In vivo CRISPR screens reveal SCAF1 and USP15 as drivers of pancreatic cancer.

Functionally characterizing the genetic alterations that drive pancreatic cancer is a prerequisite for precision medicine. Here, we perform somatic CRISPR/Cas9 mutagenesis screens to assess the transforming potential of 125 recurrently mutated pancreatic cancer genes, which revealed USP15 and SCAF1 as pancreatic tumor suppressors. Mechanistically, we find that USP15 functions in a haploinsufficient manner and that loss of USP15 or SCAF1 leads to reduced inflammatory TNFα, TGF-ß and IL6 responses and increased sensitivity to PARP inhibition and Gemcitabine. Furthermore, we find that loss of SCAF1 leads to the formation of a truncated, inactive USP15 isoform at the expense of full-length USP15, functionally coupling SCAF1 and USP15. Notably, USP15 and SCAF1 alterations are observed in 31% of pancreatic cancer patients. Our results highlight the utility of in vivo CRISPR screens to integrate human cancer genomics and mouse modeling for the discovery of cancer driver genes with potential prognostic and therapeutic implications.

Pharmacodynamical research of extracts and compounds in traditional Chinese medicines for Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease (AD). Currently, there is no cure for PD, and medications can only control the progression of the disease. Various experimental studies have shown the significant efficacy of TCM in treating PD, and combination with western medicine can enhance the effects and reduce toxicity. Thus, exploring effective anti-PD compounds from TCM has become a popular research fields. This review summarizes commonly used TCM extracts and natural products for the treatment of PD, both domestically and internationally. Furthermore, it delves into various mechanisms of TCM in treating PD, such as anti-oxidative stress, anti-inflammatory, anti-apoptotic, improve mitochondrial dysfunction, inhibits α-synuclein (α-Syn) misfolding and aggregation, regulating neurotransmitters, regulates intestinal flora, enhances immunity, and so on. The results reveal that most TCMs exert their neuroprotective effects through anti-inflammatory and anti-oxidative stress actions, thereby slowing down the progression of the disease. These TCM may hold the key to improving PD therapy and have tremendous potential to be developed as novel anti-PD drugs.

Diastereomers of Steroidal Alkaloids with Cytotoxic Activities against Non-Small-Cell Lung Cancer from the Bulbs of Fritillaria sinica.

Eleven new steroidal alkaloids, along with nine known related compounds, were isolated from the bulbs of Fritillaria sinica. Seven pairs of diastereomers were identified, including six and four 20-deoxy cevanine-type steroidal alkaloid diastereomers with molecular weights of 413 and 415, respectively. Structures were elucidated based on spectroscopic data analysis, chemical derivatization, and single-crystal X-ray diffraction analysis. Compounds 5, 9, 11, 12, 16, and 20 exhibited significant in vitro cytotoxic activity against non-small-cell lung cancer with CC50 values from 6.8 ± 3.9 to 12 ± 5 µM.

Undescribed steroidal alkaloids from the bulbs of Fritillaria sinica.

Eight undescribed steroidal alkaloid derivatives, including three cevanine-type isosteroidal alkaloids (two N-oxide glycosides and one D-ring aromatization) (1-3), one verazine-type steroidal alkaloid derivative (4), three solanidine-type steroidal alkaloid glycosides (5-7), and one veratramine-type analogue (8), along with three known compounds (9-11) were isolated from the bulbs of Fritillaria sinica. Their structures were elucidated by comprehensive analysis of spectroscopic data, acidic hydrolysis, and X-ray crystal diffractions. In the in vitro bioassay, the anti-cancer effect, anti-oxidation and anti-inflammatory activities for the isolates were evaluated at a concentration of 10 µM.

Microbial transformation of osthole by human intestinal fungi and anti-osteoporosis activities of its metabolites.

Osthole is one of the major constituents in Cnidium monnieri (L.) Cuss. and possesses anti-osteoporosis activity. In this work, the biotransformation of osthole was performed based on the human intestinal fungi Mucor circinelloides. Six metabolites including three new metabolites (S2, S3, S4) were obtained, and their chemical structures were elucidated by spectroscopic data analysis. The major biotransformation reactions involved hydroxylation and glycosylation. In addition, all metabolites were evaluated for their anti-osteoporosis activity using MC3T3-E1 cells. The results demonstrated that S4, S5 and S6 could significantly promote MC3T3-E1 cell growth compared to osthole.

Fingerprint profiling and gut microbiota regulation of polysaccharides from Fritillaria species.

Few studies reported the quality evaluation and gut microbiota regulation effect of polysaccharides from Fritillaria species. In this study, polysaccharides extracted from ten Fritillaria species were compared and distinguished through multi-levels evaluation strategy and data fusion. Furthermore, the gut microbiota regulation effect of polysaccharides among different species was analyzed and evaluated. The fingerprint profiling of IR, molecular weight distribution of polysaccharides, chromatogram of partially hydrolyzed polysaccharides (oligosaccharides) and completely hydrolyzed polysaccharides (monosaccharides) were similar, and no exclusive signals were observed. However, the signal strength of functional group, oligosaccharides abundance and monosaccharides proportion showed obvious differences in inter- and intra-species. Glucan may be the main component of polysaccharides in Fritillaria species, CIRR derived from CIR, PRZ, DEL, TAI, UNI possessed higher total polysaccharides content, polymerization degree, oligosaccharides abundance (DP 2-4), and glucose content than the others. Meanwhile, data fusion model was established for identification of affinis and multi-original species, the accuracy of which proved to be 100 %. In addition, Fritillaria polysaccharides could increase the bacterial community richness and diversity, regulate the gut microbiota composition and possessed potential therapeutic effects on gastrointestinal diseases and nervous system diseases.

Advances in the chemical constituents, pharmacological properties and clinical applications of TCM formula Yupingfeng San.

Yupingfeng San (YPFS) is a famous and commonly used traditional Chinese medicine (TCM) formula for the treatment of chronic obstructive pulmonary disease, asthma, respiratory tract infections, and pneumonia in China. It is composed of three Chinese herbs, including Astragali Radix, Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix. In this review, the relevant references on YPFS were searched in the Web of Science, PubMed, China National Knowledge Infrastructure (CNKI), and other databases. Literatures published from 2000 to 2022 were screened and summarized. The constituents in YPFS could be classified into nine groups according to their structures, including flavonoids, saponins, essential oils, coumarins, lactones, amino acids, organic acids, saccharides, chromones and others. The importance of chemical constituents in YPFS were demonstrated for specific pathological processes including immunoregulatory, anti-inflammatory, anti-tumor and pulmonary diseases. This article systematically reviewed the up-to-date information on its chemical compositions, pharmacology and safety, that could be used as essential data and reference for clinical applications of YPFS.

Identification of Pinelliae Rhizoma and its counterfeit species based on enzymatic signature peptides from toxic proteins.

BACKGROUND: Pinelliae Rhizoma (PR), a toxic medication, with long history, is commonly used for eliminating phlegm. Due to the shortage of wild resources and the relative lacking of cultivation technology, it is often confused with its counterfeit species in the market, such as Typhonii Rhizoma (TR), Arisaematis Rhizoma (AR) and tubers of Typhonium flagelliforme (TF) and Pinellia pedatisecta (PP). PURPOSE: It was aimed to screen signature enzymatic peptides from toxic proteins to identify PR and its four counterfeit species. STUDY DESIGN: A comparative proteogenomics strategy based on open-source transcriptome data was applied for screening signature peptides from toxic proteins, which were applied for species authentication of PR and its counterfeit species. METHODS: Firstly, the open-source transcriptome data was used for constructing the annotated protein database, which was used for peptides identification. Secondly, the toxicity of different fractions of PR were evaluated by the rat peritoneal inflammation model. Furthermore, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to profile the main proteins bands of five species, whose sequences were identified based on the in-gel digestion experiment by using ultra-high-performance liquid chromatography/quadrupole-Orbitrap mass spectrometry. Finally, the label-free proteomic analysis was performed to character the proteins and screen the signature peptides of five species, which were validated in commercially available products by dynamic multi reaction monitor (DMRM). RESULTS: The results in this study confirmed that protein was the main toxic components of PR. Both Pinellia ternata agglutinin (PTA) and trypsin inhibitor (TI) like proteins are the main proteins, which were characterized by proteomic analysis based on four annotated protein database. Meanwhile, seven signature peptides from toxic proteins were screened and validated with good repeatability and specificity in commercial products. CONCLUSION: Seven signature enzymatic peptides from toxic protein screened by the comparative proteogenomics strategy based on open-source transcriptome data achieved good identification ability of PR and its four counterfeit species.

Behavioural Psychology of Unique Family Firms Toward R&D Investment in the Digital Era: The Role of Ownership Discrepancy.

This study examines the R&D investment behaviour of different types of family-controlled firms with the moderating role of ownership discrepancy between cash-flow rights and excess voting rights by using the sufficiency conditions' theoretical framework of ability and willingness developed by De Massis. It uses data from family firms that have issued A-shares from 2008 to 2018. They used pooled OLS regression for data analysis and Tobit regression for robustness checks. This study classifies family firm types into two categories, namely, the lone-controller family firms (LCFFs) and the multi-controller family firms (MCFFs), with each being further classified as "excess" or "no excess" voting rights. Both LCFFs without excess voting rights and MCFFs with excess voting rights have the "ability" and "willingness" toward R&D investment. LCFFs with excess voting rights and MCFFs without excess voting rights only have the ability but low willingness to invest in R&D. The study also establishes that Chinese family-controlled firms are heterogeneous toward risky investment. To the best of our knowledge, this study is the first to differentiate Chinese family firms by their unique ownership structure characteristics in investigating the effect of the family firm structure on R&D investment. The study is a novel attempt to test the willingness and ability framework of LCFFs and MCFFs. Previous studies based on agency theory have tacitly assumed that ability and willingness exist in family-controlled firms. However, this study challenges this implicit assumption.

Deep chemical identification of phytoecdysteroids in Achyranthes bidentata Blume by UHPLC coupled with linear ion trap-Orbitrap mass spectrometry and targeted isolation.

Achyranthes bidentata Blume is widely used as a traditional Chinese medicine with the effects of nourishing the liver and kidneys and strengthening muscles and bones. In this work, a rapid and simple strategy was developed for characterizing phytoecdysteroids by ultra-high-performance liquid chromatography coupled with liner ion trap-Orbitrap mass spectrometry using electrospray ionization in the negative mode. As a result, 47 phytoecdysteroids were unambiguously or tentatively characterized. Among them, seven known compounds were identified according to the reference standards along with molecular formula, retention time and fragmentation patterns, while others were mostly potential new compounds. Through targeted isolation, the structures of three new compounds were determined by NMR spectra, which were consistent with LC-MS characterization. The present study provides an efficient method to deeply characterize phytoecdysteroids.

Chemical profiling of Huashi Baidu prescription, an effective anti-COVID-19 TCM formula, by UPLC-Q-TOF/MS.

Huashi Baidu prescription (HSBDF), recommended in the Guideline for the Diagnosis and Treatment of Novel Coronavirus (2019-nCoV) Pneumonia (On Trials, the Seventh Edition), was clinically used to treat severe corona virus disease 2019 (COVID-19) with cough, blood-stained sputum, inhibited defecation, red tongue etc. symptoms. This study was aimed to elucidate and profile the knowledge on its chemical constituents and the potential anti-inflammatory effect in vitro. In the study, the chemical constituents in extract of HSBDF were characterized by UPLC-Q-TOF/MS in both negative and positive modes, and the pro-inflammatory cytokines were measured by enzyme-linked immunosorbent assays (ELISA) to determine the effects of HSBDF in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The results showed that a total of 217 chemical constituents were tentativedly characterized in HSBDF. Moreover, HSBDF could alleviate the expression levels of IL-6 and TNF-α in the cell models, indicating that the antiviral effects of HSBDF might be associated with regulation of the inflammatory cytokines production in RAW264.7 cells. We hope that the results could be served as the basic data for further study of HSBDF on anti-COVID-19 effect.

A systematic strategy integrating solid-phase extraction, full scan range splitting, mass defect filter and precursor ion list for comprehensive metabolite profiling of Danqi Tongmai tablet in rats.

In vivo metabolite profiling of herbal medicines remains a challenge due to the complex chemical composition and drastic interference from biological matrix. In this study, a systematic strategy was established for comprehensive metabolite profiling of Danqi Tongmai (DQTM) tablet, a combination of salvianolic acids and notoginsenosides, in rats after oral administration. This strategy was composed of six steps. Firstly, the rat plasma and tissue samples were collected at multiple time points to increase the representativeness of samples. Secondly, different sample preparation methods were systematically investigated including protein precipitation, liquid-liquid extraction and solid-phase extraction to obtain superior extraction efficiency for both salvianolic acids and notoginsenosides. Thirdly, the MS acquisition method was optimized by splitting the full scan range into two separate segments to improve the detection capability for minor components. Fourthly, an extended polygonal mass defect filter (EP-MDF) model was constructed to filter potential metabolites of salvianolic acids and notoginsenosides, and remove large amounts of interference ions. Fifthly, ion intensity-based time point-staggered precursor ion list (IITPS-PIL) was generated to trigger more targeted MS/MS acquisition for potential metabolites at the highest concentration. Finally, the absorbed prototypes and metabolites were comprehensively characterized by reference standards and MS/MS fragmentation. The proposed strategy significantly improved the detection ability for trace prototypes and metabolites in vivo. A total of 370 components, including 94 prototypes (38 confirmed with reference standards) and 276 metabolites, were tentatively characterized in rat plasma and tissue samples after oral administration of DQTM. Collectively, this paper provided an applicable reference for comprehensive metabolite profiling of herbal medicines in complex biological samples.

CHIP control degradation of mutant ETF:QO through ubiquitylation in late-onset multiple acyl-CoA dehydrogenase deficiency.

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common form of lipid storage myopathy. The disease is mainly caused by mutations in electron-transfer flavoprotein dehydrogenase gene (ETFDH), which leads to decreased levels of ETF:QO in skeletal muscle. However, the specific underlying mechanisms triggering such degradation remain unknown. We constructed expression plasmids containing wild type ETF:QO and mutants ETF:QO-A84T, R175H, A215T, Y333C, and cultured patient-derived fibroblasts containing the following mutations in ETFDH: c.250G>A (p.A84T), c.998A>G (p.Y333C), c.770A>G (p.Y257C), c.1254_1257delAACT (p. L418TfsX10), c.524G>A (p.R175H), c.380T>A (p.L127P), and c.892C>T (p.P298S). We used in vitro expression systems and patient-derived fibroblasts to detect stability of ETF:QO mutants then evaluated their interaction with Hsp70 interacting protein CHIP with active/inactive ubiquitin E3 ligase carboxyl terminus using western blot and immunofluorescence staining. This interaction was confirmed in vitro and in vivo by co-immunoprecipitation and immunofluorescence staining. We confirmed the existence two ubiquitination sites in mutant ETF:QO using mass spectrometry (MS) analysis. We found that mutant ETF:QO proteins were unstable and easily degraded in patient fibroblasts and in vitro expression systems by ubiquitin-proteasome pathway, and identified the specific ubiquitin E3 ligase as CHIP, which forms complex to control mutant ETF:QO degradation through poly-ubiquitination. CHIP-dependent degradation of mutant ETF:QO proteins was confirmed by MS and site-directed mutagenesis of ubiquitination sites. Hsp70 is directly involved in this process as molecular chaperone of CHIP. CHIP plays an important role in ubiquitin-proteasome pathway dependent degradation of mutant ETF:QO by working as a chaperone-assisted E3 ligase, which reveals CHIP's potential role in pathological mechanisms of late-onset MADD.

Traditional Chinese medicine (TCM) as a source of new anticancer drugs.

Covering: up to July 2020Drugs derived from traditional Chinese medicine (TCM) include both single chemical entities and multi-component preparations. Drugs of both types play a significant role in the healthcare system in China, but are not well-known outside China. The research and development process, the molecular mechanisms of action, and the clinical evaluation associated with some exemplificative anticancer drugs based on TCM are discussed, along with their potential of integration in western medicine.

Structurally diverse diterpenoid alkaloids from the lateral roots of Aconitum carmichaelii Debx. and their anti-tumor activities based on in vitro systematic evaluation and network pharmacology analysis.

Thirty-seven diterpenoid alkaloids (DAs) with diverse structures were isolated and identified from the lateral roots of Aconitum carmichaelii Debx., comprising eight C20-DAs and twenty-nine C19-DAs. Besides the 31 known DAs identified by comparing the 1H NMR and 13C NMR data with those reported in the literature, the structures of four new compounds (1, 14, 17, and 25), and two other compounds (26 and 37) which were reported to be synthesized previously, were also elucidated based on the comprehensive analysis of their HR-ESI-MS, 1D and 2D NMR spectra, including 1H-1H COSY, HSQC and HMBC and NOESY/ROESY. Among them, compound 1 represents the first example of a C20-DA glucoside. Besides, the anti-tumor activities of all the isolated compounds against human non-small-cell lung cancer A549 and H460 cells were systematically evaluated by MTT methods. The results revealed that all of the C19-DAs possessed moderate activities against both of the two cell lines with IC50 values ranging from 7.97 to 28.42 µM, and their structure-activity relationships indicated the active sites of C-8, C-10, and C-14 positions and the nitrogen atom in the C19-DA skeleton. In addition, all of the isolated DAs, with chemical structures confirmed, were further applied for network pharmacology analysis, in order to give an insight into the possible mechanisms of their anti-tumor activities. As a result, 173 potential targets and three most important pathways related to non-small-cell lung carcinoma were finally unearthed.

Two New Aristolochic Acid Analogues from the Roots of Aristolochia contorta with Significant Cytotoxic Activity.

Twelve compounds, including two new aristolochic acid analogues with a formyloxy moiety (9-10) and 10 known aristolochic acid derivates (1-8 and 11-12), were obtained from the roots of Aristolochiacontorta. Their structures were elucidated using extensive spectroscopic methods. Their cytotoxic activity in human proximal tubular cells HK-2 was evaluated by the MTT method, which has been widely used to assess cell viability. Among these molecules, compounds 3 and 9 were found to be more cytotoxic. Furthermore, molecular modeling was used to evaluate, for the first time, the interactions of compounds 3 and 9 with the target protein organic anionic transporter 1 (OAT1) that plays a key role in mediating aristolochic acid nephropathy. Structure-activity relationships are briefly discussed.

Quantitative Proteomics Analysis of Systemic Responses and Biological Mechanisms of ShuFengJieDu Capsule Using H1N1-Infected RAW264.7 Cells.

Emerging infectious diseases (EIDs) are a significant burden on global economies and public health to any country in the world. With the extensive application of traditional Chinese medicines (TCMs) for EID treatment, the underlying molecular mechanisms have caught more attention than before. The ShuFengJieDu capsule (abbreviated as SFJD) is a TCM prescription used for treating upper respiratory infection (URI) with symptoms of fever, sore throat, headaches, nasal congestion, and cough for more than 30 years in China. SFJD is also widely used for the prevention and treatment of viral infectious diseases, especially for the EIDs. In this study, a bioactivity-integrated method of ultraperformance liquid chromatography quadrapole/time-of-flight mass spectrometry combined with methyl thiazolyl tetrazolium assay was applied to screen potential antivirus compounds in SFJD on the H1N1-infected RAW264.7 cell models. Three compounds (forsythoside E, verbenalin, and emodin) exert the advantages of protective effects in cell vitality during H1N1 infection. The isobaric tags for relative and absolute quantification (iTRAQ)-coupled liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis and the subsequent quantitative proteome analysis were performed to investigate the potential molecular mechanisms triggered by these three bioactive compound-triggered molecular mechanisms in H1N1-infected RAW264.7 cells. Dysregulated proteins were involved in regulating the levels of proinflammatory cytokines, the IFN (interferon)-stimulated gene signal in the Type I IFN, TBK/IRF3, and MAPK/NF-κB signaling pathways. In conclusion, we identified the main bioactive compounds in SFJD exerting antiviral effects and illuminated that Type I IFN and MAPK/NF-κB signaling pathways are involved in the anti-H1N1 infection effects of SFJD. Our study not only provides solid theoretical support for the clinic application of SFJD but also sheds light on the novel research methods for TCM study.

Therapeutic Mechanistic Studies of ShuFengJieDu Capsule in an Acute Lung Injury Animal Model Using Quantitative Proteomics Technology.

ShuFengJieDu capsule (SFJDC), a traditional Chinese medicine (TCM) that contains eight medicinal herbs, has been extensively utilized for the treatment of acute lung injury (ALI) and respiratory infections for more than 30 years in China. SFJDC has also been listed in the official guidelines of the China Food and Drug Administration (CFDA) due to its stable clinical manifestations. However, the underlying mechanism of SFJDC during ALI repair remains unclear. In the present study, we explored the protective and therapeutic mechanisms of SFJDC in a rat model by performing qualitative and label-free quantitative proteomics studies. After establishing lipopolysaccharide (LPS)-induced ALI rat models, we profiled macrophage cells isolated from freshly resected rat lung tissues derived from ALI models and ALI rat lung tissue sections using a high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) shotgun proteomics approach to identify changes in the expression levels of proteins of interest. On the basis of our proteomics results and the results of a protein dysregulation analysis of ALI rat lung tissues and rat lung macrophages, AKT1 was selected as a putative key factor that may play an important role in mediating the effects of SFJDC treatment during ALI progression. Follow-up validation studies demonstrated that AKT1 expression effectively regulates various ALI-related molecules, and Gene Ontology analysis indicated that SFJDC-treated ALI rat macrophages were influenced by AKT1-based networks. Gain- and loss-of-function analyses following lentivirus-AKT1 or lentivirus-si-AKT1 infection in macrophages also indicated that AKT1 was essential for the development of ALI due to its ability to regulate oxidative stress, apoptosis, or inflammatory responses. In summary, SFJDC effectively modulated anti-inflammatory and immunomodulation activity during ALI, potentially due to AKT1 regulation during ALI progression. New insights into SFJDC mechanisms may facilitate the development of novel pharmaceutical strategies to control the expression of inflammatory factors.

Reduced SLC27A2 induces cisplatin resistance in lung cancer stem cells by negatively regulating Bmi1-ABCG2 signaling.

Platinum-based chemotherapies have long been used as a standard treatment in non-small cell lung cancer (NSCLC). However, cisplatin resistance is a major problem that restricts the use of cisplatin. Lung cancer stem cells (LCSCs) represent a subpopulation that is responsible for chemo-resistance. We aim to investigate the biological function of SLC27A2 and its underlying mechanisms in regulating chemo-resistance to cisplatin in LCSCs. Here, our findings testified that CD166+ cells which were derived from fresh primary NSCLC samples displayed stem cell-like features and were resistant to chemotherapy drug cisplatin. In patient cohort, we found the presence of a variable fraction of CD166+ cells in 24 out of 25 primary NSCLC samples. Significantly, SLC27A2 expression was reduced in CD166+ LCSCs. Reduced SLC27A2 correlated chemo-response and poor patient survival. Our results indicated that enhanced SLC27A2 expression sensitized CD166+ LCSCs to cisplatin by in vitro and in vivo experiments. Microarray profiling showed that the expression of Bmi1 and ABCG2 was enhanced in p-SLC27A2-LCSCs compared with that in pc3.1DNA-LCSCs. Furthermore, we demonstrated that reduced SLC27A2 induced chemo-resistance in CD166+ LCSCs by negatively regulating Bmi1-ABCG2 signaling, and ABCG2 was a direct transcriptional target of Bmi1. Thus, this study widens the window for identification and targeting of a cisplatin-resistant population and contributes to the development of potential therapeutics to improve the current treatment modalities in NSCLC. © 2015 Wiley Periodicals, Inc.

CD44 is functionally crucial for driving lung cancer stem cells metastasis through Wnt/ß-catenin-FoxM1-Twist signaling.

A novel paradigm in tumor biology suggests that non-small cell lung cancer (NSCLC) metastasis is driven by lung cancer stem cell-like cells (LCSCs), but the underlying mechanisms remain unclear. Here, we aim to investigate biological function of CD44 in regulating metastatic trait of LCSCs and its underlying mechanisms. In this study, we found that CD133+ CD44+ cells which were derived from primary lung adenocarcinoma (LAC) possessed cancer stem cell-like features. Furthermore, CD44 was demonstrated functionally crucial to drive metastatic potential of CD133+ CD44+ LCSCs by in vitro and in vivo experiments. In patient cohorts, high level of CD44 predicted increased probability of metastasis. Significantly, microarray revealed that FoxM1 and key proteins of Wnt/ß-catenin pathway were up-regulated in CD133+ CD44+ LCSCs compared with those in CD133+ CD44- cells. Then, we demonstrated that CD44 promoted metastatic activity in CD133+ CD44+ LCSCs through Wnt/ß-catenin pathway and FoxM1 was the downstream target of Wnt/ß-catenin pathway. Meanwhile, our findings indicated that FoxM1 promoted metastatic activity in CD133+ CD44+ LCSCs by inducing EMT and Twist was a direct transcriptional target of FoxM1. Collectively, CD44, both a functional biomarker and therapeutic target, promoted CD133+ CD44+ LCSCs metastasis by Wnt/ß-catenin-FoxM1-Twist signaling. This study provided support for the missing link between EMT and CSCs surface-marker and supplied a promising approach for elimination of LCSCs by targeting CD44-Wnt/ß-catenin-FoxM1-Twist signaling. © 2015 Wiley Periodicals, Inc.