Project description:We analyzed the differentially expressed proteins in 37 resected tissue samples from osteosarcoma patients using paraffin- embedded specimens and 4D data-independent acquisition (DIA) quantitative mass spectrometry to investigate biomarkers for predicting the efficacy of doxorubicin/cisplatin chemotherapy
Project description:Chemoresistance is one of the major causes of poor prognosis in osteosarcoma. Thus far, the therapeutic alternative for osteosarcoma is quite limited, thereby increasing sensitivity of the currently used drugs is an effective way to improve outcome of patients. In this study, through a Kinome-wide CRISPR screen, we identified PRKDC as a critical determinant of Doxorubicin (DOX) sensitivity in osteosarcoma. Clinical sample analysis and functional experiments demonstrated that PRKDC is hyperactivated in osteosarcoma and that knockdown of PRKDC significantly increased sensitivity of osteosarcoma to DOX. Mechanistically, PRKDC could recruit and bind with GDE2 to augment the stability of GNAS. The increased GNAS protein subsequently activate Akt S473 and T308 phosphorylation and mediated DOX sensitivity. We also showed that the PRKDC inhibitor AZD7648 and DOX could synergize with each other and intensely suppressed the growth of osteosarcoma in mice xenograft models and human organoids. In conclusion, these results revealed a novel PRKDC-GDE2-GNAS-Akt regulatory axis in DOX sensitivity and provided targetable candidates for improving therapeutic efficacy of DOX in osteosarcoma.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
Project description:A genome-wide CRISPR knockout screen investigating resistance to doxorubicin and cytarabine (Dox/AraC) in human AML cell lines identified gene knockout of cyclin dependent kinase inhibitor 2A (CDKN2A) as contributing to resistance. To investigate the therapeutic potential of upregulating CDKN2A expression using WM-1119 and further elucidate its mechanism of action, we performed RNA-seq on WM-1119- (or vehicle control) treated OCI-AML3 cells with or without CRISPR mediated genetic depletion of CDKN2A.
Project description:Genome-wide CRISPR-Cas9 knockout screen using TKOv1 sgRNA library was performed in isogenic RBM10-proficient and RBM10-deficient HCC827 cells.
Project description:To search for factors regulating paternally imprinted genes (PEGs), we performed a genome-wide loss-of-function CRISPR/Cas9 screen in haploid parthenogenetic ESCs. This by staining a pooled CRISPR library with a PEG10 antibody and next FACS-sorted for cells that presented de-novo PEG10 expression.
Project description:Background: Cellular apoptosis is a central mechanism the chemotherapy leveraged to treat human cancers. Cytosine methylations installed on both DNA and mRNA are documented to regulate apoptosis independently. However, the interplay or crosstalk between them in cellular apoptosis has not yet been explored. Methods: Cisplatin and doxorubicin were used to induce cell apoptosis in three differed types of osteosarcoma cell lines, respectively. Bisulfite-PCR, MS-PCR, and MeDIP experiments were employed to evaluate promoter cytosine methylation, and MeRIP were performed to investigate mRNA cytosine methylation. MeRIP-seq revealed the common target genes regulated by DNMT1 and NSun2. Particular chimeric reporter constructs were generated and dual luciferase assays dissected corresponding regulations via DNA and/or mRNA methylation. Cell viability assay, apoptosis analysis by flow cytometry, and tumor xenograft model were utilized to determine osteosarcoma drug resistance. Results: Promoter methylation by DNMT1 dialogues with mRNA methylation by NSun2 to coordinately regulate osteosarcoma cell apoptosis. DNMT1 was induced in osteosarcoma cell apoptosis triggered by chemotherapeutic drugs, whereas NSun2 expression was dampened. DNMT1 was found to represses NSun2 expression by methylating NSun2 promoter. Moreover, DNMT1 and NSun2 regulate anti-apoptotic genes AXL, NOTCH2, and YAP1 through DNA and mRNA methylation, respectively. Upon exposure to cisplatin or doxorubicin, DNMT1 elevation drastically reduced these anti-apoptotic gene expressions via enhanced promoter methylation coupled with NSun2 ablation-mediated attenuation of mRNA methylation, thus rendering osteosarcoma to apoptosis. Conclusions: Our findings establish a crosstalk of importance between DNA and RNA cytosine methylations in determining osteosarcoma resistance to apoptosis during chemotherapy, shedding new light on future treatment of osteosarcoma, and add additional layers to the control of gene expression at different epigenetic levels.
Project description:To identify candidate genes that are up-regulated in chemoresistant (cisplatin and doxorubicin) hepatospheres as compared with their differentiated counterparts Affymetrix Human Genome U133 Plus GeneChip 2.0 PLC/PRF/5 HCC cells were grown as spheroids and treated with cisplatin and doxorubicin combination. Differentiated counterparts were generated by addition of FBS in the spheroids and allowed to attach.
