Cancer stem cells (CSCs) represent a major source of treatment resistance and tumor progression. However, regulation of CSCs stemness is not entirely understood. Here, we report that TSPAN8 expression is upregulated in breast CSCs, promotes the expression of the stemness gene NANOG, OCT4, and ALDHA1, and correlates with therapeutic resistance. Mechanistically, TSPAN8 interacts with PTCH1 and inhibits the degradation of the SHH/PTCH1 complex through recruitment of deubiquitinating enzyme ATXN3. This results in the translocation of SMO to cilia, downstream gene expression, resistance of CSCs to chemotherapeutic agents, and enhances tumor formation in mice. Accordingly, expression levels of TSPAN8, PTCH1, SHH, and ATXN3 are positively correlated in human breast cancer specimens, and high TSPAN8 and ATXN3 expression levels correlate with poor prognosis. These findings reveal a molecular basis of TSPAN8-enhanced Sonic Hedgehog signaling and highlight a role for TSPAN8 in promoting cancer stemness.

Cancer stem cells (CSCs) are known to mediate metastasis and recurrence and are therefore a promising therapeutic target. In this study, we found that dihydrotanshinone (DHTS) inhibits CSC formation. DHTS inhibited mammosphere formation in a dose-dependent manner and showed significant tumor growth inhibition in a xenograft model. This compound reduced the CD44

The properties of mesenchymal stem cells (MSCs), especially their self-renewal and ability to differentiate into different cell lines, are widely discussed. Considering the fact that MSCs isolated from perinatal tissues reveal higher differentiation capacity than most adult MSCs, we examined mesenchymal stem cells isolated from Wharton's jelly of umbilical cord (WJ-MSCs) in terms of pluripotency markers expression. Our studies showed that WJ-MSCs express some pluripotency markers-such as NANOG, OCT-4, and SSEA-4-but in comparison to iPS cells expression level is significantly lower. The level of expression can be raised under hypoxic conditions. Despite their high proliferation potential and ability to differentiate into different cells type, WJ-MSCs do not form tumors in vivo, the major caveat of iPS cells. Owing to their biological properties, high plasticity, proliferation capacity, and ease of isolation and culture, WJ-MSCs are turning out to be a promising tool of modern regenerative medicine.

BACKGROUND: Curcumin is a polyphenolic compound with potent chemopreventive and anti-cancer efficacy.
PURPOSE: To explore the potential anti-metastasis efficacy of curcumin in breast cancer stem-like cells (BCSCs), which are increasingly considered to be the origin of the recurrence and metastasis of breast cancer.
METHODS: A CCK8 assay was performed to evaluate cell viability, and a colony formation assay was conducted to determine cell proliferation in MCF-7 and MDA-MB-231 adherent cells. Transwell and wound healing assays were used to detect the effect of curcumin on cell migration and invasion in MDA-MB-231 cells. Mammospheres were cultured with serum free medium (SFM) for three generations and the BCSC surface marker CD44
RESULTS: Curcumin exhibited anti-proliferative and colony formation inhibiting activities in both the MCF-7 and MDA-MB-231 cell lines. It also suppressed the migration and invasion of MDA-MB-231 cells. The CD44
CONCLUSION: The results of the present study suggest that the inhibitor effects of curcumin on breast cancer cells may be related to resistance to cancer stem-like characters and the EMT process. These data indicate that curcumin could function as a type of anti-metastasis agent for breast cancer.

Scientific evidence linking vitamin D with various cancer types is growing, but the effects of vitamin D on ovarian cancer stem cell‑like cells (CSCs) are largely unknown. The present study aimed to examine whether vitamin D was able to restrain the stemness of ovarian cancer. A side population (SP) from malignant ovarian surface epithelial cells was identified as CSCs, in vitro and in vivo. Furthermore, 1α,25‑dihydroxyvitamin D3 [1α,25(OH)2D3] treatment inhibited the self‑renewal capacity of SP cells by decreasing the sphere formation rate and by suppressing the mRNA expression levels of cluster of differentiation CD44, NANOG, OCT4, SOX2, Krüppel‑like factor 4 and adenosine triphosphate binding cassette subfamily G member 2. Additionally, 1α,25(OH)2D3 treatment decreased the expression of Cyclin D1, whereas it increased the expression of β‑catenin and vitamin D receptor (VDR). Notably, immunofluorescence staining verified that 1α,25(OH)2D3 promoted the expression of β‑catenin in the cytoplasm. Furthermore, vitamin D3 delayed the onset of tumor formation derived from injection of ovarian CSCs to nude mice, by reducing CD44 and enhancing β‑catenin expressions in vivo. In conclusion, 1α,25(OH)2D3 restrains the stem cell‑like properties of ovarian cancer cells by enhancing the expression of VDR, by promoting the expression of β‑catenin in the cytoplasm, and by suppressing the expression of CD44. These findings provide a novel insight into the functions of vitamin D in diminishing the stemness of cancer CSCs.

PURPOSE: Cancer stem cells (CSC) and epithelial-mesenchymal transition (EMT) pathways are crucial for cancer progression. However, synergistic interactions between CSC and EMT are not clear in non-small cell lung cancer (NSCLC). The objective of this study was to investigate CSC markers such as CD44, NANOG, and ALDH1 expression and its correlation with EMT markers in NSCLC patients. Its association with survival was also determined.
METHODS: CD44, NANOG, and ALDH1 protein expression was evaluated in 267 resected NSCLC and its correlation with e-cadherin, β-catenin, p120 catenin, vimentin, SNAIL, and TWIST expressions was determined based on immunohistochemical and mRNA expression data from The Cancer Genome Atlas (TCGA) database. Survival analyses also were performed based on immunohistochemistry and mRNA expression data from Gene Expression Omnibus dataset.
RESULTS: ALDH1 expression in lung adenocarcinoma was positively correlated with the epithelial-like phenotype, low vimentin and low TWIST in immunohistochemical and mRNA expression data. NANOG and ALDH1 expressions measured by immunohistochemical and mRNA expression profiling data of adenocarcinomas were associated with a favorable prognosis. ALDH1 was an independent favorable prognostic marker for overall survival or recurrence-free survival in adenocarcinoma (P = 0.026 and P = 0.033, respectively). The epithelial-like phenotype expressing P120-catenin and beta-catenin was associated with a favorable prognosis; however, the TWIST-expressing mesenchymal-like phenotype was correlated with an unfavorable prognosis.
CONCLUSIONS: NANOG and ALDH1 protein or mRNA expression showed improved prognosis in adenocarcinoma alone. ALDH1 expression correlated with an epithelial-like phenotype.

OBJECTIVE: The aim of this study was to analyze the expression, biological role and clinical relevance of cancer stem cell markers in high-grade serous carcinoma (HGSC).
METHODS: mRNA expression by qRT-PCR of NANOG, OCT4, SOX2, SOX4, SOX9, LIN28A and LIN28B was analyzed in 134 HGSC specimens (84 effusions, 50 surgical specimens). Nanog, OCT3/4, SOX2 and SOX9 protein expression by immunohistochemistry was analyzed in 52 HGSC effusions. Nanog protein expression in exosomes from 80 HGSC effusions was studied by Western Blotting. OVCAR3 cells underwent CRISPR/Cas9 Nanog knockout (KO), and the effect of Nanog KO on migration, invasion, proliferation and proteolytic activity was analyzed in OVCAR3 and OVCAR8 cells.
RESULTS: OCT4 mRNA was overexpressed in effusions compared to solid specimens (p = 0.046), whereas SOX9 was overexpressed in the ovarian tumors compared to effusions and solid metastases (p = 0.003). Higher SOX2 and SOX9 expression was associated with primary (intrinsic) chemoresistance (p = 0.009 and p = 0.02, respectively). Higher SOX9 levels were associated with shorter overall survival in univariate (p = 0.04) and multivariate (p = 0.049) analysis. OCT3/4, SOX2 and SOX9 proteins were found in HGSC cells, whereas Nanog was detected only in exosomes. Higher SOX2 protein expression was associated with shorter overall survival in univariate analysis (p = 0.049). OVCAR cells exposed to OVCAR3 NANOG KO exosomes had reduced migration, invasion and MMP9 activity.
CONCLUSIONS: SOX2 and SOX9 mRNA levels in HGSC effusions may be markers of clinically aggressive disease. Nanog is secreted in HGSC exosomes in effusions and modulates tumor-promoting cellular processes in vitro.

BACKGROUND/AIM: We recently investigated the contribution of the iPS-related genes SOX2, OCT4, and Nanog to de-differentiation by assaying for their mRNA levels. Given that mRNA expression does not always correlate with the protein levels, the aim of this study was to retrospectively determine the expression of these four iPS-related factors in human OSCC specimens by immunohistochemistry and examine their association with patient prognosis.
MATERIALS AND METHODS: iPS cell-related gene expression in 89 OSCC patients by tissue microarray, and its correlation with clinicopathological factors, differentiation, metastasis, and poor prognoses were investigated.
RESULTS: No evidence of statistically significant relationships was found between the expression of iPS cell-related genes and clinicopathological parameters. However, our data indicated that KLF4 expression was associated with survival, and poor tumor differentiation. In addition, high expression of KLF4 was an independent poor prognostic factor (p=0.004) for OSCC patients.
CONCLUSION: In preoperative biopsies, higher KLF4 and poor differentiation may be clinically effective predictors for the prognosis of oral cancer.

BACKGROUND: Breast cancer stem cells have self-renewal capability and are resistant to conventional chemotherapy. PD-L1 could promote the expression of stemness markers (OCT4 and Nanog) in breast cancer stem cells. However, the mechanisms by which PD-L1 regulates the stemness of breast cancer cells and PD-L1 is regulated in breast cancer cells are still unclear.
METHODS: Lentivirus infection was used to construct stable cell lines. The correlation between PD-L1 and stemness markers expression was evaluated in clinical samples. Additionally, luciferase reporter assay combined with RNA-Fluorescence in situ hybridization (RNA-FISH) and RNA-binding protein immunoprecipitation (RIP) assays were used to verify the direct binding of miR-873 on PD-L1. Furthermore, flow cytometry, mammosphere formation combined with nude mouse tumor xenograft model were carried out to examine the effects of miR-873/PD-L1 axis on the stemness of breast cancer cells. Finally, MTT assay was performed to determine the effects of miR-873/PD-L1 axis on drug resistance.
FINDINGS: PD-L1 expression was positively correlated with the expression of stemness markers, and overexpression of PD-L1 contributed to chemoresistance and stemness-like properties in breast cancer cells via activating PI3K/Akt and ERK1/2 pathways. Mechanistically, miR-873 inhibited PD-L1 expression through directly binding to its 3'-untranslated region (UTR), and miR-873 attenuated the stemness and chemoresistance of breast cancer cells which was dependent on PD-L1 and the downstream PI3K/Akt and ERK1/2 signaling. Notably, the promotion of PD-L1 on the stemness and chemoresistance was enhanced by recombinant PD-1 (rPD-1), this effect was attenuated by PD-1/PD-L1 inhibitor.
INTERPRETATION: miR-873/PD-L1 regulatory axis might serve as a therapeutic target to enhance the chemo-sensitivity and eliminate the stemness of breast cancer cells. FUND: This work was supported by the National Nature Science Foundation of China, No. 81702957, China Postdoctoral Science Foundation, No. 2017M620230, the Postdoctoral Research Funding Scheme of Jiangsu Province (2017), No. 1701197B, and the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Melanoma is a malignant tumor. The acquisition of stemness of melanoma cells aggravates the malignant transformation, which can be regulated by microRNAs (miRNAs, miR). MiR-363-3p is a key tumor-related miRNA, but its role in stemness and melanoma cells is still unknown. Presently, miR-363-3p, induced by hypoxia inducible factor (HIF)-2α, played a positive role in the stemness of melanoma cells. The levels of miR-363-3p and HIF-2α were upregulated in melanoma cell lines. Overexpression of HIF-2α significantly increased the levels of miR-363-3p. However, both HIF-2α knockdown and miR-363-3p inhibition inhibited the levels of the stemness markers (CD133, CD271, Jarid1B, and Nanog). Furthermore, the levels of miR-363-3p and HIF-2α were upregulated in fluorescence activated cell sorting (FACS)-sorted CD271high/+ cells. Whereas miR-363-3p depletion reduced the proportion and the spheroidization of the CD271high/+ cells, decreased the levels of CD133, CD271, Jarid1B and Nanog with restrained proliferative activity of CD271high/+ cells. Additionally, miR-363-3p was confirmed a key downstream of HIF-2α. Intriguingly, cyclin-dependent kinase inhibitor 1A [CDKN1A, p21(Cip1/Waf1)], a key inhibitor of S-phase DNA synthesis and cell cycle progression, was confirmed a target gene of miR-363-3p by luciferase reporter gene assay. The protein levels of CD133, CD271, Jarid1B and Nanog were upregulated with enhanced proliferative activity of CD271high/+ cells by inhibition of p21 in melanoma cells. In conclusion, miR-363-3p is induced by HIF-2α to promote the stemness of melanoma cells via inhibiting p21. The present study provides novel insights that HIF-2α/miR-363-3p/p21 signaling may be a potential target of research and therapy of melanoma.

Breast cancer (BC) is a common cancer and leading cause of cancer‑associated mortality in women. Abnormal expression of long non‑coding RNA FOXD2 adjacent opposite strand RNA 1 (FOXD2‑AS1) was associated with the development of a number of tumors. However, whether FOXD2‑AS1 is dysregulated in BC and its underlying mechanisms remain unclear. In the present study, it was identified that FOXD2‑AS1 expression was upregulated in BC tissue, cell lines and sphere subpopulation. Additionally, the abnormal upregulation of FOXD2‑AS1 predicted poor prognosis in patients with BC. Furthermore, downregulation of FOXD2‑AS1 decreased cell proliferation, and migratory and invasive abilities in BC cells, and decreased the growth of transplanted tumors in vivo. Downregulation of FOXD2‑AS1 decreased the percentage of CD44 antigen+/signal transducer CD24- in breast cancer stem cell (BCSC) cells, and decreased the expression of numerous stem factors, including Nanog, octamer‑binding transcription factor 4 (Oct4), and sex determining region Y‑box 2 (SOX2), and inhibited the epithelial‑mesenchymal transition process. FOXD2‑AS1 was identified to be primarily located in the cytoplasm. Using bioinformatics analysis, a reporter gene assay and reverse transcription‑polymerase chain reaction assays, it was demonstrated that microRNA (miR)‑150‑5p was able to bind directly with the 3'‑untranslated region of FOXD2‑AS1 and PFN2 mRNA. miR‑150‑5p mimics decreased the cell proliferation, migration and invasion of BC cells. FOXD2‑AS1 knockdown significantly inhibited the miR‑150‑5p inhibitor‑induced increase in Nanog, Oct4 and SOX2 expression. The miR‑150‑5p inhibitor‑induced increase in N‑cadherin, and decrease in E‑cadherin and vimentin was inhibited by FOXD2‑AS1 knockdown. Profilin 2 (PFN2) expression was significantly upregulated in BC tissues. Additionally, the abnormal upregulation of PFN2 was associated with poor prognosis in patients with BC. FOXD2‑AS1 and PFN2 expression was positively correlated. Collectively, the present results demonstrated the role of the FOXD2‑AS1/miR‑150‑5p/PFN2 axis in the development of BC, and provides novel targets for the treatment of BC, and potential biomarkers for diagnosis and prognosis of BC.

Background: Recent several studies have showed that the nanog overexpression leads to poor prognosis in some kinds of cancer including hepatocellular carcinoma and gastrointestinal luminal cancer. However, the correlations between prognosis and clinic-pathological features and nanog overexpression in lung cancer are still not well-known. Thus, we performed a meta-analysis to evaluate the role of nanog in lung cancer.
Methods: An electronic retrieval for related studies was conducted in PubMed, Cochrane Library, Web of Science, EMBASE databases, Chinese CNKI, and the Chinese Wan Fang database up to May 2018. The relationships between nanog overexpression and overall survival (OS) and disease-free survival (DFS) as well as clinic-pathological features in lung cancer were investigated. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated by STATA12.
Results: 11 studies containing 1422 patients were identified in our meta-analysis. The overexpression of nanog showed decreased OS (HR = 1.83, 95% CI = 1.49-2.25,
Conclusions: Our results suggested that nanog overexpression, a hazard factor of differentiation, lymph node metastasis, and tumor size, may predicate decreased OS and DFS for lung cancer.

BACKGROUND: Docetaxel was used to treat metastatic CRPC patients. However, Doc resistance in prostate cancer (PCa) hinders its clinical application.
OBJECTIVE: To understand the underlying mechanisms by which Doc resistance is developed and to find novel therapeutic target to cure Doc resistant PCa has clinical importance.
METHODS: We established Doc resistant cell lines and explored the role of Ezh2 in the development of Doc resistance by overexpressing its cDNA or using its inhibitor.
RESULTS: We found that Ezh2 was induced in our established Doc resistant (DocR) cells, which was attributable to the silenced expression of miR-101-3p and miR-138-5p. Blockage of Ezh2 activity by either inhibitor or miRNA mimics could overcome Doc resistance by suppressing Doc-induced cancer stem cells populations. Mechanistically, Ezh2 activity was required for the induced expression of Nanog, Sox2 and CD44 upon Doc treatment.
CONCLUSIONS: Targeting Ezh2 could overcome Doc resistance.

Cancer stem cells (CSCs) have been shown as a distinct population of cancer cells strongly implicated with resistance to conventional chemotherapy. Metformin, the most widely prescribed drug for diabetes, was reported to target cancer stem cells in various cancers. In this study, we sought to determine the effects of metformin on head and neck squamous cell carcinoma (HNSCC). CSCs and non-stem HNSCC cells were treated with metformin and cisplatin alone, and in combination, and cell proliferation levels were measured through MTS assays. Next, potential targets of metformin were explored through computational small molecule binding analysis. In contrast to the reported effects of metformin on CSCs in other cancers, our data suggests that metformin protects HNSCC CSCs against cisplatin in vitro. Treatment with metformin resulted in a dose-dependent induction of the stem cell genes CD44, BMI-1, OCT-4, and NANOG. On the other hand, we observed that metformin successfully decreased the proliferation of non-stem HNSCC cells. Computational drug⁻protein interaction analysis revealed mitochondrial complex III to be a likely target of metformin. Based on our results, we present the novel hypothesis that metformin targets complex III to reduce reactive oxygen species (ROS) levels, leading to the differential effects observed on non-stem cancer cells and CSCs.

PURPOSE: Recent evidence suggests that small subpopulations of stem-like cells are accountable for tumour initiation, progression and metastasis. Until now, studies were focused exclusively on the characterization of these cell populations within the tumour itself, while tumour margins were neglected, although it is known that the histological and molecular status of tumour margins may play a significant role in the course of the disease. Therefore, the aims of the study were to isolate cells from oral squamous cell carcinomas and their respective margins, to characterize these cells using specific markers, to assess their self-renewal potential and determine their chemoresistance.
METHODS: Cell cultures were obtained from 12 tissue specimens (6 tumours and 6 margins). Total RNA was extracted and gene expression analysis was done by real-time PCR (RT-PCR). Flow cytometry, immunocytometry, sphere formation and MTT assays were also applied.
RESULTS: With minor differences, cells originating from both tumours and tumour margins showed the presence of stem cell markers CD133, Nanog, Sox2, CD44, and Oct4, had the capacity to form spheroids and showed chemoresistance.
CONCLUSIONS: Subpopulations of margin cells appeared to have stemness properties which might raise the question of re-evaluation of optimal surgical management.

NANOG is an essential transcriptional factor for the maintenance of embryonic stem cells (ESCs) and cancer stem cells (CSCs) in prostate cancer (PCa). However, the regulation mechanism of NANOG protein stability in cancer progression is still elusive. Here, we report that NANOG is degraded by SPOP, a frequently mutated tumor suppressor of PCa. Cancer-associated mutations of SPOP or the mutation of NANOG at S68Y abrogates the SPOP-mediated NANOG degradation, leading to elevated PCa cancer stemness and poor prognosis. In addition, SPOP-mediated NANOG degradation is controlled by the AMPK-BRAF signal axis through the phosphorylation of NANOG at Ser68, which blocked the interaction between SPOP and NANOG. Thus, our study provides a regulation mechanism of PCa stemness controlled by phosphorylation-mediated NANOG stability, which helps to identify novel drug targets and improve therapeutic strategy for PCa.

BACKGROUND: Endometrial cancer is one of the most common gynecological malignancies and has exhibited an increasing incidence rate in recent years. Cancer stem cells (CSCs), which are responsible for tumor growth and chemoresistance, have been confirmed in endometrial cancer. However, it is still challenging to identify endometrial cancer stem cells to then target for therapy.
METHODS: Flow cytometry was used to identify the endometrial cancer stem cells. Sphere formation assay, western blotting, qRT-PCR assay, cell viability assay, xenograft assay and immunohistochemistry staining analysis were utilized to evaluate the effect of SPARC-related modular calcium binding 2 (SMOC-2) on the cells proliferation and drug resistance. Cell viability assay, qRT-PCR assay, immunofluorescence staining, Co-IP assay and luciferase reporter gene assay were performed to explore the possible molecular mechanism by which SMOC-2 activates WNT/β-catenin pathway.
FINDINGS: We found the expression of SPARC-related modular calcium binding 2 (SMOC-2), a member of SPARC family, was higher in endometrial CSCs than that in non-CSCs. SMOC-2 was also more highly expressed in spheres than in monolayer cultures. The silencing of SMOC-2 suppressed cell sphere ability; reduced the expression of the stemness-associated genes SOX2, OCT4 and NANOG; and enhanced chemosensitivity in endometrial cancer cells. By co-culture IP assay, we demonstrated that SMOC-2 directly interacted with WNT receptors (Fzd6 and LRP6), enhanced ligand-receptor interaction with canonical WNT ligands (Wnt3a and Wnt10b), and finally, activated the WNT/β-catenin pathway in endometrial cancer. SMOC-2 expression was closely correlated with CSC markers CD133 and CD44 expression in endometrial cancer tissue.
INTERPRETATION: Taken together, we conclude that SMOC-2 might be a novel endometrial cancer stem cell signature gene and therapeutic target for endometrial cancer. FUND: National Natural Science Foundation of China, Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission, Scientific and Technological Innovation Act Program of Fengxian Science and Technology Commission, Natural Science Foundation of Shanghai.

AIMS: A major obstacle for effective cancer treatment by radiation therapy is the development of radio-resistance and identification of underlying mechanisms and activated pathways will lead to better combination therapies.
MAIN METHODS: Irradiated MCF-7 and MDA-MB-231 breast cancer cell lines were characterised following different recovery periods. Proliferation was assessed by MTT, BrdU and clonogenic assays and apoptosis by Annexin V/ propidium iodide staining and flow cytometry. Gene expression was monitored by real time PCR/ELISA/antibody labelling and migration using transwell inserts.
KEY FINDINGS: Breast cancer cell lines exposed to 6 Gy followed by recovery period for 7 days (D7-6 G) had increased ability for proliferation as well as apoptosis. D7-6 G from both cell lines had increased expression of transforming growth factor isoforms (TGF)-β1, β2 and β3, their receptors TGF-βR1 and TGF-βR2 which are known for such dual effects. The expression of downstream transcription factors Snail, Zeb-1 and HMGA2 also showed a differential pattern in D7-6 G cells with upregulation of at least two of these transcription factors. D7-6 G cells from both cell lines displayed hybrid epithelial-mesenchymal (E/M) phenotype with increased expression of E/M markers and migration. D7-6 G cells had increased expression of cancer stem cells markers Oct4, Sox2, and Nanog; aldehyde dehydrogenase expression and activity; proportion of CD44
SIGNIFICANCE: Blocking of TGF-β signalling may therefore be an effective strategy for overcoming radio resistance induced by radiation exposure.

Accurate and efficient delivery of genome editing plasmids to targeted cells is of critical importance in genome editing. Herein, we prepared a multifunctional delivery vector with a combination of ligand-mediated selectivity and peptide-mediated transmembrane function to effectively deliver plasmids to targeted cancerous cells. In the delivery system, the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) plasmid is combined with protamine with membrane and nuclear translocating activities and co-precipitated with CaCO

Glioma is the most common sub-type of brain tumor. Due to the presence of stem-like cells, it is characterized by poor prognosis, aggressive ability and high post-surgical recurrence rates. Hence, there is critical need to identify molecular mechanisms of glioma stem-like cells. We found a novel lncRNA in the ZNF281 gene and named it lncRNA-ZNF281. We detected the expression of lncRNA-ZNF281 in glioma stem-like cells (U251s), the glioma cell line (U251) and also in normal brain tissue. The expression of lncRNA-ZNF281 was lower in glioma stem-like cells (U251s) and this indicates that lncRNA-ZNF281 can regulate the self-renewal capacity of glioma stem-like cells and stem cell marker expression. Most significantly, lncRNA-ZNF281 inhibits the invasion of glioma stem-like cells by regulating the expression of the NF-κB1 signaling pathway. Our data demonstrates that lncRNA-ZNF281 inhibits the self-renewing ability and invasion of GSCs in vitro and in vivo and can reduce tumorigenicity in the glioma stem-like cell (U251s). The underlying mechanisms may involve the regulation of stem cell markers (CD133, Nestin, OCT4 and Nanog) to reduce the self-renewal ability and regulate the NF-κB1 signaling pathway and inhibit U251s glioma stem-like cell invasion. These finding suggest that lncRNA-ZNF281 could be a successful new therapeutic target in glioma.

Background: Beta-hydroxybutyrate (BHB) as a ketone body is the metabolic fuel in oxidative phosphorylation pathway. So far the effects of BHB on the biology of tumor cells is contradictory. Therefore, we investigated the effect of BHB on viability, metabolism, proliferation and migration of 5FU treated SW480 colon cancer cell line. Methods: we treated the SW480 cells with IC50 dose of 5-fluorouracil (5FU) for 72 h to isolate a subpopulation of 5FU treated cells that were resistant to it. Effects of BHB on cell viability was investigated by MTT assay. Measurement of oxygen consumption rate (OCR) in parallel with extracellular acidification rate (ECAR) upon BHB treatment was used for determination of metabolic profile of these cells. Investigating the relationship between metabolic phenotype and the status of differentiation and stemness was done by analyzing the expression of PGC-1α, c-MYC, NANOG, ALPi and KRT20 genes by qRT-PCR. Clonogenic and scratch assay were performed to determine the proliferation and migration abilities of incubated with BHB compared to untreated cells. Results: BHB increased cell viability in SW480 and 5FU treated SW480 cells. The results showed a significantly decreased ECAR and increased OCR in both cell types following BHB treatment reflecting the superiority of oxidative phosphorylation profile compared to glycolysis in both cell types. Also, treatment with BHB increases the expression of genes normally associated with stemness and mitochondrial biogenesis and decreases the expression of genes related to glycolytic program and differentiation in 5FU treated cells. Self-renewal and migration potential of BHB treated cells increased significantly. Conclusion: These findings suggest that BHB utilization via oxidative mitochondrial metabolism can fuel proliferation, migration and stemness in 5FU treated SW480 colon cancer cells.

BACKGROUND: Emerging evidence have illustrated the vital role of long noncoding RNAs (lncRNAs) long intergenic non-protein coding RNA 00511 (LINC00511) on the human cancer progression and tumorigenesis. However, the role of LINC00511 in breast cancer tumourigenesis is still unknown. This research puts emphasis on the function of LINC00511 on the breast cancer tumourigenesis and stemness, and investigates the in-depth mechanism.
METHODS: The lncRNA and RNA expression were measured using RT-PCR. Protein levels were measured using western blotting analysis. CCK-8, colony formation assays and transwell assay were performed to evaluate the cell proliferation ability and invasion. Sphere-formation assay was also performed for the stemness. Bioinformatic analysis, chromatin immunoprecipitation (ChIP) and luciferase reporter assays were carried to confirm the molecular binding.
RESULTS: LINC00511 was measured to be highly expressed in the breast cancer specimens and the high-expression was correlated with the poor prognosis. Functionally, the gain and loss-of-functional experiments revealed that LINC00511 promoted the proliferation, sphere-formation ability, stem factors (Oct4, Nanog, SOX2) expression and tumor growth in breast cancer cells. Mechanically, LINC00511 functioned as competing endogenous RNA (ceRNA) for miR-185-3p to positively recover E2F1 protein. Furthermore, transcription factor E2F1 bind with the promoter region of Nanog gene to promote it transcription.
CONCLUSION: In conclusion, our data concludes that LINC00511/miR-185-3p/E2F1/Nanog axis facilitates the breast cancer stemness and tumorigenesis, providing a vital insight for them.

BACKGROUND: It has been proposed that mesenchymal stromal cells (MSCs) promote tumor progression by interacting with tumor cells and other stroma cells in the complex network of the tumor microenvironment. We characterized MSCs isolated and expanded from tumor tissues of pediatric patients diagnosed with neuroblastomas (NB-MSCs) to define interactions with the tumor microenvironment.
METHODS: Specimens were obtained from 7 pediatric patients diagnosed with neuroblastoma (NB). Morphology, immunophenotype, differentiation capacity, proliferative growth, expression of stemness and neural differentiation markers were evaluated. Moreover, the ability of cells to modulate the immune response, i.e. inhibition of phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cytotoxic function, was examined. Gene expression profiles, known to be related to tumor cell stemness, Wnt pathway activation, epithelial-mesenchymal transition (EMT) and tumor metastasis were also evaluated. Healthy donor bone marrow-derived MSCs (BM-MSC) were employed as controls.
RESULTS: NB-MSCs presented the typical MSC morphology and phenotype. They showed a proliferative capacity superimposable to BM-MSCs. Stemness marker expression (Sox2, Nanog, Oct3/4) was comparable to BM-MSCs. NB-MSC in vitro osteogenic and chondrogenic differentiation was similar to BM-MSCs, but NB-MSCs lacked adipogenic differentiation capacity. NB-MSCs reached senescence phases at a median passage of P7 (range, P5-P13). NB-MSCs exhibited greater immunosuppressive capacity on activated T lymphocytes at a 1:2 (MSC: PBMC) ratio compared with BM-MSCs (p = 0.018). NK cytotoxic activity was not influenced by co-culture, either with BM-MSCs or NB-MSCs. Flow-cytometry cell cycle analysis showed that NB-MSCs had an increased number of cells in the G0-G1 phase compared to BM-MSCs. Transcriptomic profiling results indicated that NB-MSCs were enriched with EMT genes compared to BM-MSCs.
CONCLUSIONS: We characterized the biological features, the immunomodulatory capacity and the gene expression profile of NB-MSCs. The NB-MSC gene expression profile and their functional properties suggest a potential role in promoting tumor escape, invasiveness and metastatic traits of NB cancer cells. A better understanding of the complex mechanisms underlying the interactions between NB cells and NB-derived MSCs should shed new light on potential novel therapeutic approaches.

Ovarian cancer is one of the most serious diseases worldwide and the fifth-most common cancer among women. Celastrol, extracted from Thunder God Vine, exerts anti-cancer effects on various cancers; however, the mechanism underlying these anti-cancer effects in ovarian cancer needs further investigation. Herein, we investigated the anti-cancer efficacy of celastrol and its underlying mechanism in human ovarian cancer cell lines A2780, OVCAR3, and SKOV3. Celastrol significantly suppressed cell proliferation and migration in a dose-dependent manner. Celastrol resulted in a G2/M cell cycle arrest, accompanied with the down-regulation of Cyclin D1, CDK2, and CDK4. Celastrol induced apoptosis primarily via up-regulation of caspase-3, caspase-8, and Bax, and down-regulation of Bcl-2. Celastrol treatment inhibited the expression of stem cell marker CD44, Nanog, Klf4, and Oct4, and reduced a portion of the CD44

Cancer stem cells (CSCs), which are found in various types of human cancer, including pancreatic cancer, possess elevated metastatic potential, lead to tumor recurrence and cause chemoradiotherapy resistance. Alterations in cellular bioenergetics through the regulation of 5' adenosine monophosphate‑activated protein kinase (AMPK) signaling may be a prerequisite to stemness. Betulinic acid (BA) is a well‑known bioactive compound with antiretroviral and anti‑inflammatory potential, which has been reported to exert anticancer effects on various types of cancer, including pancreatic cancer. The present study aimed to investigate whether BA could inhibit pancreatic CSCs via regulation of AMPK signaling. The proliferation of pancreatic cancer cells was examined by MTT and colony formation assays. The migratory and invasive abilities of pancreatic cancer cells were assessed using wound‑scratch and Transwell invasion assays. In addition, the expression levels of candidate genes were measured by reverse transcription‑quantitative polymerase chain reaction and western blotting. The results revealed that BA inhibited the proliferation and tumorsphere formation of pancreatic cancer cells, suppressed epithelial‑mesenchymal transition (EMT), migration and invasion, and reduced the expression of three pluripotency factors [SRY‑box 2 (Sox2), octamer‑binding protein 4 (Oct4) and Nanog]. Furthermore, immunohistochemical analysis confirmed that there was a significant inverse association between the expression levels of phosphorylated (P)‑AMPK and Sox2 in pancreatic cancer, and it was revealed that BA may activate AMPK signaling. Notably, knockdown of AMPK reversed the suppressive effects of BA on EMT and stemness of pancreatic cancer cells. In addition, BA reversed the effects of gemcitabine on stemness and enhanced the sensitivity of pancreatic cancer cells to gemcitabine. Collectively, these results indicated that BA may effectively inhibit pluripotency factor expression (Sox2, Oct4 and Nanog), EMT and the stem‑like phenotype of pancreatic cancer cells via activating AMPK signaling. Therefore, BA may be considered an attractive therapeutic candidate and an effective inhibitor of the stem‑like phenotype in pancreatic cancer cells. Further investigation into the development of BA as an anticancer drug is warranted.

Migration of human glioma cells (hGCs) within the brain parenchyma makes glioblastoma one of the most aggressive and lethal tumors. Studies of the cellular and molecular mechanisms underlying hGC migration are hindered by the limitations of existing glioma models. Here we developed a dorsal root ganglion axon-oligodendrocyte-hGC co-culture to study in real time the migration and interaction of hGCs with their microenvironment. hGCs interact with myelinated and non-myelinated axons through the formation of pseudopodia. Isolation of pseudopodia-localized polysome-bound RNA reveals transcripts of Lck, Paxillin, Crk-II, and Rac1 that undergo local translation. Inhibition of Lck phosphorylation using a small-molecule inhibitor (Lck-I), blocks the phosphorylation of Paxillin and Crk-II, the formation of pseudopodia and the migration of hGCs. In vivo intraventricular administration of the Lck-I using an orthotopic xenograft glioma model, results in statistically significant inhibition of tumor size and significant down-regulation of Nanog-targeted genes, which are associated with glioblastoma patient survival. Moreover, treatment of human glioma stem cells (hGSCs) with Lck-I, results in significant inhibition of self-renewal and tumor-sphere formation. The involvement of Lck in different levels of glioma malignant progression, such as migration, tumor growth, and regulation of cancer stemness, makes Lck a potentially important therapeutic target for human glioblastomas.

BACKGROUND: Hypoxic tumor microenvironment and maintenance of stemness contribute to drug resistance in breast cancer. However, whether Hypoxia-inducible factor-2α (HIF-2α) in hypoxic tumor microenvironment mediates conversion to a stem cell phenotype and chemoresistance of breast tumors has not been elucidated.
METHODS: The mRNA and protein expressions of HIF-1α, HIF-2α, Wnt and Notch pathway were determined using qRT-PCR and western blot. Cell viability and renew ability were assessed by MTT, Flow cytometric analysis and soft agar colony formation.
RESULTS: In our study, acute hypoxia (6-12 h) briefly increased HIF-1α expression, while chronic hypoxia (48 h) continuously enhanced HIF-2α expression and induced the resistance of breast cancer cells to Paclitaxel (PTX). Furthermore, HIF-2α overexpression induced a stem cell phenotype, the resistance to PTX and enhanced protein expression of stem cell markers, c-Myc, OCT4 and Nanog. Most importantly, Wnt and Notch signaling, but not including Shh, pathways were both activated by HIF-2α overexpression. Dickkopf-1 (DKK-1), a Wnt pathway inhibitor, and L685,458, an inhibitor of the Notch pathway, reversed the resistance to PTX and stem phenotype conversion induced by HIF-2α overexpression. In addition, HIF-2α overexpression enhanced tumorigenicity and resistance of xenograft tumors to PTX, increased activation of the Wnt and Notch pathways and induced a stem cell phenotype in vivo.
CONCLUSION: In conclusion, HIF-2α promoted stem phenotype conversion and induced resistance to PTX by activating Wnt and Notch pathways.

The poor treatment outcomes of pancreatic cancer are linked to an enrichment of cancer stem cells (CSCs) in these tumors, which are resistant to chemotherapy and promote metastasis and tumor recurrence. The present study investigated an extract from the root of the medicinal plant Rauwolfia vomitoria (Rau) for its activity against pancreatic CSCs. In vitro tumor spheroid formation and CSC markers were tested, and in vivo tumorigenicity was evaluated in nude mice. Rau inhibited the overall proliferation of human pancreatic cancer cell lines with a 50% inhibitory concentration (IC50) ranging between 125 and 325 µg/ml, and showed limited cytotoxicity towards normal epithelial cells. The pancreatic CSC population, identified using cell surface markers or a tumor spheroid formation assay, was significantly reduced, with an IC50 value of ~100 µg/ml treatment for 48 h and ~27 µg/ml for long-term tumor spheroid formation. The levels of CSC-related gene Nanog and nuclear β-catenin were decreased, suggesting suppression of the Wnt/β-catenin signaling pathway. In vivo, 20 mg/kg of Rau administered five times per week by oral gavage significantly reduced the tumorigenicity of PANC-1 cells in immunocompromised mice. Taken together, these data showed that Rau preferentially inhibited pancreatic cancer stem cells. Further investigation is warranted to examine the potential of Rau as a novel treatment for pancreatic cancer.

Glioma is a devastating cancer with a dismal prognosis and there is an urgent need to discover novel glioma-specific antigens for glioma therapy. Previous studies have identified CD163-positive tumour cells in certain solid tumours, but CD163 expression in glioma remains unknown. In this study, via an analysis of public datasets, we demonstrated that CD163 overexpression in glioma specimens correlated with an unfavourable patient prognosis. CD163 expression was increased in glioma cells, especially primary glioma cells. The loss of CD163 expression inhibited both cell cycle progression and the proliferation of glioblastoma multiforme (GBM) cell lines and primary glioma cells. CD163 interacted directly with casein kinase 2 (CK2) and CD163 silencing reduced AKT/GSK3β/β-catenin/cyclin D1 pathway activity via CK2. Moreover, CD163 was upregulated in CD133-positive glioma stem cells (GSCs), and CD163 downregulation decreased the expression of GSC markers, including CD133, ALDH1A1, NANOG and OCT4. The knockdown of CD163 impaired GSC stemness by inhibiting the CK2/AKT/GSK3β/β-catenin pathway. Finally, a CD163 antibody successfully induced complement-dependent cytotoxicity against glioma cells. Our findings indicate that CD163 contributes to gliomagenesis via CK2 and provides preclinical evidence that CD163 and the CD163 pathway might serve as a therapeutic target for glioma.

BACKGROUND: Cancer stem cells (CSC) are characterized by deregulated self-renewal, tumorigenicity, metastatic potential, aberrant stemness signaling pathways, resistance to conventional therapy, and the ability to give rise to a progeny of proliferating cells that constitute the bulk of tumors. Targeting CSC will provide novel treatments for cancer. Different investigations have focused on developing complementary approaches that involve natural compounds that decrease chemo-resistance and reduce the side effects of conventional therapies. Since, it has been reported that molecular iodine (I
METHODS: HeLa and SiHa cervical cancer cells were treated with 200uM I
RESULTS: In the present study, monolayer and CSC-enriched cultures (cervospheres) from cervical cancer-derived cell lines, HeLa and SiHa, showed that 200uM I
CONCLUSIONS: All this data led us to suggest a clinical potential use of I