CD133 Protein Levels Research Articles

Perillaldehyde inhibits bone metastasis and receptor activator of nuclear factor-κB ligand (RANKL) signaling-induced osteoclastogenesis in prostate cancer cell lines

ABSTRACT Perillaldehyde (PAH), one of the active ingredients of the traditional Chinese medicine (TCM) plant Perilla frutescens, is widely used and exerts crucial anti-cancer activities. The aim of current study is to illustrate the potential mechanisms of PAH-mediated regulation of bone metastasis and osteoclastogenesis in prostate cancer (PCa) cell lines. Effects of PAH on proliferation, invasion and migration of PC-3 cells were assessed with the Cell Counting Kit-8 (CCK-8) assay and Transwell assays, respectively. Effects of PAH on stem cell characteristics of PC-3 cells were evaluated by cell-matrix adhesion assay, colony formation assay, spheroid formation assay, as well as western blot . The anti-metastasis and anti-osteoclastogenesis activity of PAH in RAW264.7 cells was examined by osteoclast differentiation assay and western blot. The protein levels of CD133 and CD44 in PC-3 cells and the activity of nuclear factor kappa B (NF-κB) signaling pathway in RAW264.7 cells were measured by western blot. PAH suppressed proliferation, invasion and migration of PC-3 cells, prevented stem cell characteristics including cell-matrix adhesion, colony formation, spheroid formation as well as CD133 and CD44 expression. PAH inhibited bone metastasis and osteoclastogenesis via repressing the activation of NF-κB pathway as well as (RANKL) – and cancer cell-induced osteoclastogenesis in PCa cells. These findings suggested the potential therapeutic effects of PAH on the metastasis of patients with PCa.

Nanos3, a cancer-germline gene, promotes cell proliferation, migration, chemoresistance, and invasion of human glioblastoma

BackgroundRadiotherapy, chemotherapy, and surgery have made crucial strides in glioblastoma treatment, yet they often fail; thus, new treatment and new detection methods are needed. Aberrant expression of Nanos3 has been functionally associated with various cancers. Here, we sought to identify the clinical significance and potential mechanisms of Nanos3 in human glioblastoma.MethodsNanos3 expression was studied in nude mouse glioblastoma tissues and glioblastoma cell lines by immunohistochemistry, Western blot, and RT-PCR. Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 gene editing assay was performed to generate the Nanos3 knockdown glioblastoma cell lines. The effects of Nanos3 on glioblastoma cells proliferation, migration, invasion, chemoresistance, germ cell characteristics, and tumor formation were analyzed by CCK8, transwell, cell survival experiments and alkaline phosphatase staining in vitro and in nude mouse models in vivo. Correlation between the expression of stemness proteins and the expression of Nanos3 was evaluated by Western blot.ResultsWe found that Nanos3 was strongly expressed in both glioblastoma cell lines and tissues. Western blot and sequencing assays showed that the Nanos3 knockdown glioblastoma cell lines were established successfully, and we discovered that Nanos3 deletion reduced the proliferation, migration, and invasion of glioblastoma cells in vitro (P < 0.05). Nanos3 knockdown enhanced the sensitivity of glioblastoma cells to doxorubicin (DOX) and temozolomide (TMZ) (P < 0.05), and Nanos3+/− glioblastoma cell lines did not show the characteristics of the germline cells. In addition, Nanos3 deletion inhibited subcutaneous xenograft tumor growth in vivo (P < 0.001). Moreover, the oncogenesis germline protein levels of CD133, Oct4, Ki67, and Dazl decreased significantly in glioblastoma cells following Nanos3 knockdown.ConclusionsBoth in vitro and in vivo assays suggest that Nanos3, which is a cancer-germline gene, initiates the tumorigenesis of glioblastoma via acquiring the oncogenesis germline traits. These data demonstrate that ectopic germline traits are necessary for glioblastoma growth.

Disruption of crosstalk between LX-2 and liver cancer stem-like cells from MHCC97H cells by DFOG via inhibiting FOXM1.

Hepatic stellate cell (HSC) line LX-2 is activated by liver cancer stem-like cells (LCSLCs) and produces various cytokines that make up most of the hepatocellular carcinoma (HCC) microenvironment. The new genistein derivative, 7-difluoromethoxyl-5,4'-di-n-octylgenistein (DFOG), shows anticancer effects in multiple malignancies by controlling forkhead box M1 (FOXM1). In this study, we aimed to assess whether DFOG disrupts the crosstalk between human HSC LX-2 cells and LCSLCs. Distinct generations of MHCC97H-derived spheres were obtained with the second generation considered as LCSLCs which displayed enhanced self-renewal ability and elevated expression levels of CD133, CD44, and EpCAM proteins, as well as tumorigenicity, as revealed by colony formation assay in vitro and tumorigenicity assay in vivo. LX-2 and MHCC97H cells were co-cultured with/without DFOG (1, 5, and 10μM, respectively) using the transwell system. FOXM1 overexpression and/or knockdown were employed for mechanistic investigations. Our results suggested that Co-CM promoted LX-2 cell transformation into liver cancer-associated HSCs. Meanwhile, FOXM1 was up-regulated and the level of hepatocyte growth factor (HGF) was increased in LX-2 cells and in the supernatant after Co-CM stimulation. Sphere and colony formation abilities in MHCC97H cells, and protein levels of CD133, CD44, and EpCAM, were also markedly elevated. DFOG dose-dependently inhibited the above effects, similar to FOXM1 knockdown in LX-2 cells. FOXM1 overexpression reversed the inhibitory effects of DFOG or FOXM1 knockdown or both on LX-2 cell activation and LCSLC feature induction in MHCC97H cells by LCSLC/LX-2 co-culture. This study demonstrated that DFOG disrupts the crosstalk between HSCs and LCSLCs to suppress LCSLC features via down-regulating FOXM1 expression and reducing HGF secretion in HSCs.

Isovitexin Suppresses Cancer Stemness Property And Induces Apoptosis Of Osteosarcoma Cells By Disruption Of The DNMT1/miR-34a/Bcl-2 Axis.

BackgroundIsovitexin (apigenin-6-C-glucoside, ISOV) is a natural flavonoid that exhibits tumor suppressive activity on various types of cancer. However, it is unknown whether the mechanism of its action in osteosarcoma (OS) is associated with epigenetic regulation and whether it involves DNA methyltransferase 1 (DNMT1), microRNAs and their targets.Materials and methodsThe present study investigated the effects of ISOV on DNMT1 activation and miR-34a and Bcl-2 expression levels in order to explain the mechanism underlying ISOV-mediated repression of proliferation and stemness. In addition, the induction of apoptosis in the spheres derived from OS cells was investigated.ResultsThe results indicated that ISOV significantly repressed survival, induced apoptosis and decreased the level of CD133, CD44, ABCG2 and ALDH1 mRNA in the spheres derived from U2OS (U2OS-SC) and MG63 cells (MG63-SC). ISOV further reduced the sphere formation rate of U2OS-SC and MG63-SC. It is important to noted that, ISOV inhibited tumor growth and reduced tumor size of U2OS-SC xenografts in nude mice, which was accompanied by decreased CD133 protein levels, elevated apoptotic index, downregulation of proliferating cell nuclear antigen (PCNA) expression, reduced DNMT1 activity and expression, increased miR-34a and decreased Bcl-2 levels. We identified that Bcl-2 as a direct functional target of miR-34a. Furthermore, ISOV exhibited a synergistic effect with 5-aza-2′-deoxycytidine, the miR-34a mimic or ABT-263 in order to repress cell survival, induce apoptosis, downregulate CD133, CD44, ABCG2 and ALDH1 mRNA expression levels and reduce sphere formation rates of U2OS-SC and MG63-SC cells.ConclusionThe findings suggested that ISOV-mediated epigenetic regulation involved the DNMT1/miR-34a/Bcl-2 axis and caused the suppression of stemness and induction of apoptosis in the spheres derived from OS cells. The data indicated that ISOV exhibited a novel efficient potential for the treatment of OS.

MicroRNA-363-3p/p21(Cip1/Waf1) axis is regulated by HIF-2α in mediating stemness of melanoma cells.

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.

Docetaxel promotes cell apoptosis and decreases SOX2 expression in CD133‑expressing hepatocellular carcinoma stem cells by suppressing the PI3K/AKT signaling pathway.

Docetaxel is a front‑line standard‑of‑care chemotherapeutic drug for the treatment of cancers. However, the underlying function and mechanism of docetaxel in human hepatocellular carcinoma (HCC) are uncertain. Therefore, the present study aimed to determine the effects of docetaxel on cell apoptosis and SOX2 expression in cultured human HCC stem cells. After human HCC stem cells were treated with docetaxel, cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT) method, the cell apoptotic rate was evaluated by flow cytometry, the expression of CD133 and sex determining region Y‑box 2 (SOX2) was determined by RT‑PCR and immunohistochemistry, and the protein levels of CD133, SOX2, phosphoinositide 3‑kinases (PI3K), AKT and phosphorylated AKT (p‑AKT) were analyzed by western blotting. The results indicated that SOX2 and CD133 were highly expressed in patients with HCC while their expression was significantly decreased after patients with HCC were treated with docetaxel. In vitro, docetaxel inhibited the proliferation while it enhanced the apoptosis of human CD133‑expressing HCC stem cells. Furthermore, lower expression of p‑AKT and SOX2 were revealed in the presence of docetaxel. Notably, docetaxel‑inhibited SOX2 expression and growth of human CD133‑expressing HCC stem cells were partially restricted following the block of the PI3K/AKT signaling pathway using the inhibitor LY294002. The present study collectively indicated that docetaxel promoted apoptosis and upregulated SOX2 expression of human HCC stem cells through the suppression of the PI3K/AKT signaling pathway.

Correlation of 18F-FDG uptake and thyroid cancer stem cells.

2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (18F-FDG PET) has the potential to detect various types of cancers, including thyroid cancer (TC), at a potentially curable stage. Increased uptake of 18F-FDG was observed in anaplastic and poorly differentiated thyroid cancer cells, and PET-positive tumors are more likely to be resistant to 131I treatment. As cancer stem cells (CSCs) possess a dedifferentiated phenotype and are resistant to many anticancer therapies, we hypothesized that the expression of CSC-related markers is correlated with the ability of tumor cells in TC to uptake FDG. The present study cohort included 12 patients with TC, who underwent 18F-FDG PET/CT imaging before surgery. Quantitative polymerase chain reaction (QPCR) and immunohistochemical (IHC) staining were performed to analyze the expression patterns of gene markers related to embryonic stem (ES) cells and CSCs in TC. The mRNA expression levels of CSC- (CD133 and CD44) and ES-related genes (Oct4 and Nanog) were higher in TC tissue than in normal thyroid tissue, whereas the mRNA expression levels of thyroid-specific genes (Tg, TSHR, and TTF1) were higher in normal thyroid tissue than in TC tissue. There was a positive and statistically significant correlation between FDG uptake (SUV<inf>max</inf>) of tumor and relative mRNA levels of CD133, CD44, Oct4, and Nanog. The IHC results demonstrated that CD133 and Nanog were expressed in TC tissue but not in normal thyroid tissue, however, CD44 expression was observed in both TC and normal thyroid tissue. Comparisons of the clinicopathological parameters between TC tissues with low and high SUV<inf>max</inf> demonstrated significant differences in protein level of CD133 but not in that of Nanog. The pre-therapeutic tumor SUV<inf>max</inf> obtained from 18F-FDG PET/CT may be a potential predictor for evaluating the proportion of CSC population in individual patients with TC.

CD133 correlation with chemoradiotherapy resistance in rectal cancer

CD133 is a type of a biomarker of rectal cancer stem cells. Recent research suggested that colorectal cancer stem cells have the radiotherapy and chemotherapy resistance. This study intends to explore CD133 expression in rectal cancer and its correlation with Chemoradiotherapy (CRT) resistance. Cancer tissues were collected from 56 rectal cancer patients receiving surgical treatment and CRT. CD133 positive expression rate in cancer tissue was detected by immunohistochemistry. Colorectal cancer cell line HT-29 was transfected with CD133 plasmid. CD133 protein level was determined by Western blot. Cell survival rate was tested by colony formation assay. Immunohistochemistry results showed that CD133 positive rate in rectal cancer patients received CRT achieved 71.4%. Its positive rate reached 89.2% in patients with ineffective CRT treatment, which was obviously higher than that in effective treatment group as 36.8% (P<0.05). CD133 was successfully transfected to HT-29 cells, resulting in CD133 overexpression compared with control (P<0.05). Cell survival detection demonstrated that it was markedly higher in CD133 overexpressed HT-29 cells receiving radiotherapy (P<0.05). CD133 positive expression rate elevated in rectal cancer patients after CRT treatment. CD133 expression may be related to cancer cell CRT resistance.

Lidamycin decreases CD133 expression in hepatocellular carcinoma via the Notch signaling pathway.

Cluster of differentiation (CD)133 is considered a molecular marker of cancer stem cells in hepatocellular carcinoma. In the present study, the effect of lidamycin (LDM) on CD133 expression in hepatocellular carcinoma (Huh7 cells) was evaluated and the potential molecular mechanism was investigated. Flow cytometry analysis, as well as sorting, sphere formation and western-blot assays, were performed in vitro to explore the effects of LDM on CD133 expression. A subcutaneous tumor model in nude mice was used to observe the effects of LDM on tumor volume and CD133 protein in vivo. To investigate the potential underlying molecular mechanism, Notch signaling pathway activity was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction. The proportion of CD133+ cells and the expression of CD133 protein were revealed to be downregulated by LDM. Sphere formation of sorted CD133+ cells was suppressed 7 days after LDM treatment. In addition, LDM inhibited tumor volume formed from sorted CD133+ cells and CD133 protein level in vivo. LDM decreased the mRNA level of NOTCH1, Hes1 (Hes family BHLH transcription factor 1) and Hey1 (Hes-related family BHLH transcription factor with YRPW motif 1) genes; consequently, the protein expression of NOTCH1, Notch intracellular domain, Hes1 and Hey1 was decreased by LDM. Downregulation of the Notch signaling pathway by LDM was enhanced through combination with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. In brief, these data suggest that LDM suppresses CD133 expression via the Notch signaling pathway, indicating the potential mechanism of LDM on CD133 and the benefits for further clinical application.

Abstract 1904: Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells

Abstract Objectives: Recently, it has been shown that a subset population of cancer cells in a tumor, the so-called cancer stem cells (CSCs), majorly contributes to chemoresistance and the ability of a tumor to form recurrences and metastases. Since the response to the standard chemotherapeutic drug 5-fluorouracil (5-FU) in patients with colorectal cancer (CRC) leads only to an inadequate response, we aimed to synthesize new hybrid molecules by combining 5-FU with the plant-derived drug thymoquinone (TQ). Methods: The cytotoxic effect of hybrids, the single drugs and the combination was investigated by crystal violet assays. HCT116, HT29 and 5-FU-resistant colorectal tumor cells were used to assess the spheroid forming capability after treatment with different concentrations of the hybrid and the single drugs by staining with the fluorescent stem cell marker CDy1 and Hoechst 3324 after 21 days of incubation. Western Blot analysis for stem cell surface marker CD133 were performed to verify these results. We have analyzed the anticancer effects of the single drugs TQ and 5-FU and the most effective hybrid on deregulation of cancer signaling pathways utilizing the NANOSTRING cancer pathway panel. In addition, we performed a single cell cloning from spheroids to generate monoclonal cell lines with enriched CSC populations. In vivo effects were studied in the chick chorioallantoic membrane (CAM) assay and in mouse xenografts. Results: The 5FU-TQ hybrid has an increased cytotoxic effect towards CRC cells when compared to single drug treatments. The spheroid formation assay revealed the capability of the hybrid to reduce sphere number and size at fairly low concentrations (10, 20 µM), however at 50 µM there was a complete suppression of spheroid growth. NANOSTRING analysis showed that gene expression of main components of the PI3K/AKT signaling pathway was significantly down-regulated after treatment with all drugs but most prominently with the new hybrid. After data processing we found that AKT3, IGF1, COL11A1, FGF10, GNG7, EPO genes were downregulated in hybrid-treated cells, while CDKN1A, BAX, JUN, MDM2, FAS were upregulated. This was verified via Western Blotting for a panel of markers. Next, we investigated the effectiveness of the hybrid to kill clones having an enriched CSC population. Indeed, CD133 protein levels were significantly decreased in hybrid-treated clones. Histological analysis of in vivo CAM grafts showed that HCT116 control cells developed a well vascularized tumor mass whereas HCT116 cells that were treated with the hybrid showed no more vital tumor cells, but extended areas of necrosis, calcification, and desmoplasia. In vivo mouse xenografts verified the potential of this hybrid as an antitumor agent reducing tumor volume significantly. Conclusion: Our findings strongly suggest that the newly synthesized 5-FU-TQ hybrid might become a promising anticancer drug by directly targeting CSCs. Citation Format: Benardina Ndreshkjana, Aysun Çapcı Karagöz, Volker Klein, Pithi Chanvorachote, Julienne K. Muenzner, Kerstin Huebner, Chuanpit Ninsontia, Sara Steinmann, Abbas Agaimy, Maamoun Fatfat, Hala Gali-Muhtasib, Svetlana B. Tsogoeva, Regine Schneider-Stock. Novel 5-fluorouracil-thymoquinone hybrid kills colon cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1904. doi:10.1158/1538-7445.AM2017-1904

MiR-200b inhibits CD133+ glioma cells by targeting the AKT pathway

MicroRNA-200b (miR-200b) is a tumor suppressor in multiple tumor types, including gastric cancer, breast cancer, ovarian cancer and glioma. The biological significance of a known normal and cancer stem cell marker, CD133, remains elusive. The aim of the present study was to identify the function and mechinism of miR-200b in suppressing CD133+ glioma cells. CD133+ glioma cells were sorted by flow cytometry. The expression of miR-200b, Ki67, GAP43, GFAP and CD133 were tested by reverse transcription-quantitative polymerase chain reaction. The binding of miR-200b to prominin 1 (PROM1) was certificated by luciferase reporter assay. Cell proliferation was analyzed by bromodeoxyuridine staining. The protein level of CD133, p-AKT, AKT and Notch1 was detected by western blot analysis. Analysis of glioma samples revealed that CD133 expression is negatively associated with miR-200b. PROM1, which is the gene that codes CD133, was certified to be a target of miR-200b. miR-200b expression inhibited the stemness properties and division of the CD133+ glioma cells. Our results identified a miR-200b/CD133/PI3K/Akt signaling axis, exploring the fundamental role of miR-200b and CD133 in glioma stem cell behavior.

Interaction between CD133 and phosphatidylinositol 3-kinase p85 promote the chemoresistance capacity of gastric cancer cells

Objective By building stable CD133 knock-down and overexpression cell lines, we will study its effects on the ability of multiple chemoresistance and the possible relevant mechanism. Methods Firstly, we established KATOⅢ-shCD133 and MKN45-CD133 cell lines, and then tested the interference and overexpression efficiency of CD133 by Western blotting. Secondly, we detected the cells sensibility to 5-fluorouracil (5-Fu) and cisplatin (DDP) by cell counting kit-8 (CCK-8), and measured the protein level of chemoresistance related factors: P-gp, B cell lymphoma/leukemia-2 (bcl-2), B cell lymphoma/leukemia-2 associated X protein (bax) and the activation level of protein kinase B (Akt) by Western blotting. Finally, we examined the interaction of CD133 and p85 by immunoprecipitation. Results By Western blotting, we found the protein level of CD133 of the knock-down or overexpression cell lines were significantly lower or higher than the control group. Under the same drug concentrations, 5-Fu and DDP showed higher inhibition rate in the CD133 konck-down group than that of the vector group with the half maximal inhibitory concentration (IC50) value [(54.030 0±0.647 9) μmol/L vs. (133.300 0±4.918 0) μmol/L, P=0.005] and [(2.312 0±0.223 6) μmol/L vs. (24.590 0±2.159 0) μmol/L, P=0.001], while the overexpression group took the opposite sides with the IC50 value [(8.102 0±0.433 1) μmol/L vs. (2.217 0±0.132 4) μmol/L, P=0.010] and [(24.487 0±2.132 0) μmol/L vs. (11.620 0±1.534 0) μmol/L, P=0.030]. After stablyknock-down the expression of CD133, the protein levels of chemoresistance related factor: P-gp, bcl-2 and p-Akt were significantly lower, and the promoting apoptosis factor bax was significantly increased; the overexpression group was still the opposite. Conclusion CD133 could obviously increase the chemoresistance capacity of gastric cancer cells, which might be realized through the activation of phosphoinositide 3-kinase (PI3K)/Akt pathway by CD133-p85 interaction. Key words: Gastric cancer; CD133; Chemoresistance; Phosphatidylinositol 3-kinase p85

The Clinical Significance of miR-34a in Pancreatic Ductal Carcinoma and Associated Molecular and Cellular Mechanisms

Background: Pancreatic ductal adenocarcinoma (PDAC) exhibits poor prognosis and resistance to chemotherapy. This study was to identify the biomarkers associated with the progression, poor prognosis and chemoresistance of PDAC. Methods: miR-34a and miR-150 levels in the plasma and tissues from PDAC patients were measured by real-time PCR. Xenograft PDAC tumor models were established in mice by inoculation of CD133+ stem cells isolated from PDAC tumors. Protein expression was measured by Western blot. Results: The plasma miR-34a and miR-150 levels were significantly lower in PDAC patients than in patients with benign pancreatic lesions and in healthy subjects. The miR-34a and miR-150 levels in the tumor tissues were significantly lower than in pancreatic tissues with benign lesions. The protein levels of CD133, Notch1, Notch2 and Notch4 receptors in PDAC tumor tissues were significantly higher than in pancreatic tissues with benign lesions. miR-34a injection significantly inhibited the tumor growth of PDAC tumors and sensitized the anticancer effects of 5-fluorouracil (5-FU). miR-34a significantly inhibited Notch1, Notch2 and Notch4 expression in xenograft tumor tissues in vivo and BxPC-3 cells in vitro. miR-34a and miR-150 significantly induced apoptosis and inhibited proliferation, invasion and migration in BxPC-3 cells. miR-34a, but not miR-150, significantly sensitized the anticancer effect of 5-FU in BxPC-3 cells in vitro. Conclusion: A loss of expression of miR-34a, but not of miR-150, is associated with disease progression and poor prognosis in PDAC patients, and may be involved in the chemoresistance of PDAC cells.

Signal transducer and activator of transcription 3-mediated CD133 up-regulation contributes to promotion of hepatocellular carcinoma.

Enhanced expression of the cancer stem cell (CSC) marker, CD133, is closely associated with a higher rate of tumor formation and poor prognosis in hepatocellular carcinoma (HCC) patients. Despite its clinical significance, the molecular mechanism underlying the deregulation of CD133 during tumor progression remains to be clarified. Here, we report on a novel mechanism by which interleukin‐6/signal transducer and activator of transcription 3 (IL‐6/STAT3) signaling up‐regulates expression of CD133 and promotes HCC progression. STAT3 activated by IL‐6 rapidly bound to CD133 promoter and increased protein levels of CD133 in HCC cells. Reversely, in hypoxic conditions, RNA interference silencing of STAT3 resulted in decrease of CD133 levels, even in the presence of IL‐6, with a concomitant decrease of hypoxia‐inducible factor 1 alpha (HIF‐1α) expression. Active STAT3 interacted with nuclear factor kappa B (NF‐κB) p65 subunit to positively regulate the transcription of HIF‐1α providing a mechanistic explanation on how those three oncogenes work together to increase the activity of CD133 in a hypoxic liver microenvironment. Activation of STAT3 and its consequent induction of HIF‐1α and CD133 expression were not observed in Toll‐like receptor 4/IL‐6 double‐knockout mice. Long‐term silencing of CD133 by a lentiviral‐based approach inhibited cancer cell‐cycle progression and suppressed in vivo tumorigenicity by down‐regulating expression of cytokinesis‐related genes, such as TACC1, ACF7, and CKAP5. We also found that sorafenib and STAT3 inhibitor nifuroxazide inhibit HCC xenograft formation by blocking activation of STAT3 and expression of CD133 and HIF‐1α proteins. Conclusion: IL‐6/STAT3 signaling induces expression of CD133 through functional cooperation with NF‐κB and HIF‐1α during liver carcinogenesis. Targeting STAT3‐mediated CD133 up‐regulation may represent a novel, effective treatment by eradicating the liver tumor microenvironment. (Hepatology 2015;62:1160‐1173)

Abstract 4224: CD133 knockdown sensitizes melanoma to kinase inhibitors

Abstract Melanoma is an aggressive and lethal form of cancer, responsible for 9,710 deaths in the US every year, as indicated by the Surveillance Epidemiology and End Results (SEER) program. Although early stages are surgically treatable, the advanced or metastatic stages are the most fatal, presumably due to resistance to chemotherapy, radiation or other therapies. Refractoriness of metastatic melanoma is due to self-renewal properties of a subpopulation of resistant cells, which may be the same as what have been termed “melanoma initiating cells” (MIC), many of which have been associated with the expression CD133. CD133 (PROM-1) is a 5-transmembrane-glycoprotein that is a cell surface marker for different cancers including late-stage cutaneous melanoma. By magnetic activated cell sorting, we isolated a CD133-positive population derived from a patient harboring a difficult-to-treat NRASQ61R/BRAFWT profile. The MIC were transfected with CD133-specific small-interfering RNA (siRNAs) to deplete CD133 protein levels in cells. Our results show that knocking down CD133 in NRASQ61R/BRAFWT mutant melanoma renders cells more sensitive to clinically employed-MEK/BRAF inhibitors. Thus, specific gene knockdown or small molecule targeting, along with the multikinase inhibitors, may be a high potency therapeutic of late-stage and recurrent melanoma. Analyses of patient-derived melanomas harboring different mutation signatures are currently in progress to determine if CD133 and MIC play broader roles in melanoma chemoresistance. Citation Format: Maryam AbdusSamad, Anirudh Gaur, Hengbo Zhou, John L. Zapas, Cynthia M. Simbulan-Rosenthal, Edward C. McCarron, Dean S. Rosenthal. CD133 knockdown sensitizes melanoma to kinase inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4224. doi:10.1158/1538-7445.AM2015-4224

Effects of stromal cells on sentitivity to imatinib in Sup-B15 Philadelphia chromosome positive acute lymphoblastic leukemia cells

目的探讨基质细胞OP9能否影响Ph+急性淋巴细胞白血病细胞株Sup-B15细胞对伊马替尼的敏感性以及作用机制。方法实验分为Sup-B15细胞组及基质细胞OP9与Sup-B15共培养组（即Sup-B15/OP9组）。采用CCK-8法测定细胞增殖抑制率；流式细胞术检测Annexin V/7-AAD标记的细胞凋亡率、CD34+CD38−细胞比例；荧光定量PCR检测细胞ALDH1、CD144、β-catenin mRNA水平；Western blot法检测细胞CD133、CD144、β-catenin蛋白表达水平；免疫共沉淀方法检测结合于CD144的β-catenin水平。结果10~45 µmol/L伊马替尼对Sup-B15、Sup-B15/OP9细胞均有增殖抑制作用，其IC50值分别为26.3和35.8 µmol/L，两组差异有统计学意义（P<0.05）。30 µmol/L伊马替尼作用于Sup-B15、Sup-B15/OP9细胞24 h，细胞总凋亡率分别为（14.24±2.11）%和（3.45±0.68）%，两组差异有统计学意义（P<0.05）。与Sup-B15组相比，Sup-B15/OP9组CD34+CD38−细胞比例增高[（3.42±0.28）%对（2.16±0.15）%，P<0.05]，ALDH1 mRNA水平增高（0.097±0.012对0.046±0.010，P<0.05），CD133蛋白表达水平升高；CD144 mRNA水平增高（0.103±0.015对0.010±0.003，P<0.05），CD144蛋白表达水平亦明显增高；β-catenin mRNA水平无明显变化（P>0.05），其蛋白总量明显上升，转位到细胞核的β-catenin蛋白明显增加，结合于CD144的β-catenin蛋白增加。结论与OP9细胞共培养后，Sup-B15细胞对伊马替尼敏感性下降，该作用可能与上调CD144表达、稳定CD144 /β-catenin信号通路、增加β-catenin核转位有关。

Expression of cancer stem cell marker during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis.

One of the theories regarding oral carcinogenesis is that the tumor growth is initiated from cancer stem cells (CSCs) that self-renew and give rise to differentiated tumor cells, like stem cells do in normal tissues. The most common methods of CSC identification are based on CSC marker expression in carcinogenesis. This study examined the expression of CD133 and CD44, the most commonly used CSC biomarkers in oral squamous cell sarcoma (SCC), with the goal of identifying molecular biomarkers whose expression is associated with the multistep oral carcinogenesis. The expression of CD133, CD44, proliferating cell nuclear antigen (PCNA), and Cytokeratin (CK) was examined by Western blot analysis and confirmed by immunohistochemistry in a 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis model. Also, the expression of aldehyde dehydrogenase 1 (ALDH1), OCT-4 and Nanog were investigated for alteration of cancer cell stemness by Western blot. Along with the progress of multistep carcinogenesis, there were slight increases of CD133 and CD44 expression in the dysplasia group compared with normal rats. However, CD133 protein level was significantly overexpressed in SCC. The expression of PCNA and CK were low in normal group, but sequentially increased in SCC. ALDH1, Nanog and OCT-4 expression were significantly increased according to SCC grade during carcinogenesis. The findings indicate that CD133 is useful in identifying oral CSCs, which suggests that CD133 may serve as a predictor to identify CSCs with a high risk of oral cancer development.

CD133 and CD133‐regulated nucleophosmin linked to 5‐fluorouracil susceptibility in human colon cancer cell line SW620

Cancer stem cells (CSCs) are known to be resistant to conventional chemotherapy and radiotherapy. Specific CSC targeting and eradication is therefore a therapeutically important challenge. CD133 is a colorectal CSC marker with unknown function(s). Assessing proteomic changes induced by CD133 may provide clues not only to new CD133 functions but also to the chemotherapy and radiation susceptibility of colon cancer cells. To identify the proteins affected by CD133, CD133-positive (CD133+), and CD133-negative (CD133-) human colon cancer cells were obtained by cell sorting. Whole proteomes were profiled from SW620/CD133+ and SW620/CD133- cells and analyzed by 2D-based proteome analysis. Nucleophosmin (NPM1) was identified as a protein regulated by CD133. CD133 protein level was not affected by NPM1, and an interaction between the two proteins was not observed. CD133 and NPM1 protein levels were positively correlated in 11 human colon cancer cell lines. The CD133+ subpopulation percentage or its value normalized against CD133 protein level was only linked to intrinsic susceptibility of human colon cancer cells to 5-fluorouracil (5-FU). However, either suppression of CD133 or NPM1 significantly increased 5-FU susceptibility of SW620. The present study suggests that CD133-regulated NPM1 protein level may provide a clue to novel CD133 function(s) linked to human colon cancer cell susceptibility to chemotherapy.

Abstract 3376: CD133 plays a critical role in epithelial-mesenchymal transition related to pancreatic cancer migration and invasion

Abstract Objective: Pancreatic cancer is an exceptionally devastating and incurable disease due to early local invasion and distant metastasis. Accumulated evidence shows that certain cells within solid tumors may reactivate a latent embryonic progress, epithelial-mesenchymal transition (EMT), during cancer progression. Through EMT, pancreatic tumor epithelial cells can acquire mesenchymal traits which facilitate migration and invasion. Previously, we reported that CD133-expressing cancer stem-like cells play an important role in tumorigenesis and chemoresistance of pancreatic cancer. In the present study we investigate whether CD133 plays a role in EMT using highly migrated cells enriched by a migration assay system. Methods: Expression of EMT characteristic markers, E-cadherin and vimentin, was examined in pancreatic cancer cell line, Capan-1. Highly migratory cell line Capan1-M9 was established by migration and invasion assay confirmed. CD133 expression was detected by FACS, real-time RT-PCR and Western blot. EMT-related genes (slug, snail, E-cadherin, vimentin, occludin, desmoplakin) were examined by real-time RT-PCR and Western blot. shRNA-CD133-GFP was transfected into Capan1-M9 by lentivirus vector to knock down endogenous CD133. Results: Typical EMT, which showed suppressed E-cadherin and elevated vimentin levels, was observed in migrating Capan-1 cells. Using a migration transwell system, we specifically established that Capan1-M9 cells showed migratory and invasive abilities two- to three-fold, compared with parental cells. FACS analysis showed that CD133 was enriched by more than 90% in Capan1-M9 cells. Furthermore, the elevated mRNA and protein levels of CD133 were confirmed in Capan1-M9 by real-time RT-PCR and Western blot, respectively. Importantly, as demonstrated by real-time RT-PCR, slug and snail EMT-related transcription factors in Capan1-M9 were upregulated by seven- and two-fold, respectively, compared with parental cells. Simultaneously, levels of vimentin mRNA expression increased two-fold. By contrast, occludin and desmoplakin cell-cell junction molecules were suppressed to half their previous level. Finally, shRNA-CD133-GFP achieved 85% knockdown of endogenous CD133, which not only inhibited migration and invasion but also suppressed slug EMT-related transcription factor expression to less than 20%. Conclusions: Our results suggest that CD133 contributes to migration and invasion of pancreatic cancer cells through a process which might trigger EMT, especially via slug upregulation. By targeting CD133-induced EMT in pancreatic cancer, transition to an invasive phenotype leading to metastasis may be preventable. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3376. doi:10.1158/1538-7445.AM2011-3376

Abstract 2459: Interferon-α contributes to combined chemotherapy for CD133+ cancer stem cells in pancreatic cancer

Abstract Objectives: Pancreatic cancer is a highly lethal disease because of high resistance to anti-cancer agents. Recent evidence suggests that a small population of cancer stem cells (CSCs) in solid tumors sustain tumor formation, self-renewal and drug resistance. CD133+ CSCs are reportedly essential for the development and perpetuation of pancreatic cancer and may account for resistance to standard chemotherapy drugs such as gemcitabine (GEM). Interferon-alpha (IFN-α) is a cytokine with pleiotropic effects, possessing direct cytotoxic and cytostatic effects on tumor cells. In the present study, we investigate the effect of IFN-α on combined chemotherapy in CD133+ pancreatic cancer cells. Methods: CD133+ and CD133− populations of Capan-1 cells, a human pancreatic cancer cell line, were isolated by FACS, and sphere formation assay and tumorigenic assay were performed. Capan-1 cells were treated by GEM with or without IFN-α and BrdU assay was used for cell-cycle analysis. Growth of xenograft tumors in nude mice was assessed after 3-weeks treatment with vehicle, GEM (1200 mg/kg/wk) alone, IFN-α (20000 U/mouse/every 2 days) alone or GEM combined with IFN-α. Results: 1) CD133+ population of Capan-1 cells showed CSC-like properties such as generation of spheres in the serum-free culture and tumorigenesis when implanted into NOD/SCID mice. 2) The growth inhibition by GEM treatment (IC50: 100 ng/mL) showed a significant difference between CD133+ and CD133− populations of Capan-1 cells, resulting in an increase of the CD133+ population. Similarly, CD133 protein levels increased in a time-dependent manner after GEM treatment by Western blot. 3) GEM treatment reduced S phase of both CD133+ and CD133− cells, and significantly induced apoptosis of CD133− cells more than that of CD133+ cells by BrdU assay. However, GEM treatment increased G0/G1 phase of CD133+ cells, indicating that CD133+ cells showed GEM resistance through the increase of G0/G1 phase. 4) All Capan-1 cells expressed IFN-α/β receptor II by FCM and immunohistochemical analyses and IFN-α (MED: 5000 U/mL) treatment reduced the CD133+ population ratio of Capan-1 cells. Importantly, IFN-α showed not only the decrease of G0/G1 phase but also the increase of S phase in CD133+ cells. Consequently, GEM combined with IFN-α treatment significantly increased the apoptotic phases in both CD133+ and CD133− cells. 5) In the in vivo experiment, GEM combined with IFN-α treatment similarly suppressed the tumor growth of Capan-1 cells. Conclusions: Our results indicate that IFN-α contributes to combined chemotherapy for CD133+ pancreatic cancer stem cells by modulating the cell cycle and/or cell proliferation, suggesting a clinical potential for treatment of pancreatic CSCs. Further investigation of the interactions between IFN-α and key regulatory pathways in CSC should be considered. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2459. doi:10.1158/1538-7445.AM2011-2459