CD133+ cells from medulloblastoma and PNET cell lines are more resistant to cyclopamine inhibition of the sonic hedgehog signaling pathway than CD133− cells

CD133 has recently been used as a reliable marker for brain tumor stem cells isolation. Sonic hedgehog (SHH) is implicated in medulloblastoma and central primitive neuroectodermal tumor (cPNET) formation. It has recently been suggested a role for the EWS/FLI1 fusion protein—typical of pPNET—in the upregulation of GLI1 and PTCH1 genes. Cyclopamine inhibits the SHH pathway in medulloblastoma cell lines, but its effect on cPNET and pPNET cell lines has not been well established yet. Our purpose was to study the effect of cyclopamine on medulloblastoma and PNET cell lines and to analyze whether CD133 expression might be able to modify this effect. We analyzed gene expression, cell viability, apoptosis, and tumorigenic capability before and after cyclopamine treatment in CD133 high-expressing and CD133 low-expressing cell lines. All medulloblastoma and PNET cell lines displayed an inhibitory effect on the expression of SHH pathway genes, on viability, and on tumorigenic potential after treatment. Nevertheless, CD133 expression made the cells more resistant to cyclopamine inhibition. These results open new doors to the understanding of CD133+ cancer stem cells as residual cells that might be responsible for treatment resistance.     

Lewis Y regulates signaling molecules of the transforming growth factor β pathway in ovarian carcinoma-derived RMG-I cells

LeY (Lewis Y) is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of LeY is frequently observed in epithelial-derived cancers and is correlated to pathological staging and prognosis. To study the role of LeY on cancer cells, a stably LeY-overexpressing cell line, RMG-I-H, was developed previously by transfection of the α1,2-fucosyltransferase gene, a key enzyme that catalyzes the synthesis of LeY, into ovarian carcinoma-derived RMG-I cells. Our studies have shown that LeY is involved in the changes in biological behavior of RMG-I-H cells. However, the mechanism is still largely unknown. In this study, we determined the structural relationship and co-localization between LeY and TβRI/TβRII, respectively, and the potential cellular signaling mechanism was also investigated. We found that both TβRI and TβRII contain the LeY structure, and the level of LeY in TβRI and TβRII in RMG-I-H cells was significantly increased. Overexpression of LeY up-regulates the phosphorylation of ERK, Akt and down-regulates the phosphorylation of Smad2/3. In addition, the phosphorylation intensity was attenuated significantly by LeY monoantibody. These findings suggest that LeY is involved in the changes in biological behavior through TGF?β receptors via Smad, ERK/MAPK and PI3K/Akt signaling pathways. We suggest that LeY may be an important composition of growth factor receptors and could be an attractive candidate for cancer diagnosis and treatment.     

Targeting the Ras–ERK pathway in pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma (PAC) stands as the poorest prognostic tumor of the digestive tract with limited therapeutic options. PAC carcinogenesis is associated with the loss of function of tumor suppressor genes such as INK4A, TP53, BRCA2, and DPC4, and only a few activated oncogenes among which K-RAS mutations are the most prevalent. The K-RAS mutation occurs early in PAC carcinogenesis, driving downstream activation of MEK and ERK1/2 which promote survival, invasion, and migration of cancer cells. In PAC models, inhibition of members of the Ras–ERK pathway blocks cellular proliferation and metastasis development. As oncogenic Ras does not appear to be a suitable drug target, inhibitors targeting downstream kinases including Raf and MEK have been developed and are currently under evaluation in clinical trials. In this review, we describe the role of the Ras–ERK pathway in pancreatic carcinogenesis and as a new therapeutic target for the treatment of PAC.     

Study on the role of Ras/p-ERK signaling pathway in the reversing effect of As2O3 on multi-drug-resistance北大核心CSCD

Objective: The aim of this in vitro study is to assess the reversing effects of arsenic trioxide (As2O3) on multi-drug-resistance (MDR) of SGC7901/adriamycin (ADM) and determine the molecular mechanism associated with Ras/p-ERK downregulation in human gastric cancer cells. Methods: The reversing effects of As2O3 before and after the treatment with an ERK agonist were determined by methyl thiazolyl tetrazolium assay. The molecular mechanism involved was further studied by examining the expressions of Ras and phosphorylated p-ERK by immunocytochemical assay. The process was repeated in pre- and post-intervention of As2O3 and granulocyte colony stimulating factor (G-CSF), respectively. Changes in the cell cycle and apoptotic rates were determined by flow cytometry. Results: SGC7901/ADM was resistant to ADM, and As2O3 could reverse the drug resistance. After the treatment of As2O3 at a dose of 0.5 μmol/L for 48 h, the the multiple of reversed drug resistance was about 6.29 approximately, which was greater compared with that of the normal cells. After administration of As2O3, the IC50 decreased dramatically, which shows that As2O3 could reverse MDR. After the G-CSF intervention, the multiple of the reversing effects was only 4.72. Immunocytochemical assay showed higher expressions of Ras in SGC7901/ADM than in the parental cell line SGC7901/S. No obvious difference in the p-ERK expressions of these cell lines was observed. As2O3 could decrease both Ras and p-ERK expressions (q-test, P<0.01). After pre-treatment with the ERK agonist, Ras and p-ERK expressions decreased (t-test, P<0.01). Flow cytometry indicated that the proportion of G0-G1 cells and apoptosis rate were higher in the As2O3 - treated group (0.1 and 0.5 μmol/L) than in the control groups. And apoptotic rate of both the cells significantly decreased in the As2O 3 group of the same dose after treatment of G-CSF. Conclusion: As2O3 can reduce the MDR of gastric cancer in vitro via the Ras/p-ERK signaling transduction pathway.     

Fibroblast activation protein protects bortezomib-induced apoptosis in multiple myeloma cells through β-catenin signaling pathway

Multiple myeloma (MM) is a malignant plasma cells proliferative disease. The intricate cross-talk of myeloma cells with bone marrow microenvironment plays an important role in facilitating growth and survival of myeloma cells. Bone marrow mesenchymal stem cells (BMMSCs) are important cells in MM microenvironment. In solid tumors, BMMSCs can be educated by tumor cells to become cancer-associated fibroblasts (CAFs) with high expression of fibroblast activation protein (FAP). FAP was reported to be involved in drug resistance, tumorigenesis, neoplastic progression, angiogenesis, invasion, and metastasis of tumor cells. However, the expression and the role of FAP in MM bone marrow microenvironment are still less known. The present study is aimed to investigate the expression of FAP, the role of FAP, and its relevant signaling pathway in regulating apoptosis induced by bortezomib in MM cells. In this study, our data illustrated that the expression levels of FAP were not different between the cultured BMMSCs isolated from MM patients and normal donors. The expression levels of FAP can be increased by tumor cells conditioned medium (TCCM) stimulation or coculture with RPMI8226 cells. FAP has important role in BMMSCs mediated protecting MM cell lines from apoptosis induced by bortezomib. Further study showed that this process may likely through β-catenin signaling pathway in vitro. The activation of β-catenin in MM cell lines was dependent on direct contact with BMMSCs other than separated by transwell or additional condition medium from BMMSCs and cytokines.     

The prognostic role of TGF-β signaling pathway in breast cancer patients

BackgroundThe transforming growth factor-β (TGF-β) pathway has dual effects on tumor growth. Seemingly, discordant results have been published on the relation between TGF-β signaling markers and prognosis in breast cancer. Improved prognostic information for breast cancer patients might be obtained by assessing interactions among TGF-β signaling biomarkers.Patients and methodsThe expression of nuclear Smad4, nuclear phosphorylated-Smad2 (p-Smad2), and the membranous expression of TGF-β receptors I and II (TβRI and TβRII) was determined on a tissue microarray of 574 breast carcinomas. Tumors were stratified according to the Smad4 expression in combination with p-Smad2 expression or Smad4 in combination with the expression of both TGF-β receptors.ResultsTumors with high expression of TβRII, TβRI and TβRII, and p-Smad2 (P = 0.018, 0.005, and 0.022, respectively), and low expression of Smad4 (P = 0.005) had an unfavorable prognosis concerning progression-free survival. Low Smad4 expression combined with high p-Smad2 expression or low expression of Smad4 combined with high expression of both TGF-β receptors displayed an increased hazard ratio of 3.04 [95% confidence interval (CI) 1.390–6.658] and 2.20 (95% CI 1.464–3.307), respectively, for disease relapse.ConclusionsCombining TGF-β biomarkers provides prognostic information for patients with stage I–III breast cancer. This can identify patients at increased risk for disease recurrence that might therefore be candidates for additional treatment.     

Chemoresistance of CD133（＋） tumor stem cells from human brain glioma

OBJECTIVE： To explore the multidrug resistance （MDR） mechanism of ABC superfamily transporters in the tumor stem eells（TSC） from human brain ghoma tissues. METHODS： Samples of glioma were obtained from 30 patients undergoing microsurgical tumor resection. The CD133（＋） cells and CD133（-） cells from these tumor specimens were isolated by magnetic activated cell sorting（MACS）. These cells were cultured, proliferated and passaged. The protein and activity expression of multidrug-resistance protein 1 （MDR1） and multidrug-resistance associated protein 1（MRP1） were analyzed between CD133（＋） and CD133（-） cells by immunocytochemistty and RT-PCR respectively.[第一段]     

The Wnt/β-catenin signaling pathway is involved in the antitumor effect of fulvestrant on rat prolactinoma MMQ cells

Although an antiestrogen treatment for estrogen-dependent diseases, such as breast cancers, has been reported, the effect of this endocrine therapy on prolactinomas and its possible mechanism are unclear. This study investigates the antitumor effect of fulvestrant, which is a new estrogen receptor antagonist, on rat prolactinoma MMQ cells and the possible roles of the Wnt/β-catenin signaling pathway that is involved in this antitumor effect. To investigate the antitumor effect of fulvestrant, the effects of exposure to gradient doses of fulvestrant (0, 0.04, 1, 25, and 625 nM) on the proliferation of cells and the secretion of prolactin (PRL) were studied. Then, the expression levels of the Wnt/β-catenin signaling pathway-related proteins β-catenin and Wnt inhibitory factor-1 (WIF-1) were measured to investigate their possible roles in the antitumor effect of fulvestrant. The cells were also treated with decitabine (10 μM) to investigate the epigenetic mechanism of WIF-1 expression. The proliferation of MMQ cells and the secretion of PRL were suppressed by fulvestrant in a dose-dependent manner (up to 57.0?±?3.9 % and 51.2?±?4.9 %, respectively). β-Catenin expression was downregulated and was positively correlated with ER-α expression (P?<?0.01). As a tumor suppressor, WIF-1 expression was upregulated and was negatively correlated with ER-α expression (P?<?0.01). Furthermore, WIF-1 expression was upregulated via the hypomethylation of the promoter by decitabine, and cellular proliferation was correspondingly suppressed (37.8?±?4.3 %). Antitumor effect of fulvestrant was partially disrupted by SB 216763 via activation of the Wnt/β-catenin pathway. In conclusion, through the Wnt/β-catenin signaling pathway, fulvestrant can suppress the proliferation of MMQ cells and the secretion of PRL.     

CD24 enhances DNA damage-induced apoptosis by modulating NF-κB signaling in CD44-expressing breast cancer cells

Cluster of differentiation 24 (CD24) is a small glycosylphosphatidylinositol-linked cell surface molecule that is expressed in a variety of human carcinomas, including breast cancer. To determine the role of CD24 in breast cancer cells, we expressed CD24 in CD24-negative/low and cluster of differentiation 44 (CD44)-positive MDA-MB-231 metastatic breast cancer cells. Forced expression of CD24 resulted in a decrease in c-Raf/mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein kinase signaling and reduced cell proliferation. Apoptosis induced by DNA damage was greatly enhanced in MDA-MB-231 CD24 cells as compared with MDA-MB-231 vec cells. CD24 expression efficiently attenuated DNA damage-induced nuclear factor-kappaB (NF-κB) signaling in MDA-MB-231 cells. However, in CD24-positive and CD44-negative/low MCF-7 cells, knockdown of CD24 did not significantly affect DNA damage-induced apoptosis nor NF-κB signaling. Silencing of CD24 in CD24/CD44-double-positive MDA-MB-468 cells partially rescued DNA damage-induced apoptosis. Transient transfection studies with 293T cells also revealed that CD24 attenuated cell viability and NF-κB signaling only when CD44 was cotransfected. These data indicate that CD24 expression potentiated DNA-induced apoptosis by suppressing antiapoptotic NF-κB signaling in CD44-expressing cells.     

β1, 4-N-acetylgalactosaminyltransferase III modulates cancer stemness through EGFR signaling pathway in colon cancer cells

Cancer stem cells are cancer cells characterized with tumor initiating capacity. β1,4-N-acetylgalactosaminyltransferase III (B4GALNT3) synthesizes GalNAcβ1-4GlcNAc (LacdiNAc) which contributes to self-renewal of mouse embryonic stem cells. We previously showed that B4GALNT3 overexpression enhances colon cancer cell malignant phenotypes in vitro and in vivo. However, the role of B4GALNT3 in cancer stemness remains unclear. We found that B4GALNT3 expression was positively correlated with advanced stages and poor survival in colorectal cancer patients. Knockdown of B4GALNT3 using small interfering (si) RNAs in colon cancer cell lines (HCT116, SW480, HCT15, and HT29 cells) decreased sphere formation and the expression of stem cell markers, OCT4 and NANOG. The expression of B4GALNT3 was upregulated in colonospheres. Interestingly, we found that B4GALNT3 primarily modified N-glycans of EGFR with LacdiNAc by Wisteria floribunda agglutinin (WFA) pull down assays. B4GALNT3 knockdown suppressed EGF-induced phosphorylation of EGFR and its downstream signaling molecules. Furthermore, EGF-induced degradation of EGFR was facilitated. In addition, EGF-induced migration and invasion were significantly suppressed by B4GALNT3 knockdown. Taken together, these data suggest B4GALNT3 regulates cancer stemness and the invasive properties of colon cancer cells through modifying EGFR glycosylation and signaling. Our results provide novel insights into the role of LacdiNAc in colorectal cancer development.     

Wnt/β-catenin signaling is a key downstream mediator of MET signaling in glioblastoma stem cells

Background

Glioblastoma (GBM) is the most lethal and common type of primary brain tumor. Recent evidence suggests that a subpopulation of GBM cells (glioblastoma stem cells [GSCs]) is critical for tumor progression, invasion, and therapeutic resistance. We and others have demonstrated that MET, a receptor tyrosine kinase, positively regulates the stemness phenotype and radioresistance of GSCs. Here, we interrogated the downstream effector pathways of MET signaling in GSCs.

Methods

We have established a series of GSCs and xenograft tumors derived from freshly dissociated specimens from patients with GBM and characterized a subpopulation enriched with MET activation (MET^high/+). Through global expression profiling and subsequent pathways analysis, we identified signaling pathways that are enriched in MET^high/+ populations, one of which is Wnt/β-catenin signaling pathway. To determine molecular interaction and the biological consequences of MET and Wnt/β-catenin signaling, we used pharmacological and shRNA-mediated genetic inhibition and performed various molecular and cellular analyses, including flow cytometry, immunohistochemistry, and clonogenicity assays.

Results

We found that Wnt/β-catenin signaling is highly active in MET^high/+ cells, compared with bulk tumor cells. We also showed that Wnt/β-catenin signaling activities in GBM are directly modulated by the addition of ligand-mediated MET activation or MET inhibition. Furthermore, the ectopic expression of active-β-catenin (S37A and S45Y) rescued the phenotypic effects caused by MET inhibition.

Conclusion

These data suggest that Wnt/β-catenin signaling is a key downstream effector of MET signaling and contributes to the maintenance of GSC and GBM malignancy.     

The Wnt/β-catenin signaling pathway in the tumor microenvironment of hepatocellular carcinoma

The Wnt/β-catenin signaling pathway regulates many aspects of tumor biology, and many studies have focused on the role of this signaling pathway in tumor cells. However, it is now clear that tumor development and metastasis depend on the two-way interaction between cancer cells and their environment, thereby forming a tumor microenvironment(TME). In this review, we discuss how Wnt/β-catenin signaling regulates cross-interactions among different components of the TME, including immune cells, stem...     

Gene expression profiling and pathway analysis identify the integrin signaling pathway to be altered by IL-1β in human pancreatic cancer cells: role of JNK

CYR61 over-expression promotes cell proliferation by inhibiting carboplatin-induced apoptosis, decreasing Bax expression, and increasing Bcl-xL, Mcl-1, and Bcl-2. At the same time, down-regulating p53 expression, while up-regulated NF-κB expression. Additionally, p21 and p53 promoter activities were reduced, while NF-κB and Bcl-2 activities increased. In parallel, CYR61-expressing cells, during carboplatin-induced apoptosis, resulted in an increase of Akt phosphorylation, while rapamycin-treated cells were not affected. Carboplatin effectively inhibited the activation of mTOR signaling cascade, which includes mTOR, 4E-BP1, p70S6K, HIF-1α, and VEGF. These results provide evidence that CYR61 promotes cell proliferation and inhibits apoptosis.