Targeting cancer stem cells by disulfiram and copper sensitizes radioresistant chondrosarcoma to radiation.

Overcoming the radiosensitivity of chondrosarcoma (CS), the second most common primary bone tumor, is needed. Radioresistance is attributed to cancer stem cells (CSCs) in many malignancies. Disulfiram (DSF), an FDA-approved anti-alcoholism drug, complexed with Cu (DSF/Cu) can radiosensitize epithelial CSCs. This prompted us to investigate the radiosensitizing effect of DSF/Cu on CS CSCs (CCSCs). The radiosensitizing effects of DSF/Cu on CCSCs were investigated in vitro using cell lines SW1353 and CS-1. Stemness was identified independently by flow cytometry for CCSCs (ALDH+CD133+), sphere-forming ability, and Western blot analysis of stemness gene protein expression. The radiosensitizing effect of DSF/Cu was studied in an orthotopic CS xenograft mouse model by analyzing xenograft growth and residual xenografts for stemness. CCSCs were found to be resistant to single-dose (IR) and fractionated irradiation (FIR). IR and FIR increased CS stemness. Combined with DSF/Cu in vitro and in vivo, IR and FIR eliminated CS stemness. RT + DSF/Cu was safer and more effective than either RT ± DSF in inhibiting growth of orthotopic CS xenografts. In conclusion, DSF/Cu radiosensitizes CCSCs. These results can be translated into clinical trials for CS patients requiring RT for improved outcomes.

Isolation and characterization of circulating pro-vascular progenitor cell subsets from human whole blood samples.

The examination of circulating pro-vascular progenitor cell frequency and function is integral in understanding aberrant blood vessel homeostasis in individuals with cardiometabolic disease. Here, we outline the characterization of progenitor cell subsets from peripheral blood using high aldehyde dehydrogenase (ALDH) activity, an intracellular detoxification enzyme previously associated with pro-vascular progenitor cell status. Using this protocol, cells can be examined by flow cytometry for ALDH activity and lineage restricted cell surface markers simultaneously. For complete details on the use and execution of this protocol, please refer to Terenzi et al. (2019) and Hess et al. (2019, 2020).

Overexpression of ALDH1 and EMT marker profile are linked with unfavorable outcome in head and neck cancer

BACKGROUND: The aim of this research was to assess the expression of aldehyde dehydrogenase 1 (ALDH1) and epithelial-mesenchymal transition (EMT) markers in head and neck squamous cell carcinoma (HNSCC), and to correlate them with the clinical and histopathological parameters of a patient cohort with follow-up over an 8-year period. MATERIAL AND METHODS: For this, seventeen HNSCC and non-neoplastic adjacent epithelium (AE) samples were subjected to laser microdissection and real-time PCR to evaluate the mRNA expression of ALDH1, E-cadherin (E-CAD), N-cadherin (N-CAD), and vimentin (VIM). Also, immunohistochemistry was performed for ALDH1, E-CAD, N-CAD, and VIM in the tumor center (TC), invasion front (IF), and AE of the seventeen samples. Mann-Whitney, Kruskal-Wallis and Chi-square tests were used to correlate the mRNA and immunohistochemical ex-pression with different variables, considering p < 0.05. Kaplan-Meier curves were produced for local recurrence, regional metastasis and treatment. RESULTS: A mRNA overexpression of ALDH1 in primary tumors was associated with regional metastasis and a high ALDH1 immunostaining was related to metastasis and a worse patient outcome. Additionally, a favorable outcome was associated with the transition phase and an unfavorable outcome was associated with EMT event. An overall 26.9 months was observed with longer survival associated with surgery and radiotherapy. CONCLUSIONS: However, due to the intense variability inherent to the indicator proteins in the EMT process, the complete profile markers related to this biological process should be continuous investigated

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Effect of Single-Dose, Oral Enzymatic Porcine Placental Extract on Pharmacokinetics of Alcohol and Liver Function in Rats.

BACKGROUND: Human placenta extract (HPE) has been used to treat a number of liver diseases. Porcine placenta is relatively safe and has been reported to have similar immune effects to HPE and used as its alternative. This study evaluates the effect of enzymatic porcine placental extract (EPPE, Uni-Placenta®) on alcohol pharmacokinetics in rat. METHODS: This study was designed to determine the effect of single-dose EPPE on the pharmacokinetics of alcohol and liver function. Results were based on serum alcohol and acetaldehyde concentrations and activities of hepatic and gastric ADH and ALDH in rats. RESULTS: The hepatic ADH in alcohol group was significantly increased and it may be enzyme-induction by alcohol. The hepatic ALDH and gastric ADH were not changed, but gastric ALDH was significantly decreased only in the high-dose EPPE group. In the alcohol pharmacokinetics parameters, the AUC was 44.5 mM∙h in the alcohol group. Otherwise, AUCs of low, middle, high, and silymarin groups were significantly decreased. Cmax was reached at 1 hour and then gradually decreased to 63% and 43% in the middle and high groups at 3 hours, respectively, and to 92% in the low groups. The pharmacokinetics and serum concentrations of acetaldehyde showed no differences between EPPE groups except the silymarin group. No histologic changes were seen in any group. CONCLUSIONS: The single-dose EPPE (0.5 to 2.5 g/kg) suppressed absorption of alcohol in the gastrointestinal tract. This may be useful in preventing hangover effects and toxicity after drinking alcohol and may also preserve liver health after alcohol ingestion.

ALDH as a Stem Cell Marker in Solid Tumors.

Aldehyde dehydrogenase (ALDH) is an enzyme that participates in important cellular mechanisms as aldehyde detoxification and retinoic acid synthesis; moreover, ALDH activity is involved in drug resistance, a characteristic of cancer stem cells (CSCs). Even though ALDH is found in stem cells, CSCs and progenitor cells, this enzyme has been successfully used to identify and isolate cell populations with CSC properties from several tumor origins. ALDH is allegedly involved in cell differentiation through its product, retinoic acid. However, direct or indirect ALDH inhibition, using specific inhibitors or retinoic acid, has shown a reduction in ALDH activity, along with the loss of stem cell traits, reduction of cell proliferation, invasion, and drug sensitization. For these reasons, ALDH and retinoic acid are promising therapeutic targets. This review summarizes the current evidence for ALDH as a CSCs marker in solid tumors, as well as current knowledge about the functional roles of ALDH in CSCs. We discuss the controversy of ALDH activity to maintain CSC stemness, or conversely, to promote cell differentiation. Finally, we review the advances in using ALDH inhibitors as anti-cancer drugs.

Aldehyde dehydrogenase serves as a biomarker for worse survival profiles in ovarian cancer patients: an updated meta-analysis.

BACKGROUND: The purpose of this comprehensive meta-analysis was to assess the association of aldehyde dehydrogenase (ALDH) expression with overall survival (OS) and disease-free survival (DFS)/progression-free survival (PFS) in ovarian cancer patients. METHODS: Systematic searches of Pubmed databases was performed to identify relevant literature published before February 28, 2018. A total of 14 studies (13 articles) with 2210 ovarian cancer patients were pooled. All included studies were performed by using Immunohistochemistry (IHC) for detection of ALDH expression. Hazard ratio (HR) and 95% confidence interval (CI) were extracted from included studies to evaluate the correlation of ALDH expression with OS and DFS/PFS. RESULTS: High expression of ALDH was associated with worse OS (HR: 1.43; 95% CI: 1.18-1.73) and poor DFS/PFS (HR: 1.55, 95% CI: 1.12-2.14). No evidence of publication bias was observed in OS (Begg's test, P = 0.113; Egger's test, P = 0.355) and DFS/PFS (Begg's test, P = 0.655; Egger's test, P = 0.189) in ovarian cancer patients. The subgroup of studies with cut-off value of low expression showed that high expression of ALDH was correlated with poor OS (HR: 1.36; 95% CI: 1.14-1.62) and DFS/PFS (HR: 1.79; 95% CI: 1.45-2.20) in ovarian cancer patients, with no observed heterogeneity (OS: I2 = 0%, P = 0.45; DFS/PFS: I2 = 0%, P = 0.55). CONCLUSION: In conclusion, high expression of ALDH is correlated with worse survival profiles in ovarian cancer patients, indicating that ALDH might act as a potential molecular biomarker for prognosis of ovarian cancer.

Effect of Tissue Factor on Colorectal Cancer Stem Cells.

BACKGROUND/AIM: Tissue factor (TF) expression increases cancer stem cell (CSC) activity in breast and lung cancer. There are ongoing studies focused on targeting CSCs via anti-TF treatment, for breast and lung cancer therapy. Herein, the aim was to determine whether targeting TF could have an anti-CSC therapeutic role in colorectal cancer (CRC). MATERIALS AND METHODS: Evaluation of colonosphere-forming efficiency (CFE) and aldehyde dehydrogenase (ALDH) expression level was used to quantify CSC activity in two CRC cell lines, after TF knockdown (TFKD) or TF over-expression (TFOE). RESULTS: TFKD resulted in increased levels of ALDH in SW620 (1.31±0.04-fold, p<0.001) and DLD-1 (1.63±0.14-fold, p=0.04) cells. CFE was increased in SW620 (1.21±0.23% vs. 2.03±0.29%, p=0.01) and DLD-1 (0.41±0.12% vs. 0.68±0.9%, p=0.01) cells. Conversely, TFOE decreased ALDH expression (0.72±0.04-fold, p=0.001) and CFE (0.33±0.05% vs. 0.66±0.14%, p=0.006) in DLD-1, but had no impact on SW620 cells. CONCLUSION: In the examined CRC cell lines, TF expression was inversely related to CSC activity suggesting that anti-TF therapies may not have a role in CRC treatment.

Co-detection of ALDH1A1, ABCG2, ALCAM and CD133 in three A549 subpopulations at the single cell level by one-step digital RT-PCR.

Cancer stem-like cells (CSCs) displaying the properties of normal stem cells have become the main culprit associated with cancer transportation and recurrence. As of now, various CSC functions and marker genes have been identified due to the heterogeneity of cancer, such as aldehyde dehydrogenase (ALDH), the second member of the ABC transporter G-subfamily (ABCG2), activated leukocyte cell adhesion molecule (ALCAM) and CD133. To investigate these markers, most conventional approaches are bulk-based strategies, which may veil the disparity of single cells' gene expression. In this study, one-step digital RT-PCR at the single cell level was developed to co-determine the expression of ALDH1A1, ABCG2, ALCAM and CD133 genes in A549 cancer stem cells that perform high ALDH activities (ALDH+ A549 cells), as well as in ALDH- A549 cells and A549 cells, with 36, 20 and 20 cell samples each. The results demonstrated that, when compared to single ALDH- or A549 cells, the majority of single ALDH+ A549 cells displayed a 1.5- and 2.0-fold increase in the gene expression of ALDH1A1 and ALCAM (P < 0.001), respectively. However, for ABCG2 and CD133, there was no significant difference (P > 0.05), which means that they are not appropriate as co-indicated markers to identify ALDH+ A549 cells. Conclusively, as a single cell level approach, one-step digital RT-PCR has potential in exploring efficient co-detection markers for the classification and identification of CSCs.

Aldehyde dehydrogenase 1B1: a novel immunohistological marker for colorectal cancer.

BACKGROUND: Aldehyde dehydrogenase (ALDH) 1A1 is an immunohistological biomarker of various solid tumours, but has not been successfully proved as a colorectal cancer (CRC) marker. We recently reported that ALDH1B1, which has functional roles in tumourigenesis, may be a better CRC marker than ALDH1A1. METHODS: Human CRC explants and cell lines were analysed to identify candidate CRC markers from eight ALDH isozymes including ALDH1A1 and ALDH1B1. A tissue microarray, including paired specimens of normal and tumour tissues, was subsequently analysed to determine if candidate ALDHs could distinguish CRC from normal tissue. RESULTS: Based on mRNA analysis, ALDH1B1 and ALDH2 were selected as suitable candidates. These were strongly and regularly expressed in tumour tissue and cell lines, including highly tumourigenic cell populations (ALDH+CD44+ cells), while other ALDHs, including ALDH1A1, showed differential or low expression. No genetic alteration of ALDH1B1 in CRC was suggested by the relationships between mRNA and protein levels/enzymatic activities, and cDNA sequences of CRC cell lines. Tissue microarray findings showed that ALDH1B1, but not ALDH2, could distinguish CRC from normal tissue. Furthermore, ratios of ALDH1B1 to ALDH1A1 or ALDH2 were found to be powerful CRC indicators. CONCLUSIONS: These results suggest that ALDH1B1 is a novel human CRC biomarker.

Cancer stem cell mediated acquired chemoresistance in head and neck cancer can be abrogated by aldehyde dehydrogenase 1 A1 inhibition.

Chemoresistance leading to disease relapse is one of the major challenges to improve outcome in head and neck cancers. Cancer Stem Cells (CSCs) are increasingly being implicated in chemotherapy resistance, this study investigates the correlation between CSC behavior and acquired drug resistance in in vitro cell line models. Cell lines resistant to Cisplatin (Cal-27 CisR, Hep-2 CisR) and 5FU (Cal-27 5FUR) with high Resistance Indices (RI) were generated (RI ≥ 3) by short-term treatment of head and neck squamous cell carcinoma (HNSCC) cell lines with chemotherapeutic drugs (Cisplatin, Docetaxel, 5FU), using a dose-incremental strategy. The cell lines (Cal-27 DoxR, Hep-2 DoxR, Hep-2 5FUR) that showed low RI, nevertheless had a high cross resistance to Cisplatin/5FU (P < 0.05). Cal-27 CisR and DoxR showed 12-14% enrichment of CD44+ cells, while CisR/5FUR showed 4-6% increase in ALDH1A1+ cells as compared to parental cells (P < 0.05). Increased expression of stem cell markers (CD44, CD133, NOTCH1, ALDH1A1, OCT4, SOX2) in these cell lines, correlated with enhanced spheroid/colony formation, migratory potential, and increased in vivo tumor burden (P < 0.05). Inhibition of ALDH1A1 in Cal-27 CisR led to down regulation of the CSC markers, reduction in migratory, self-renewal and tumorigenic potential (P < 0.05) accompanied by an induction of sensitivity to Cisplatin (P < 0.05). Further, ex vivo treatment of explants (n = 4) from HNSCC patients with the inhibitor (NCT-501) in combination with Cisplatin showed a significant decrease in proliferating cells as compared to individual treatment (P = 0.001). This study hence suggests an ALDH1A1-driven, CSC-mediated mechanism in acquired drug resistance of HNSCC, which may have therapeutic implications. © 2016 Wiley Periodicals, Inc.

EGFL6 Regulates the Asymmetric Division, Maintenance, and Metastasis of ALDH+ Ovarian Cancer Cells.

Little is known about the factors that regulate the asymmetric division of cancer stem-like cells (CSC). Here, we demonstrate that EGFL6, a stem cell regulatory factor expressed in ovarian tumor cells and vasculature, regulates ALDH+ ovarian CSC. EGFL6 signaled at least in part via the oncoprotein SHP2 with concomitant activation of ERK. EGFL6 signaling promoted the migration and asymmetric division of ALDH+ ovarian CSC. As such, EGFL6 increased not only tumor growth but also metastasis. Silencing of EGFL6 or SHP2 limited numbers of ALDH+ cells and reduced tumor growth, supporting a critical role for EGFL6/SHP2 in ALDH+ cell maintenance. Notably, systemic administration of an EGFL6-neutralizing antibody we generated restricted tumor growth and metastasis, specifically blocking ovarian cancer cell recruitment to the ovary. Together, our results offer a preclinical proof of concept for EGFL6 as a novel therapeutic target for the treatment of ovarian cancer. Cancer Res; 76(21); 6396-409. ©2016 AACR.

Re-purposing of curcumin as an anti-metastatic agent for the treatment of epithelial ovarian cancer: in vitro model using cancer stem cell enriched ovarian cancer spheroids.

Malignant epithelial ovarian cancer (EOC) spheroids high frequently are detected in the malignant ascites of the patients with the extensive peritoneal metastasis of ovarian cancer, which represent a significant obstacle to efficacious treatment. Clinical data also suggested that EOC spheroids play a putative role in the development of chemoresistance. Since standard surgery and conventional chemotherapy is the only available treatment, there is an urgent need to identify a more effective therapeutic strategy. Recent studies demonstrated that curcumin exerts an anticancer effect in a variety of human cancers including ovarian cancer. This study evaluates anti-peritoneal metastasis and chemoresistance of curcumin related to the EOC spheroids. In this study, we confirm that the high invasive EOC cells forming the spheroids express a high level of a cancer stem cell (CSC) marker, aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which was significantly down-regulated by curcumin treatment. Curcumin treatment markedly enhances the sensitivity of EOC spheroids to cisplatin in a dose-dependent manner. Our experiments provided evidence that curcumin could abolish the sphere-forming capacity of EOC cells in a dose-dependent manner. Moreover, curcumin substantially suppressed the growth of the pre-existed EOC spheroids, inhibited the adhesion of EOC spheroids to ECM as well as the invasion of EOC spheroids to the mesothelial monolayers. We propose to re-purpose curcumin as anti-metastatic and chemoresistant agent for EOC management in combination with conventional regimen. Further preclinical studies are necessary to validate the anti-cancer effect of curcumin in patients with EOC.

Aldehyde dehydrogenase is used by cancer cells for energy metabolism.

We found that non-small-cell lung cancer (NSCLC) cells express high levels of multiple aldehyde dehydrogenase (ALDH) isoforms via an informatics analysis of metabolic enzymes in NSCLC and immunohistochemical staining of NSCLC clinical tumor samples. Using a multiple reaction-monitoring mass spectrometry analysis, we found that multiple ALDH isozymes were generally abundant in NSCLC cells compared with their levels in normal IMR-90 human lung cells. As a result of the catalytic reaction mediated by ALDH, NADH is produced as a by-product from the conversion of aldehyde to carboxylic acid. We hypothesized that the NADH produced by ALDH may be a reliable energy source for ATP production in NSCLC. This study revealed that NADH production by ALDH contributes significantly to ATP production in NSCLC. Furthermore, gossypol, a pan-ALDH inhibitor, markedly reduced the level of ATP. Gossypol combined with phenformin synergistically reduced the ATP levels, which efficiently induced cell death following cell cycle arrest.

Aldehyde dehydrogenase isoform 1 (ALDH1) expression as a predictor of radiosensitivity in laryngeal cancer

Background: Aldehyde dehydrogenase isoform 1 (ALDH1) has been shown to be a marker of cancer stem cells (CSCs). These stem cells may be responsible for tumour perpetuation as well as local and distant invasion. Several studies have shown that CSCs are more chemoradiotherapy (CRT)-resistant and may be responsible for tumour recurrence. Other studies, in contrast, have found ALDH1 expression to be indicative of a better prognosis. Methods: We retrospectively evaluated 84 patients diagnosed and treated for laryngeal cancer between 2006 and 2011. All patients underwent curative-intent radiotherapy or CRT at our institution. 57 of the 84 tumour samples contained sufficient material for ALDH1 assessment. Results: ALDH1 expression was detected in 17.5 % (10/ 57) of the tissue samples. None of the tumours from stage I patients tested positive for ALDH1. The relapse rate in ALDH1 + patients was 10 versus 51.2 % for ALDH1-. No differences in overall survival were observed between the groups; however, disease-free survival was 90 % for the ALDH1 ? group versus 48.9 % for ALDH1- patients (p = 0.034). Conclusion: The patients in this study with ALDH1 ? tumours had better outcomes than their counterparts with ALDH1- tumours. This finding suggests that not all CSCs are resistant to conventional cancer treatments. It may also imply that new methods of correctly identifying these cells are needed (AU)

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Targeting epithelial-mesenchymal transition and cancer stem cells for chemoresistant ovarian cancer.

Chemoresistance is the main challenge for the recurrent ovarian cancer therapy and responsible for treatment failure and unfavorable clinical outcome. Understanding mechanisms of chemoresistance in ovarian cancer would help to predict disease progression, develop new therapies and personalize systemic therapy. In the last decade, accumulating evidence demonstrates that epithelial-mesenchymal transition and cancer stem cells play important roles in ovarian cancer chemoresistance and metastasis. Treatment of epithelial-mesenchymal transition and cancer stem cells holds promise for improving current ovarian cancer therapies and prolonging the survival of recurrent ovarian cancer patients in the future. In this review, we focus on the role of epithelial-mesenchymal transition and cancer stem cells in ovarian cancer chemoresistance and explore the therapeutic implications for developing epithelial-mesenchymal transition and cancer stem cells associated therapies for future ovarian cancer treatment.

Proteomic analysis of soybean root including hypocotyl during recovery from drought stress.

UNLABELLED: Soybean is a nutritionally important crop that exhibits reductions in growth and yield under drought stress. To investigate soybean responses during post-drought recovery, a gel-free proteomic technique was used. Two-day-old soybeans were treated with drought stress for 4days and recovered for 4days. Root including hypocotyl was collected during the drought treatment and recovery stage. Seedling growth was suppressed by drought stress, but recovered following stress removal. The malondialdehyde content increased under drought stress, but decreased during the recovery stage. A total of 792 and 888 proteins were identified from the control and recovering seedlings, respectively. The identified proteins were related to functional categories of stress, hormone metabolism, cell wall, secondary metabolism, and fermentation. Cluster analysis indicated that abundances of peroxidase and aldehyde dehydrogenase were highly changed in the seedlings during the post-drought recovery. The activity of peroxidase decreased under drought conditions, but increased during recovery. In contrast, the activity of aldehyde dehydrogenase was increased in response to drought stress, but decreased during the recovery stage. These results suggest that peroxidase and aldehyde dehydrogenase play key roles in post-drought recovery in soybean by scavenging toxic reactive oxygen species and reducing the load of harmful aldehydes. BIOLOGICAL SIGNIFICANCE: Post-drought recovery response mechanisms in soybean root including hypocotyl were analyzed using gel-free proteomic technique. A total of 643 common proteins between control and drought-stressed soybeans changed significantly in abundance over time. The proteins that changed during post-drought recovery were assigned to protein, stress, hormone metabolism, secondary metabolism, cell wall, redox, and glycolysis categories. The analysis revealed that peroxidase and aldehyde dehydrogenase were increased in protein abundance under drought stress. The enzyme activity of peroxidase decreased under drought but increased during recovery. The activity of aldehyde dehydrogenase was increased under drought stress but decreased during recovery stage. Peroxidase and aldehyde dehydrogenase reduce the toxic reactive oxygen species and aldehydes from the plant, respectively, and help to recover from drought stress. The study provides information about post-drought recovery mechanism in soybean.

Cancer stem cells, metabolism, and therapeutic significance.

Cancer stem cells (CSCs) have attracted much attention of the research community in the recent years. Due to their highly tumorigenic and drug-resistant properties, CSCs represent important targets for developing novel anticancer agents and therapeutic strategies. CSCs were first described in hematopoietic malignancies and subsequently identified in various types of solid tumors including brain, breast, lung, colon, melanoma, and ovarian cancer. CSCs possess special biological properties including long-term self-renewal capacity, multi-lineage differentiation, and resistance to conventional chemotherapy and radiotherapy. As such, CSCs are considered as a major source of residual disease after therapy leading to disease occurrence. Thus, it is very important to understand the cellular survival mechanisms specific to CSCs and accordingly develop effective therapeutic approaches to eliminate this subpopulation of cancer cells in order to improve the treatment outcome of cancer patients. Possible therapeutic strategies against CSCs include targeting the self-renewal pathways of CSCs, interrupting the interaction between CSCs and their microenvironment, and exploiting the unique metabolic properties of CSCs. In this review article, we will provide an overview of the biological characteristics of CSCs, with a particular focus on their metabolic properties and potential therapeutic strategies to eliminate CSCs.

Chemotherapy induces stemness in osteosarcoma cells through activation of Wnt/ß-catenin signaling.

Development of resistance represents a major drawback in osteosarcoma treatment, despite improvements in overall survival. Treatment failure and tumor progression have been attributed to pre-existing drug-resistant clones commonly assigned to a cancer stem-like phenotype. Evidence suggests that non stem-like cells, when submitted to certain microenvironmental stimuli, can acquire a stemness phenotype thereby strengthening their capacity to handle with stressful conditions. Here, using osteosarcoma cell lines and a mouse xenograft model, we show that exposure to conventional chemotherapeutics induces a phenotypic cell transition toward a stem-like phenotype. This associates with activation of Wnt/ß-catenin signaling, up-regulation of pluripotency factors and detoxification systems (ABC transporters and Aldefluor activity) that ultimately leads to chemotherapy failure. Wnt/ß-catenin inhibition combined with doxorubicin, in the MNNG-HOS cells, prevented the up-regulation of factors linked to transition into a stem-like state and can be envisaged as a way to overcome adaptive resistance. Finally, the analysis of the public R2 database, containing microarray data information from diverse osteosarcoma tissues, revealed a correlation between expression of stemness markers and a worse response to chemotherapy, which provides evidence for drug-induced phenotypic stem cell state transitions in osteosarcoma.

TMPRSS4 induces cancer stem cell-like properties in lung cancer cells and correlates with ALDH expression in NSCLC patients.

Metastasis involves a series of changes in cancer cells that promote their escape from the primary tumor and colonization to a new organ. This process is related to the transition from an epithelial to a mesenchymal phenotype (EMT). Recently, some authors have shown that migratory cells with an EMT phenotype share properties of cancer stem cells (CSCs), which allow them to form a new tumor mass. The type II transmembrane serine protease TMPRSS4 is highly expressed in some solid tumors, promotes metastasis and confers EMT features to cancer cells. We hypothesized that TMPRSS4 could also provide CSC properties. Overexpression of TMPRSS4 reduces E-cadherin and induces N-cadherin and vimentin in A549 lung cancer cells, supporting an EMT phenotype. These changes are accompanied by enhanced migration, invasion and tumorigenicity in vivo. TMPRSS4 expression was highly increased in a panel of lung cancer cells cultured as tumorspheres (a typical assay to enrich for CSCs). H358 and H441 cells with knocked-down TMPRSS4 levels were significantly less able to form primary and secondary tumorspheres than control cells. Moreover, they showed a lower proportion of ALDH+ cells (examined by FACS analysis) and lower expression of some CSC markers than controls. A549 cells overexpressing TMPRSS4 conferred the opposite phenotype and were also more sensitive to the CSC-targeted drug salinomycin than control cells, but were more resistant to regular chemotherapeutic drugs (cisplatin, gemcitabine and 5-fluorouracil). Analysis of 70 NSCLC samples from patients revealed a very significant correlation between TMPRSS4 expression and CSC markers ALDH (p = 0.0018) and OCT4 (p = 0.0004), suggesting that TMPRSS4 is associated with a CSC phenotype in patients' tumors. These results show that TMPRSS4, in addition to inducing EMT, can also promote CSC features in lung cancer; therefore, CSC-targeting drugs could be an appropriate treatment for TMPRSS4+ tumors.