CRISPR-Cas9 combinatorial KO of epigenetic regulators in human ovarian cancer cells
Ontology highlight
ABSTRACT: We sequenced polyA mRNA from OVCAR8-ADR-Cas9 cells in which one or two of 3 epigenetic regulators (BRD4, KDM4C, KDM6B) had been knocked out to examine how global gene expression was affected and evaluate potential synergistic effects at a molecular level. Gene expression data (RNA-Seq) in OVCAR8-ADR-Cas9 cells infected with control vector or vectors expressing gRNAs targeting one of 4 epigenetic regulators (BRD4, KDM4C, KDM6B) with biological replicates.
Project description:We sequenced polyA mRNA from OVCAR8-ADR-Cas9 cells in which one or two of 3 epigenetic regulators (BRD4, KDM4C, KDM6B) had been knocked out to examine how global gene expression was affected and evaluate potential synergistic effects at a molecular level.
Project description:Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and therapeutic targets.
Project description:Chemoresistance in breast cancer has been a great interest in past studies, however, the development of rational therapeutic strategies targeting chemoresistant cells is still a challenge for clinical oncology.The resistant property of MCF7/ADR cells was confirmed by long term culture with Dox, cell viability, and PARP cleavage assays. Microarray analysis was performed to compare the global differences of gene expression between MCF-7 and MCF-7/ADR cells. MCF-7 and MCF-7/ADR gene expression profiles were analyzed. Total RNA were prepared for analysis with Affymetrix Human U133 Plus 2.0 arrays according to the manufacturerâs instructions.
Project description:The cell surface receptor, Epithelial Cell Adhesion Molecule (EpCAM), is overexpressed in a variety of cancers and has been carefully examined for its functional role in carcinogenesis and its potential as a therapeutic target. However, little is known about the upstream molecular pathways that regulate EpCAM expression. To elucidate the mechanisms by which EpCAM expression is controlled, we designed a combinatorial screening strategy that utilized pooled shRNA gene knockdown with magnetically-activated cell separation for selection of cells with reduced EpCAM protein expression on the cell surface. The pooled RNAi screen was analyzed by comparing EpCAM-enriched and EpCAM-depleted cell populations for shRNA abundance using competitive hybridization to microarrays. Our RNAi screen and further validation efforts revealed that at least four genes (GRM1, SLITRK5, EFNA5, and IL23A) are important for EpCAM expression in the ovarian cancer cell line OVCAR8. Further investigations into the ephrin ligand, EFNA5, suggest that its activation by the Eph receptor, EPHA4, contributes to the regulation of EpCAM expression via a reverse signaling mechanism. Our screening strategy revealed novel mechanisms for the regulation of EpCAM expression and provides insight into the biology of this cancer-associated gene. Importantly, this study also demonstrates the utility of pooled RNAi screening in the identification of genes required for the regulation of cell surface protein expression. Two condition experiment; EpCAM-Bound or EpCAM-Unbound OVCAR8 cells vs Reference population of transduced cells without phenotypic selection; biological triplicate
Project description:Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. Here we report our comparative N-glycoproteomics study of MCF‐7/ADR vs. MCF-7 cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptides levels.
Project description:Background: Cancer stem cells are presumed to have virtually unlimited proliferative and self-renewal abilities and to be highly resistant to chemotherapy, a feature that is associated with overexpression of ATP-binding cassette transporters. We investigated whether prolonged continuous selection of cells for drug resistance enriches cultures for cancer stem-like cells. Methods: Cancer stem cells were defined as CD44+/CD24– cells that could self-renew (ie, generate cells with the tumorigenic CD44+/CD24– phenotype), differentiate, invade, and form tumors in vivo. We used doxorubicin-selected MCF-7/ADR cells, weakly tumorigenic parental MCF-7 cells, and MCF-7/MDR, an MCF-7 subline with forced expression of ABCB1 protein. Cells were examined for cell surface markers and side-population fractions by microarray and flow cytometry, with in vitro invasion assays, and for ability to form mammospheres. Xenograft tumors were generated in mice to examine tumorigenicity (n = 52). The mRNA expression of multidrug resistance genes was examined in putative cancer stem cells and pathway analysis of statistically significantly differentially expressed genes was performed. All statistical tests were two-sided. Results: Pathway analysis showed that MCF-7/ADR cells express mRNAs from ABCB1 and other genes also found in breast cancer stem cells (eg, CD44, TGFB1, and SNAI1). MCF-7/ADR cells were highly invasive, formed mammospheres, and were tumorigenic in mice. In contrast to parental MCF-7 cells, more than 30% of MCF-7/ADR cells had a CD44+/CD24– phenotype, could self-renew, and differentiate (ie, produce CD44+/CD24– and CD44+/CD24+ cells), and overexpressed various multidrug resistance-linked genes (including ABCB1, CCNE1, and MMP9). MCF-7/ADR cells were statistically significantly more invasive in Matrigel than parental MCF-7 cells (MCF-7 cells = 0.82 cell per field and MCF-7/ADR = 7.51 cells per field, difference = 6.69 cells per field, 95% confidence interval = 4.82 to 8.55 cells per field, P<.001). No enrichment in the CD44+/CD24– or CD133+ population was detected in MCF-7/MDR. Conclusion: The cell population with cancer stem cell characteristics increased after prolonged continuous selection for doxorubicin resistance. PARALLEL study design with 4 samples Parental MCF-7 cell line versus Doxorubicin Resistant MCF-7 cell sublines Biological replicates: 2 parental controls, 2 drug resistant, independently grown and harvested. agent:Selection agent is multi-step doxorubicin selection: MCF7226ng, MCF7262ng biological replicate: MCF71, MCF72 biological replicate: MCF226ng, MCF7262ng
Project description:The ovarian cancer biomarker HE4 has been shown to have multiple effects on ovarian cancer pathogenesis. In order to reveal gene regulation upstream of HE4 biological effects in ovarian cancer, HE4 was overexpressed in OVCAR8 wild type ovarian cancer cells and compared to null vector transfected cells. To gain a more comprehensive understanding of HE4 effects and elucidate similarities/differences between short-term HE4 exposure versus stable overexpression, we also compared untreated wild type cells with cells exposed to recombinant HE4 protein.
Project description:Currently, drug resistance of anti-cancer therapy has become the main cause of low survival rate and poor prognosis. Full understanding of drug resistance mechanisms is an urgent request for further development of anti-cancer therapy and improvement of prognosis. Here we present our N-glycoproteomics study of putative N-glycoprotein biomarkers of drug resistance in doxorubicin resistance breast cancer cell line michigan cancer foundation-7 (MCF-7/ADR) relative to parental michigan cancer foundation-7 (MCF-7) cells. Intact N-glycopeptides (IDs) from MCF-7/ADR and MCF-7 cells were enriched with zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), labeled with stable isotopic diethylation (SIDE), and analyzed with C18-RPLC-MS/MS (HCD with stepped normalized collision energies); these IDs were identifed with database search engine GPSeeker, and the diferentially expressed intact N-glycopeptides (DEGPs) were quantifed with GPSeekerQuan. With target-decoy searches and control of spectrum-level FDR≤1%, 322 intact N-glycopeptides were identifed; these intact N-glycopeptides come from the combination of 249 unique peptide backbones (corresponding to 234 intact N-glycoproteins) and 90 monosaccharide compositions (corresponding to 248 putative N-glycosites). The sequence structures of 165 IDs were confrmed with structure-diagnostic fragment ions. With the criteria of observation at least twice among the three technical replicates,≥1.5-fold change and p value<0.05, 20 DEGPs were quantifed, where fve of them were up-regulated and 15 of them were down-regulated; the corresponding intact N-glycoproteins as putative markers of drug resistance were discussed.
Project description:p85beta is one of the regulatory subunits of the PI3K. Apart from its canonical role in binding to and regulating the p110 catalytic subunit, the protein may bind to other interacting proteins to perform other biological functions. In this study, the protein binding partners of p85beta were determined by immunoprecipitation followed by mass spectrometry using samples of ovarian cancer cells OVCAR8.