MiRNA sequencing signature in acquired radioresistant and normal colorectal cancer cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE159528 | GEO | 2021/01/10
REPOSITORIES: GEO
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Project description:The molecular mechanisms underlying exceptional radioresistance in pancreatic cancer remain elusive. In the present study, we established a stable radioresistant pancreatic cancer cell line MIA PaCa-2-R by exposing the parental MIA PaCa-2 cells to fractionated ionizing radiation (IR). Systematic proteomics and bioinformatics comparison of protein expression in MIA PaCa-2 and MIA PaCa-2-R cells revealed that several growth factor- and cytokine-mediated pathways, including the OSM/STAT3, PI3K/AKT and MAPK/ERK pathways, were activated in the radioresistant cells, leading to enhanced cell migration, invasion and epithelial-mesenchymal transition (EMT), and inhibition of apoptosis. We focused functional analysis on one of the most upregulated proteins in the radioresistant cells, CD73, which is a cell surface protein that is overexpressed in a variety types of cancer. Ectopic overexpression of CD73 in the parent cells resulted in radioresistance and conferred resistance to IR-induced apoptosis. Knockdown of CD73 resensitized the radioresistant cells to IR and IR-induced apoptosis. The effect of CD73 on radioresistance and apoptosis is independent of the enzymatic activity of CD73. Further studies suggest that CD73 confers acquired radioresistance in pancreatic cancer cells at least in part through inactivating proapoptotic protein BAD via phosphorylation of BAD at Ser-136. Furthermore, we found that knockdown of CD73 in the radioresistant cells alone reverted the gene expression and phenotype of the radioresistant cells from those of mesenchymal-like cells to the ones of epithelial cells, demonstrating that CD73 upregulation is required for maintaining EMT in the radioresistant cells. Our results support the notion that the enhanced growth factor/cytokine signaling that promotes epithelial-mesenchymal plasticity, and acquisition of cancer stem-like cell properties contributes to acquired radioresistance in the residual surviving cells after fractionated irradiation, and that CD73 is a novel downstream factor of those enhanced signaling and acts to confers acquired radioresistance and maintains EMT in the radioresistant pancreatic cancer cells.
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Project description:Background: Radiotherapy plays an important role in the multimodal treatment of breast cancer. The response of a breast tumour to radiation depends not only on its innate radiosensitivity but also on tumour repopulation by cells that have developed radioresistance. Development of effective cancer treatments will require further molecular dissection of the processes that contribute to resistance. Methods: Radioresistant cell lines were established by exposing MDA-MB-231, MCF-7 and ZR-751 parental cells to increasing weekly doses of radiation. The development of radioresistance was evaluated through proliferation and colony formation assays. Phenotypic characterisation included migration and invasion assays and immunohistochemistry. Intrinsic differences and changes in response to radiation between parental and radioresistant cells were investigated by whole-transcriptome gene expression analysis. Gene enrichment and pathway-focused analyses identified signalling networks differentially activated in radioresistant cells, which were confirmed by western blotting. Results: Proliferation and colony formation assays confirmed radioresistance. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition, and limited activation of DNA damage and apoptotic pathways in response to 2 Gy ionising radiation. Significantly, acquisition of radioresistance in MCF-7 and ZR-751 cell lines resulted in a loss of expression of both ERα and PgR and an increase in EGFR expression; based on gene analysis they changed subtype classification from their parental luminal A to HER2-overexpressing (MCF-7 RR) and normal-like (ZR-751 RR) subtypes, indicating the extent of phenotypic changes and cellular plasticity involved in this process. Whole-transcriptome gene expression analysis identified down-regulation of ER signalling genes and up-regulation of genes associated with PI3K, MAPK and WNT pathway activity in radioresistant cell lines derived from ER+ cells; this was confirmed by western blot, which showed increased p-AKT and p-ERK expression following radiation. Conclusions: This is the first study to date that extensively describes the development and characterisation of three novel radioresistant breast cancer cell lines through both genetic and phenotypic analysis. More changes were identified between parental cells and their radioresistant derivatives in the ER+ (MCF-7 and ZR-751) compared with the ER- cell line (MDA-MB-231) model; however, multiple and likely interrelated mechanisms were identified that may contribute to the development of acquired resistance to radiotherapy.
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