Phase I trial of human umbilical cord-derived mesenchymal stem cells for treatment of severe bronchopulmonary dysplasia.

Severe bronchopulmonary dysplasia (BPD) is a chronic lung disorder that primarily affects premature babies with extremely low birth weight and involves in multiple organ system; no effective pharmacotherapy for this disease exists, and mortality remains high. Based on the evidence from previous preclinical studies and phase I clinical trials, this study aims to test the safety of intravenous application of a single dose of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) in patients with severe BPD. The Mesenchymal Stem cells for Bronchopulmonary Dysplasia Treatment (MSBDT) trial is a single center, open-label, dose-escalation phase I clinical trial. Severe BPD patients were enrolled in Children Hospital of Chongqing Medical University, Chongqing, China. The first six patients were treated with low-dose hUC-MSCs (1 × 106 cells/kg) and the next seven patients were treated with high-dose hUC-MSCs (5 × 106 cells/kg). This study is registered with ClinicalTrials.gov, number NCT03558334. No prespecified infusion-associated adverse events, immediate complication, respiratory or cardiovascular compromise were observed during infusion and 24 h after infusion. No significant changes in safety laboratory values were observed. One death event occurred in the low-dose group on study day 10, and one death event occurred in the high-dose group on study day 24, while, after review in detail, the two cases are not believed to be infusion-associated events. In conclusion, intravenous application of a single dose of hUC-MSCs was tolerated in thirteen patients with severe BPD.

The myo-inositol biosynthesis rate-limiting enzyme ISYNA1 suppresses the stemness of ovarian cancer via Notch1 pathway.

Cancer stem cells (CSCs) play a central role in ovarian cancer (OC), understanding regulatory mechanisms governing their stemness is critical. Here, we report ISYNA1, the rate-limiting enzyme in myo-inositol biosynthesis, as a suppressor of OC regulating cancer stemness. We identified ISYNA1 as a differentially expressed gene in normal ovary and ovarian cancer tissues, as well as OC cells and OCSCs. Low ISYNA1 expression correlated with poor prognosis in OC patients. In addition, ISYNA1 was negatively correlated with cancer stem cell (CSC) markers, and ISYNA1-related pathways were enriched in Wnt, Notch, and other critical cancer pathways. ISYNA1 deficiency promoted OC cell growth, migration, and invasion ability in vitro and in vivo. Knockdown of ISYNA1 increased stemness of OC cells, including self-renewal, CSC markers expression, ALDH activity, and proportion of CD44+/CD117+ CSCs. Conversely, ectopic overexpression of ISYNA1 suppresses cell proliferation, migration, invasion and stemness of OC cells. Mechanistically, ISYNA1 inhibits OC stemness by regulating myo-inositol to suppress Notch1 signaling. In summary, these data provide evidence that ISYNA1 act as a tumor suppressor in OC and a regulator of stemness, providing insight into potentially targetable pathways for ovarian cancer therapy.

Zinc finger and SCAN domain containing 1, ZSCAN1, is a novel stemness-related tumor suppressor and transcriptional repressor in breast cancer targeting TAZ.

Introduction: Cancer stem cells (CSCs) targeted therapy holds the potential for improving cancer management; identification of stemness-related genes in CSCs is necessary for its development. Methods: The Cancer Genome Atlas (TCGA) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets were used for survival analysis. ZSCAN1 correlated genes was identified by Spearman correlation analysis. Breast cancer stem-like cells (BCSLCs) were isolated by sorting CD44+CD24- cells from suspension cultured breast cancer (BC) spheroids. The sphere-forming capacity and sphere- and tumor-initiating capacities were determined by sphere formation and limiting dilution assays. The relative gene expression was determined by qRT-PCR, western blot. Lentivirus system was used for gene manipulation. Nuclear run-on assay was employed to examine the levels of nascent mRNAs. DNA pull-down and Chromatin immunoprecipitation (ChIP) assays were used for determining the interaction between protein and target DNA fragments. Luciferase reporter assay was used for evaluating the activity of the promoter. Results and discussion: ZSCAN1 is aberrantly suppressed in BC, and this suppression indicates a bad prognosis. Ectopic expression of ZSCAN1 inhibited the proliferation, clonogenicity, and tumorigenicity of BC cells. ZSCAN1-overexpressing BCSLCs exhibited weakened stemness properties. Normal human mammary epithelial (HMLE) cells with ZSCAN1 depletion exhibited enhanced stemness properties. Mechanistic studies showed that ZSCAN1 directly binds to -951 ~ -925bp region of WWTR1 (encodes TAZ) promoter, inhibits WWTR1 transcription, thereby inhibiting the stemness of BCSCs. Our work thus revealed ZSCAN1 as a novel stemness-related tumor suppressor and transcriptional repressor in BC.

Nano-Econazole Enhanced PD-L1 Checkpoint Blockade for Synergistic Antitumor Immunotherapy against Pancreatic Ductal Adenocarcinoma.

Insufficienct T lymphocyte infiltration and unresponsiveness to immune checkpoint blockade therapy are still major difficulties for the clinical treatment of pancreatic ductal adenocarcinoma (PDAC). Although econazole has shown promise in inhibiting PDAC growth, its poor bioavailability and water solubility limit its potential as a clinical therapy for PDAC. Furthermore, the synergistic role of econazole and biliverdin in immune checkpoint blockade therapy in PDAC remains elusive and challenging. Herein, a chemo-phototherapy nanoplatform is designed by which econazole and biliverdin can be co-assembled (defined as FBE NPs), which significantly improve the poor water solubility of econazole and enhance the efficacy of PD-L1 checkpoint blockade therapy against PDAC. Mechanistically, econazole and biliverdin are directly released into the acidic cancer microenvironment, to activate immunogenic cell death via biliverdin-induced PTT/PDT and boost the immunotherapeutic response of PD-L1 blockade. In addition, econazole simultaneously enhances PD-L1 expression to sensitize anti-PD-L1 therapy, leading to suppression of distant tumors, long-term immune memory effects, improved dendritic cell maturation, and tumor infiltration of CD8+ T lymphocytes. The combined FBE NPs and α-PDL1 show synergistic antitumor efficacy. Collectively, FBE NPs show excellent biosafety and antitumor efficacy by combining chemo-phototherapy with PD-L1 blockade, which has promising potential in a precision medicine approach as a PDAC treatment strategy.

Luteolin directly binds to KDM4C and attenuates ovarian cancer stemness via epigenetic suppression of PPP2CA/YAP axis.

Long-term use of low-toxic natural products holds the promise for eradicating cancer stem cells. In this study, we report that luteolin, a natural flavonoid, attenuates the stemness of ovarian cancer stem cells (OCSCs) by directly binding to KDM4C and epigenetic suppression of PPP2CA/YAP axis. Ovarian cancer stem like cells (OCSLCs) isolated by suspension culture and CD133 + ALDH+ cell sorting was employed as OCSCs model. The maximal non-toxic dose of luteolin suppressed stemness properties, including sphere-forming capacity, the expression of OCSCs markers, sphere-initiating and tumor-initiating capacities, as well as the percentage of CD133 + ALDH+ cells of OCSLCs. Mechanistic study showed that luteolin directly binds to KDM4C, blocks KDM4C-induced histone demethylation of PPP2CA promoter, inhibits PPP2CA transcription and PPP2CA-mediated YAP dephosphorylation, thereby attenuating YAP activity and the stemness of OCSLCs. Furthermore, luteolin sensitized OCSLCs to traditional chemotherapeutic drugs in vitro and in vivo. In summary, our work revealed the direct target of luteolin and the underlying mechanism of the inhibitory effect of luteolin on the stemness of OCSCs. This finding thus suggests a novel therapeutic strategy for eradicating human OCSCs driven by KDM4C.

Protein degradation: expanding the toolbox to restrain cancer drug resistance.

Despite significant progress in clinical management, drug resistance remains a major obstacle. Recent research based on protein degradation to restrain drug resistance has attracted wide attention, and several therapeutic strategies such as inhibition of proteasome with bortezomib and proteolysis-targeting chimeric have been developed. Compared with intervention at the transcriptional level, targeting the degradation process seems to be a more rapid and direct strategy. Proteasomal proteolysis and lysosomal proteolysis are the most critical quality control systems responsible for the degradation of proteins or organelles. Although proteasomal and lysosomal inhibitors (e.g., bortezomib and chloroquine) have achieved certain improvements in some clinical application scenarios, their routine application in practice is still a long way off, which is due to the lack of precise targeting capabilities and inevitable side effects. In-depth studies on the regulatory mechanism of critical protein degradation regulators, including E3 ubiquitin ligases, deubiquitylating enzymes (DUBs), and chaperones, are expected to provide precise clues for developing targeting strategies and reducing side effects. Here, we discuss the underlying mechanisms of protein degradation in regulating drug efflux, drug metabolism, DNA repair, drug target alteration, downstream bypass signaling, sustaining of stemness, and tumor microenvironment remodeling to delineate the functional roles of protein degradation in drug resistance. We also highlight specific E3 ligases, DUBs, and chaperones, discussing possible strategies modulating protein degradation to target cancer drug resistance. A systematic summary of the molecular basis by which protein degradation regulates tumor drug resistance will help facilitate the development of appropriate clinical strategies.

SOX2 maintains the stemness of retinoblastoma stem-like cells through Hippo/YAP signaling pathway.

PURPOSE: To explore the mechanisms underlying stemness maintenance of retinoblastoma (RB) stem cells (RSCs). METHODS: The retinoblastoma stem-like cells (RSLCs) were isolated by single cell cloning in combination of examination of sphere-forming capacities. The stemness of the cells were characterized by the sphere-forming capacity and the expression levels of RSCs markers. Gene manipulation was performed by lentivirus system. Transcriptional regulation was identified by qRT-PCR, luciferase reporter, nuclear run-on and DNA pull-down assay. Spearman analysis was employed for correlation analysis of genes in tumor tissues of RB patients. RESULTS: The isolated RSLCs exhibited enhanced sphere-forming capacity and constantly higher levels of CD44, ABCG2, SOX2 and PAX6, but not CD133. SOX2 positively regulated the stemness of RSLCs. SOX2 directly binds to the promoters of WWTR1 and YAP and transcriptionally activates WWTR1 and YAP. Knockdown of WWTR1 or YAP partially abolished the effect of SOX2 on the stemness of RSLCs. CONCLUSIONS: SOX2, as a key deriver, maintains RB stemness by activating Hippo/YAP signaling. Inhibition of Hippo/YAP signaling would be an effective strategy for human RB caused by SOX2 upregulation.

Machine learning-based prediction of survival prognosis in cervical cancer.

BACKGROUND: Accurately forecasting the prognosis could improve cervical cancer management, however, the currently used clinical features are difficult to provide enough information. The aim of this study is to improve forecasting capability by developing a miRNAs-based machine learning survival prediction model. RESULTS: The expression characteristics of miRNAs were chosen as features for model development. The cervical cancer miRNA expression data was obtained from The Cancer Genome Atlas database. Preprocessing, including unquantified data removal, missing value imputation, samples normalization, log transformation, and feature scaling, was performed. In total, 42 survival-related miRNAs were identified by Cox Proportional-Hazards analysis. The patients were optimally clustered into four groups with three different 5-years survival outcome (≥ 90%, ≈ 65%, ≤ 40%) by K-means clustering algorithm base on top 10 survival-related miRNAs. According to the K-means clustering result, a prediction model with high performance was established. The pathways analysis indicated that the miRNAs used play roles involved in the regulation of cancer stem cells. CONCLUSION: A miRNAs-based machine learning cervical cancer survival prediction model was developed that robustly stratifies cervical cancer patients into high survival rate (5-years survival rate ≥ 90%), moderate survival rate (5-years survival rate ≈ 65%), and low survival rate (5-years survival rate ≤ 40%).

SOX4 maintains the stemness of cancer cells via transcriptionally enhancing HDAC1 revealed by comparative proteomics study.

BACKGROUND: Cancer stem cells (CSCs) are the root of human cancer development and the major cause of treatment failure. Aberrant elevation of SOX4, a member of SOX (SRY-related HMG-box) family transcription factors, has been identified in many types of human cancer and promotes cancer development. However, the role of SOX4 in CSCs, especially at a proteome-wide level, has remained elusive. The aim of this study is to investigate the effect of SOX4 on the stemness of CSCs and reveal the underlying mechanisms by identification of SOX4-induced proteome changes through proteomics study. RESULTS: Overexpression of SOX4 promotes sphere formation and self-renewal of colorectal cancer cells in vitro and in vivo and elevates the expression levels of CSCs markers. Through iTRAQ-based quantitative proteomics analysis, 215 differentially expressed proteins (128 upregulated, 87 downregulated) in SOX4-overexpressing HCT-116 spheres were identified. The bioinformatic analysis highlighted the importance of HDAC1 as the fundamental roles of its impacted pathways in stem cell maintenance, including Wnt, Notch, cell cycle, and transcriptional misregulation in cancer. The mechanistic study showed that SOX4 directly binds to the promoter of HDAC1, promotes HDAC1 transcription, thereby supporting the stemness of colorectal cancer cells. HDAC1 hallmarks colorectal cancer stem cells and depletion of HDAC1 abolished the stimulatory effect of SOX4. Furthermore, SOX4-HDAC1 axis is conserved in multiple types of cancer. CONCLUSIONS: The results of this study reveal SOX4-induced proteome changes in HCT-116 spheres and demonstrates that transcriptional activation of HDAC1 is the primary mechanism underlying SOX4 maintaining CSCs. This finding suggests that HDAC1 is a potential drug target for eradicating SOX4-driven human CSCs.

Aberrant methylation of GADD45A is associated with decreased radiosensitivity in cervical cancer through the PI3K/AKT signaling pathway.

Epigenetic inactivation of GADD45A is a common occurrence in different types of cancer. However, little is known regarding its association with radiosensitivity in cervical cancer (CC). Thus, the present study aimed to investigate the association between aberrant GADD45A methylation and radiosensitivity in CC. SiHa, HeLa and CaSki CC cells were treated with 5-azacytidine (5-azaC), with or without irradiation. The expression levels of GADD45A and AKT related molecules were detected via reverse transcription-quantitative PCR and western blot analyses. The methylation status of GADD45A was assessed via methylation-specific PCR and cell proliferation assays, while clonogenic assays and flow cytometric analysis were performed to assess the function of the genes (GADD45A and AKT) in the CC cell lines. The results demonstrated that methylation of GADD45A was significantly higher in the radioresistant tissues (63.16%) compared with the radiosensitive samples (33.33%). In addition, the surviving fraction of SiHa cells following irradiation with 2 Gy was demonstrated to be highest amongst the three CC cells (CaSki, 57±9.5%; HeLa, 70±4% and SiHa, 75±10%). The survival rate of SiHa cells following treatment with 5-azaC and ionizing radiation (IR) significantly decreased as the radiation dose increased, compared with treatment with IR alone. Following overexpression of GADD45A or treatment with 5-azaC, the radiosensitivity of SiHa cells significantly increased compared with both the control vector and PBS treated groups. In addition, the AKT inhibitor, MK-2206, increased the radiosensitivity of SiHa cells. Notably, aberrant methylation of GADD45A was associated with decreased radiosensitivity in CC, and the PI3K/AKT signaling pathway was essential for radioresistance, which was mediated through downregulation of GADD45A.

SKP1 promotes YAP-mediated colorectal cancer stemness via suppressing RASSF1.

BACKGROUND: Cancer stem cells (CSCs) have been recognized as an important drug target, however, the underlying mechanisms have not been fully understood. SKP1 is a traditional drug target for cancer therapy, while, whether SKP1 promotes colorectal cancer (CRC) stem cells (CRC-SCs) and the underlying mechanisms have remained elusive. METHODS: Human CRC cell lines and primary human CRC cells were used in this study. Gene manipulation was performed by lentivirus system. The mRNA and protein levels of target genes were examined by qRT-PCR and western blot. The sphere-forming and in vitro migration capacities were determined by sphere formation and transwell assay. The self-renewal was determined by limiting dilution assay. The tumorigenicity and metastasis of cancer cells were examined by xenograft model. The promoter activity was examined by luciferase reporter assay. Nuclear run-on and Chromatin immunoprecipitation-PCR (ChIP-PCR) assay were employed to examine the transcription and protein-DNA interaction. Co-immunoprecipitation assay was used to test protein-protein interaction. The relationship between gene expression and survival was analyzed by Kaplan-meier analysis. The correlation between two genes was analyzed by Spearman analysis. Data are represented as mean ± SD and the significance was determined by Student's t test. RESULTS: SKP1 was upregulated in CRC-SCs and predicted poor prognosis of colon cancer patients. Overexpression of SKP1 promoted the stemness of CRC cells reflected by increased sphere-forming, migration and self-renewal capacities as well as the expression of CSCs markers. In contrast, SKP1 depletion produced the opposite effects. SKP1 strengthened YAP activity and knockdown of YAP abolished the effect of SKP1 on the stemness of CRC cells. SKP1 suppressed RASSF1 at both mRNA and protein level. Overexpression of RASSF1 abolished the effect of SKP1 on YAP activity and CRC stemness. CONCLUSION: Our results demonstrated that SKP1 suppresses RASSF1 at both mRNA and protein level, attenuates Hippo signaling, activates YAP, and thereby promoting the stemness of CRC cells.

Baicalin Attenuates YAP Activity to Suppress Ovarian Cancer Stemness.

PURPOSE: This study aims to reveal the mechanism underlying baicalin-suppressing ovarian cancer stemness. METHODS: OVCAR-3 and the primary ovarian cancer cells were used for cell model. The ovarian cancer stem cells were isolated by suspension culture. Cell viability and clonogenicity were examined by CCK-8 assay and colony formation assay. The self-renewal of the cells was evaluated by the determination of sphere-forming capacity and the frequency of in vitro sphere-forming and in vivo tumor-initiating cells. The mRNA and protein levels were relatively quantified by qRT-PCR and Western blot. The transcription regulation of target genes was tested by luciferase reporter assay and a modified nuclear rn-on qRT-PCR assay. RESULTS: Treatment with a non-toxic dose of baicalin significantly inhibited the spherogenicity of ovarian cancer cells. Moreover, a non-toxic dose of baicalin treatment suppressed the frequency of sphere-forming and tumor-initiating ovarian cancer cells. Furthermore, the expression of ovarian cancer stem cell markers (CD133 and ALDH1A1) was inhibited by a non-toxic dose of baicalin treatment. Baicalin inhibits YAP activity and suppresses RASSF6, a positive regulator of YAP, at the transcriptional level. Overexpression of both YAP and RASSF6 abolished the inhibitory effect of baicalin on the proliferation and stemness of ovarian cancer cells. CONCLUSION: The results in this study demonstrated that baicalin suppresses the stemness of ovarian cancer cells by attenuating YAP activity via inhibiting RASSF6 at the transcriptional level. This finding revealed baicalin as a novel YAP inhibitor that could serve as an anti-cancer drug for eradicating ovarian cancer stem cells.

PTPRU, As A Tumor Suppressor, Inhibits Cancer Stemness By Attenuating Hippo/YAP Signaling Pathway.

BACKGROUND: PTPRU is an important signaling molecule that regulates a variety of cellular processes; however, the role of PTPRU in cancer development has remained elusive. Here, we report that PTPRU serves as a tumor suppressor that inhibits cancer stemness by attenuating Hippo/YAP signaling pathway. METHODS: Primary cancer cells and cell line cells were used in the study. The gene expression data were downloaded from R2 analysis and visualization platform and Kaplan-Meier analysis was performed to study the relationship between survival and PTPRU expression. qRT-PCR and Western blot were employed to study the expression of target genes in tissues and cells. Sphere and colony formation, proliferation, migration activities and the expression of stem cell and EMT markers were employed for characterizing the stemness. Gene manipulation was achieved by lentivirus-mediated gene delivery system. Luciferase reporter gene assay was used to study the transcriptional activity of the promoter, and ChIP-qPCR was employed to study the target binding sequence of the protein. Spearman correlation analysis was performed to study the correlation between two genes. Student's t-test was used for determination of the significance between two experimental groups. RESULTS: PTPRU is downregulated in colorectal and gastric cancer tissues and cancer stem cells. High expression of PTPRU predicts poor prognosis. Overexpression of PTPRU attenuates the stemness of gastric cancer stem cells and knockdown of PTRPU improves the maintenance of the stemness of cancer stem cells. Mechanistic analysis showed that PTPRU inhibits Hippo/YAP signaling by suppressing the expression of YAP in a transcriptional level. Overexpression of YAP restored PTPRU-induced inhibited stemness of gastric cancer stem cells. CONCLUSION: PTPRU serves as a tumor suppressor that inhibits the stemness of cancer stem cell by inhibiting Hippo/YAP signaling pathway.

Mathematical models of amino acid panel for assisting diagnosis of children acute leukemia.

BACKGROUND: The altered concentrations of amino acids were found in the bone marrow or blood of leukemia patients. Metabolomics technology combining mathematical model of biomarkers could be used for assisting the diagnosis of pediatric acute leukemia (AL). METHODS: The concentrations of 17 amino acids was measured by targeted liquid chromatograph-tandem mass spectrometry in periphery blood collected using dried blood spots. After evaluation, the mathematical models were further evaluated by prospective clinical validation cohort for AL diagnosis. RESULTS: The concentrations of 13 in 17 amino acids were statistically different between the periphery blood dried serum dots measured by targeted LC-MS/MS. The receiver operating characteristic analysis for the models of amino acid panel showed that the area under curve for AL diagnosis were 0.848, 0.834 and 0.856 by SVM, RF and XGBoost. The Kappa values in further prospectively evaluated clinical cohort were 0.697, 0.703 and 0.789 (p > 0.05) respectively, and the accuracies for the models were 84.86%, 85.20% and 89.46% respectively with further clinical validation. CONCLUSIONS: The established mathematical model is a faster, cheaper and more convenient way than conventional methods, and no significant difference on the effect of diagnosis comparing with conventional methods. The mathematical model can be clinically useful for assisting pediatric AL diagnosis.

miR-520b Promotes Breast Cancer Stemness Through Hippo/YAP Signaling Pathway.

INTRODUCTION: The breast cancer stem cells contribute to the initiation, progression, recurrence, metastasis as well as resistance of breast cancer. However, the mechanisms underlying the maintenance of breast cancer stemness have not been fully understood. MATERIALS AND METHODS: TCGA and GEO data were used for measuring miR-520b expression in breast cancer tissues. Kaplan-meier analysis was used for determining the relationship between miR-520b expression level and the prognosis of patients. Genetic manipulation was performed by lentivirus system and miR-520b inhibitor was used for knockdown of miR-520b. qRT-PCR and Western blot were employed to determine the mRNA and protein levels, respectively. The stemness and EMT (Epithelial to mesenchymal transition) were assessed by sphere-formation and transwell assay as well as the expression of the related markers. The target genes of miR-520b were identified using the online database starBase V3.0. RESULTS: miR-520b is upregulated in cancer tissues of breast cancer patients and predicts poor prognosis. Upregulation of miR-520b was found in breast cancer stem cells. Ectopic expression of miR-520b promotes the stemness of the breast cancer cells, conversely, depletion of miR-520b attenuates the stemness of these cells. miR-520b positively regulates Hippo/YAP signaling pathway and overexpression of LAST2 abolished the effect of miR-520b on the stemness of breast cancer cells. CONCLUSION: miR-520b promotes the stemness of breast cancer patients by activating Hippo/YAP signaling via targeting LATS2.

NFATC2 is a novel therapeutic target for colorectal cancer stem cells.

BACKGROUND: Colorectal cancer stem cells (CRC-SCs) contribute to the initiation and progression of colorectal cancer (CRC). However, the underlying mechanisms for the propagation of CRC-SCs have remained elusive. PURPOSE: The objective of this study was to study the role of NFATC2 in maintenance of the stemness in CRC-SCs. METHOD: The expression levels of mRNA and protein were determined by qRT-PCR and western-blot, respectively. CRC-SCs were isolated by spheroid formation assay and flowcytometry. The sphere-forming and self-renewal abilities of CRC-SCs were determined by spheroid formation assay. The tumorigenicity of CRC-SCs was determined by cell-derived xenograft model. Gene manipulation was performed by lentivirus-mediated delivery system. RESULTS: We first found that NFATC2 is upregulated in primary CRC-SCs. Overexpression of NFATC2 promotes self-renewal and the expression of stem cell markers of CRC-SCs. Conversely, knockdown of NFATC2 attenuates stem cell-like properties of CRC-SCs. Mechanistic analysis indicated that NFATC2 upregulates the expression of AJUBA, downregulates the phosphorylation level of YAP, and therefore activates the transcriptional activities of YAP and promotes the stemness of CRC-SCs. CONCLUSION: Our findings demonstrate NFATC2 as an oncogene that can promote the stemness of CRC-SCs. This work suggests a novel therapeutic strategy against CRC caused by aberrant expression of NFATC2.

Luteolin attenuates Wnt signaling via upregulation of FZD6 to suppress prostate cancer stemness revealed by comparative proteomics.

The mechanisms underlying luteolin-induced inhibition of prostate cancer (PCa) stemness have remained elusive. Here, we report that luteolin suppresses PCa stemness through Wnt signaling by upregulation of FZD6 (frizzled class receptor 6). Luteolin inhibits PCa cell proliferation, migration, self-renewal as well as the expression of prostate cancer stem cell markers in vitro. Through iTRAQ-based quantitative proteomics study, we identified 208 differentially expressed proteins in luteolin-treated PC-3 cells. Subsequent mechanistic analysis revealed that luteolin inhibits Wnt signaling by transcriptional upregulation of FZD6, and thereby suppressing the stemness of PCa cells. Furthermore, we identified FZD6 as a tumor suppressor that can abolish PCa stemness. In summary, our findings demonstrate that suppression of Wnt signaling by upregulation of FZD6 is a mechanism underlying luteolin-induced inhibition of PCa stemness. Our work suggests a new therapeutic strategy against human prostate cancer caused by aberrant activation of Wnt signaling.

Luteolin inhibits colorectal cancer cell epithelial-to-mesenchymal transition by suppressing CREB1 expression revealed by comparative proteomics study.

The cyclic AMP (cAMP) response element binding protein 1 (CREB1) is a promising target for cancer therapy. Here, we report that luteolin, a natural product, inhibits the expression of CREB1 at the transcriptional level and blocks epithelial-to-mesenchymal transition (EMT) of colorectal cancer cells. Treatment of colorectal cancer cells with luteolin induced mesenchymal-to-epithelial transition, reduced the expressions of mesenchymal markers and inhibited cell mobility in vitro. Through comparison of the proteomic profile of HCT-116 cells with and without luteolin treatment, we identified 366 differentially expressed proteins. Bioinformatics analysis revealed that downregulation of CREB1 plays a central role in this process. Immunoblot analysis verified that the protein levels of CREB1 and its downstream target genes were decreased in luteolin-treated cells. Moreover, forced expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells, suggesting the important role of CREB1 in this process. Furthermore, luciferase reporter assay and examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX) revealed that luteolin inhibits the expression of CREB1 at the transcriptional level. In summary, our results demonstrated that suppressing the expression of CREB1 is crucial in the mechanism-of-action of luteolin inhibiting EMT of colorectal cancer cells. SIGNIFICANCE: It is no doubt that understanding the mechanism-of-action of natural products at the molecular level is important for their translational application. Proteomics is a powerful platform to explore the effects of natural products on the cells. In this study, we compared the proteomic profile of HCT-116 colorectal cancer cells with and without luteolin treatment to investigate the mechanism-of-action of luteolin against colorectal cancer cells. Subsequent bioinformatics analysis revealed that CREB1 could be one of the main targets of luteolin against colorectal cancer cells. Downregulation of CREB1 by luteolin affects glucagon signaling pathway and cAMP signaling pathway. The proteomics findings were verified with mechanistic analyses. We first identified that luteolin decreased the mRNA and protein levels of CREB1 and its downstream target genes. We then found that luteolin inhibits CREB1 expression at the transcriptional level by real-time PCR and luciferase reporter assay which confirmed by examination of the half-life of CREB1 following inhibition of new protein synthesis by cycloheximide (CHX). Finally, we generated CREB1-overexpressing stable cell line and showed that ectopic expression of CREB1 abolished the inhibitory effect of luteolin on colorectal cancer cells and restored the expression levels of CREB1 target genes in colorectal cancer cells, and thereby demonstrated the critical role of CREB1 in the mechanism-of-action of luteolin against colorectal cancer. In summary, we revealed a novel mechanism-of-action of luteolin against colorectal cancer cell by the combination of proteomics discovery and mechanistic analyses.