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Expression data from CD133+ and CD133- glioma cells

ABSTRACT: Given the very substantial heterogeneity of most human cancers, it is likely that most cancer therapeutics will be active in only a small fraction of any population of patients. As such, the development of new therapeutics, coupled with methods to match a therapy with the individual patient, will be critical to achieving significant gains in disease outcome. One such opportunity is the use of expression signatures to identify key oncogenic phenotypes that can serve not only as biomarkers but also as a means of identifying therapeutic compounds that might specifically target these phenotypes. Given the potential importance of targeting tumors exhibiting a stem-like phenotype, we have developed an expression signature that reflects common biological aspects of various stem-like characteristics. The Consensus Stemness Ranking (CSR) signature is upregulated in cancer stem cell enriched samples, at advanced tumor stages and is associated with poor prognosis in multiple cancer types. Using two independent computational approaches we utilized the CSR signature to identify clinically useful compounds that could target the CSR phenotype. In vitro assays confirmed selectivity of several predicted compounds including topoisomerase inhibitors and resveratrol towards breast cancer cell lines that exhibit a high-CSR phenotype. Importantly, the CSR signature could predict clinical response of breast cancer patients to a neoadjuvant regimen that included a CSR-specific agent. Collectively, these results suggest therapeutic opportunities to target the CSR phenotype in a relevant cohort of cancer patients. CD133+ and CD133- cells were separated from two glioma xenograft tumors. Both CD133+ and CD133- glioma cells were cultured in serum-free media for 48 hours in the presence of absence of laminin.

ORGANISM(S): Homo sapiens

SUBMITTER: Nevins Joseph

PROVIDER: S-ECPF-GEOD-24716 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Tumor stem cells, postulated to be the source cells for malignancies, have been identified in several cancers using cell-surface expression of markers including CD133, a pentaspan membrane protein. CD133+ve cells form neurospheres, exhibit self-renewal and differentiation, and are tumorigenic. However, despite its association with stem cells, a causal relationship of CD133 to tumorigenesis remains to be defined. Hypothesizing that specific epigenetic and transcription factors implicated in driving the stem cell state may concurrently regulate CD133 expression in stem cells, we analyzed the structure and regulation of CD133 promoter in glioma stem cells and glioma cell lines. Initially, a minimal promoter region was identified by analyzing the activity of CD133 promoter-driven luciferase-expressing 5'-and 3'-deletion-constructs upstream of the transcription start site. This region contained a CpG island that was hypermethylated in CD133-ve glioma stem cells (GSC) and glioma cells but unmethylated in CD133+ve ones. Of several predicted TF-binding sites in this region, the role of tandem Sp1 (-242 and -221) and two Myc (-541 and -25)-binding sites were examined. Overexpression of Sp1 or Myc increased CD133 minimal promoter-driven luciferase activity and CD133 levels in GSC and in glioma cell line. Mithramycin, a Sp1 inhibitor, decreased minimal promoter activity and downregulated CD133 levels in GSC. Gel-shift assays demonstrated direct binding of Sp1 to their predicted sites that was competitively inhibited by oligonucleotide-binding-site sequences and supershifted by anti-Sp1 confirming the interaction. Sp1 and Myc-antibody chromatin immunoprecipitation (ChIP) analysis in GSC showed enrichment of regions with Sp1 and Myc-binding sites. In CD133-ve cells, ChIP analysis showed binding of the methyl-DNA-binding proteins, MBD1, MBD2 and MeCP2 to the methylated CpG island and repression of transcription. These results demonstrate that Sp1 and Myc regulate CD133 transcription in GSC and that promoter methylation and methyl-DNA-binding proteins cause repression of CD133 by excluding transcription-factor binding.

Project description:Energy metabolic reprogramming (EMR) allows for the rearrangement of a series of metabolic genes and proteins when tumor cells adapt to their microenvironment. EMR is characterized by changes in the metabolic pattern and metabolic intermediates to meet the needs of tumor cells for their malignant proliferation and infiltrative growth. The present study investigated the role of low-dose paclitaxel (PTX) in changing the expression levels of key genes and proteins during glycolysis in CD133+ U251 glioma cells and explored the relevant regulatory mechanisms of action at the molecular level. CD133 immunomagnetic beads were applied to malignant CD133+ U251 glioma cells, which were then divided into a negative control and an experimental group treated with 1, 2, 4 or 8 µM PTX for 72 h. Cell Counting Kit-8 (CCK-8) was used to measure U251 cell proliferation. RNA and protein were extracted from the malignant glioma cells in all groups to observe changes in the expression levels of key glycolytic enzymes, such as glucose transporter 1 (GLUT1), pyruvate kinase M (PKM) and lactate dehydrogenase A (LDHA), using reverse transcription-quantitative PCR and western blot assays. Transwell migration assays were performed to quantify the effects of PTX solution on U251 cells. CD133+ U251 glioma cells were isolated successfully. CD1133+ cells had a higher rate of proliferation compared with CD1133- cells. In CD1133+ cells treated with PTX, a dose-dependent reduction in the expression levels of the key glycolytic enzymes GLUT1, PKM and LDHA was observed at both the mRNA and protein levels. PTX solution also inhibited cell migration. Differences between the control and experimental groups were statistically significant (P<0.05). Since glycolysis plays an indispensable role in the proliferation and migration of stem cell-like glioma cells, PTX may inhibit tumor cell growth by downregulating the gene and protein expression levels of glycolytic enzymes in CD133+ glioma cells.

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Expression data from CD133+ and CD133- glioma cells

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