DPPA3-HIF1a axis controls colorectal cancer chemoresistance by imposing a slow cell-cycle phenotype
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE207862 | GEO | 2023/08/02
REPOSITORIES: GEO
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Project description:Tumor relapse is linked to rapid chemoresistance and represents a bottleneck for cancer therapy success. Engagement of a reduced proliferation state is a non-mutational mechanism exploited by cancer cells to bypass therapy-induced cell death. Through combining pulse-chase experiments in engineered CRC cells and transcriptomic analyses, we identified DPPA3 as a master regulator of slow-cycling phenotype in CRC. We find that DPPA3 stabilizes HIF1a even in normoxia thus limiting nuclear CCNB1 levels and represses DNA replication and cell cycle programs resulting in a slow cell-cycle phenotype. Down-regulation of HIF1a partially restores a chemosensitive proliferative phenotype in DPPA3-overexpressing cancer cells. In cohorts of patient samples, we find that DPPA3 is a predictive biomarker of CRC chemotherapeutic resistance and tumor relapse. Our work demonstrates that slow-cycling cancer cells exploit a DPPA3/HIF1a axis to support tumor persistence under therapeutic stress and provides key insights on the molecular regulation of tumor cell slow-cycliness and chemoresistance. This dataset comprises DNA methylation data of SW1222 CRC cells subjected to DPPA3 overexpression for 5 days.
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Project description:DPPA3 mutants were overexpressed using a doxycycline system to identify regions in DPPA3 necessary for DNA methylation maintenance suppression
2020-10-22 | E-MTAB-9653 | biostudies-arrayexpress
Project description:SUMMARY: Dormant or slow cycling tumour cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. Since their isolation from human tumors has been a long-stalled technical challenge preventing a direct study of their functional properties, we have adapted the pulse-chase expression and labeling approach using H2BeGFP (Tumbar et al., 2004, DOI:10.1126/science.1092436) to a doxycycline-inducible all-in-one lentiviral vector. In this manner we were able to label and isolate by FACS live slow cycling cancer cells (SCCC) from human colorectal, glioma and melanoma patient-derived tumors as well as from minitumors grown in matrigel (SW1222-cells) or in sphere culture (e225, e216 glioma models). We determined the differentially expressed transcriptional profiles in paired comparisons of slow-cycling cancer cell (SCCC) versus rapid cycling cancer cell (RCCC) sub-populations. H2BeGFP-retention revealed slow cycling as a transient cell state rather than a cellular entity. SCCC showed cancer-initiation potential and enhanced chemoresistance. Cells in this slow cycling status presented a distinctive non-genetic and cell-autonomous gene expression profile shared across different tumour types. We identified TET2 epigenetic enzyme as key factor controlling SCCC numbers and survival. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labelled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We demonstrate the enhanced chemoresistance of SCCC, revealed 5hmC as a biomarker for their clinical identification, and identified TET2 as a potential drug-target for SCCC elimination that could extend patients' survival.Experimental Design: Cells from human patient-derived xenografts (PDX) and human cancer cell lines were infected with a doxycycline (DOX)-inducible all-in-one lentivirus coding for the histone2B-eGFP fusion protein. The models were as following: T70 (colorectal cancer); MMPG3 and MMLN9 (melanoma); e225 and e216 (glioma) and human SW1222 colorectal cancer cell line as an infected pool as well as a clone (Clone2) and the following SW1222 derivate cell lines: SW1222-shCTRL (stable expression of nonsilencing shRNA), SW1222-shTET2 (stable expression of TET2-specific shRNA).H2BeGFP-infected cells were isolated and grown/propagated and injected subcutaneously in the flanks of immunocompromised NOD-Scid mice as described (Puig et al., Clin. Cancer Res. 2013; doi: 10.1158/1078-0432.CCR-12-1740.). H2BeGFP-infected SW1222 cells and derivate cell lines were grown in matrigel drops as minitumors. H2BeGFP-infected patient-derived melanoma MMPG3 and MMLN9 minitumor and glioma models e216 and e225 were grown as sphere cultures in their respective conditions as indicated.H2BeGFP expression was induced by DOX treatment in all experimental systems in a pulse-chase fashion and H2BeGFP fusion protein integrated into the host genome labeling all cells. Consecutive cell divisions of proliferating cells diluted and finally eliminated the fluorescent mark in an exponential decay fashion (RCCC or sRCCC), whereas slow-cycling cells (SCCC) retained the fluorescent stain, allowing FACS separation and isolation. Total RNA was separately extracted from both sub-populations of cells of each system and submitted to microarray analyses for characterization.
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Project description:We identified slow-cycling cells (SCCs) in Ewing sarcoma using a label retention assay with CFSE. We labeled cells of SK-ES-1, an Ewing sarcoma cell line, with CFSE. After 5 days culture, we isolated cells retaining strong fluorescence (upper, ~10%) as SCCs and other cells (lower, ~90%) as non-slow-cycling cells (non-SCCs) using FACS AriaTM Ⅲ cell sorter.
2022-10-17 | GSE204671 | GEO
Project description:Genome-wide DNA demethylation is a unique feature of mammalian development and naïve pluripotent stem cells. So far, it was unclear how mammals specifically achieve global DNA hypomethylation, given the high conservation of the DNA (de-)methylation machinery among vertebrates. We found that DNA demethylation requires TET activity but mostly occurs at sites where TET proteins are not bound suggesting a rather indirect mechanism. Among the few specific genes bound and activated by TET proteins was the naïve pluripotency and germline marker Dppa3 (Pgc7, Stella), which undergoes TDG dependent demethylation. The requirement of TET proteins for genome-wide DNA demethylation could be bypassed by ectopic expression of Dppa3. We show that DPPA3 binds and displaces UHRF1 from chromatin and thereby prevents the recruitment and activation of the maintenance DNA methyltransferase DNMT1. We demonstrate that DPPA3 alone can drive global DNA demethylation when transferred to amphibians (Xenopus) and fish (medaka), both species that naturally do not have a Dppa3 gene and exhibit no post-fertilization DNA demethylation. Our results show that TET proteins are responsible for active and - indirectly also for - passive DNA demethylation; while TET proteins initiate local and gene-specific demethylation in vertebrates, the recent emergence of DPPA3 introduced a unique means of genome-wide passive demethylation in mammals and contributed to the evolution of epigenetic regulation during early mammalian development.
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