in the expression of Wnt/ß-catenin targeting genes. Our results suggest that GSCs require the low expression of Cx43 for maintaining their malignant phenotype, and upregulation of Cx43 might be a potential strategy for treatment of malignant glioma.