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Comparative Analysis of the Development of Acquired Radioresistance in Canine and Human Mammary Cancer Cell Lines.

ABSTRACT: Research using in vitro canine mammary cancer cell lines and naturally-occurring canine mammary tumors are not only fundamental models used to advance the understanding of cancer in veterinary patients, but are also regarded as excellent translational models of human breast cancer. Human breast cancer is commonly treated with radiotherapy; however, tumor response depends on both innate radiosensitivity and on tumor repopulation by cells that develop radioresistance. Comparative canine and human studies investigating the mechanisms of radioresistance may lead to novel cancer treatments that benefit both species. In this study, we developed a canine mammary cancer (REM-134) radioresistant (RR) cell line and investigated the cellular mechanisms related to the development of acquired radioresistance. We performed a comparative analysis of this resistant model with our previously developed human breast cancer radioresistant cell lines (MCF-7 RR, ZR-751 RR, and MDA-MB-231 RR), characterizing inherent differences through genetic, molecular, and cell biology approaches. RR cells demonstrated enhanced invasion/migration capabilities, with phenotypic evidence suggestive of epithelial-to-mesenchymal transition. Similarities were identified between the REM-134 RR, MCF-7 RR, and ZR-751 RR cell lines in relation to the pattern of expression of both epithelial and mesenchymal genes, in addition to WNT, PI3K, and MAPK pathway activation. Following the development of radioresistance, transcriptomic data indicated that parental MCF-7 and ZR-751 cell lines changed from a luminal A classification to basal/HER2-overexpressing (MCF-7 RR) and normal-like/HER2-overexpressing (ZR-751 RR). These radioresistant subtypes were similar to the REM-134 and REM-134 RR cell lines, which were classified as HER2-overexpressing. To our knowledge, our study is the first to generate a canine mammary cancer RR cell line model and provide a comparative genetic and phenotypic analysis of the mechanisms of acquired radioresistance between canine and human cancer cell lines. We demonstrate that the cellular processes that occur with the development of acquired radioresistance are similar between the human and canine cell lines; our results therefore suggest that the canine model is appropriate to study both human and canine radioresistant mammary cancers, and that treatment strategies used in human medicine may also be applicable to veterinary patients.

SUBMITTER: Gray M

PROVIDER: S-EPMC7396503 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Research using in vitro canine mammary carcinoma cell lines and studies involving naturally occurring canine mammary tumours are not only fundamental models used to advance the understanding of cancer in veterinary patients but are also regarded as excellent translational models for human breast cancer. In human medicine, following disease classification and staging breast cancer is typically treated with multimodal therapies including radiotherapy. 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. Comparative canine and human studies investigating the molecular mechanisms of radioresistance may lead to the development of effective cancer treatments that benefit both species. Methods: Human (MDA-MB-231, MCF-7 and ZR-751) and canine (REM-134) radioresistant cell lines were established by exposing 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 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. Results: Colony formation assays identified a range of intrinsic radiosensitivities in the human and canine parental cell lines with the REM-134 cell line showing significantly greater radioresistance compared to the human cell lines. Colony formation and proliferation assays confirmed radioresistance in all developed radioresistant cell lines. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. Gene analysis identified that following the acquisition of radioresistance in MCF-7 and ZR-751 cell lines the cell lines 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. MDA-MB-231 and MDA-MB-231 RR cell lines both classified as basal, whereas the REM-134 and REM-134 RR cell lines with enriched for HER2 overexpression. 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: To our knowledge, our study is the first to develop a canine radioresistant cell line and provide a comparative genetic and phenotypic analysis of the mechanisms of acquired radioresistance between canine and human cell lines. The cellular process that occur with the development of acquired radioresistance are similar between the human and canine cell lines; this suggests that not only is the canine model an appropriate model to study human disease but that treatment strategies used in human medicine may also be applicable to veterinary patients.

Project description:BackgroundRadiotherapy 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.MethodsRadioresistant 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. Transcriptomic data were also generated for preliminary hypothesis generation involving pathway-focused analyses.ResultsProliferation and colony formation assays confirmed radioresistance. Radioresistant cells exhibited enhanced migration and invasion, with evidence of epithelial-to-mesenchymal-transition. 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 transcriptomic data 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. Radioresistant cell lines derived from ER+ cells also showed a shift from ER to EGFR signalling pathways with increased MAPK and PI3K activity.ConclusionsThis 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.

Project description:The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional level of Hypoxia Inducible Factor (HIF) dependent transcriptional regulation has emerged involving modulation of a specific set of miRNAs including miR-210. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. We therefore hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed Non-Small Cell Lung Cancer (NSCLC)-derived cell lines (A549 and H1975) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). MiR-210 expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. The cells were subjected to radiation levels ranging from 0 to 10Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of resistance than control cells in hypoxia. pmiR-210 cells under hypoxia showed a low mortality rate due to a decrease in apoptosis and an ability to grow even at 10Gy. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we show here that genomic double strand breaks (DSB) foci disappeared faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells (pmiR-210/HIF-1-) abolished radioresistance of cells showing that this mechanism was dependent upon HIF-1. In conclusion, miR-210 appears to be a major component in the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could even be used by tumor cells in conditions where hypoxia may not be present anymore and strongly suggests that strategies targeting miR-210 would enhance tumor radiosensitization. To identify the set of transcripts targeted by miR-210, we overexpressed has-miR-210 or a control miRNA (ce-miR-67) in human lung adenocarcinoma A549 cells by transduction using lentivirus. The resulting pmiR-67 and pmiR-210 that stably overexpress miR-210 were selected by puromycin. RNA samples were harvested at 2 different times following cell plating (24 hours or 48 hours). Experiments were performed in dye-swap.

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Comparative Analysis of the Development of Acquired Radioresistance in Canine and Human Mammary Cancer Cell Lines.

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