Abstract
BackgroundBreast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. Currently, molecular understanding of radiation carcinogenesis in mammary gland is hindered due to the scarcity of in vivo long-term follow up data. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure.MethodsSix to eight week old female C57BL/6J mice were exposed to 2 Gy of whole body γ radiation and mammary glands were surgically removed 2-month after radiation. RNA was isolated and microarray hybridization performed for gene expression analysis. Ingenuity Pathway Analysis (IPA) was used for biological interpretation of microarray data. Real time quantitative PCR was performed on selected genes to confirm the microarray data.ResultsCompared to untreated controls, the mRNA levels of a total of 737 genes were significantly (p<0.05) perturbed above 2-fold of control. More genes (493 genes; 67%) were upregulated than the number of downregulated genes (244 genes; 33%). Functional analysis of the upregulated genes mapped to cell proliferation and cancer related canonical pathways such as ‘ERK/MAPK signaling’, ‘CDK5 signaling’, and ‘14-3-3-mediated signaling’. We also observed upregulation of breast cancer related canonical pathways such as ‘breast cancer regulation by Stathmin1’, and ‘HER-2 signaling in breast cancer’ in IPA. Interestingly, the downregulated genes mapped to fewer canonical pathways involved in cell proliferation. We also observed that a number of genes with tumor suppressor function (GPRC5A, ELF1, NAB2, Sema4D, ACPP, MAP2, RUNX1) persistently remained downregulated in response to radiation exposure. Results from qRT-PCR on five selected differentially expressed genes confirmed microarray data. The PCR data on PPP4c, ELF1, MAPK12, PLCG1, and E2F6 showed similar trend in up and downregulation as has been observed with the microarray.ConclusionsExposure to a clinically relevant radiation dose led to long-term activation of mammary gland genes involved in proliferative and metabolic pathways, which are known to have roles in carcinogenesis. When considered along with downregulation of a number of tumor suppressor genes, our study has implications for breast cancer initiation and progression after therapeutic radiation exposure.
Highlights
Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer
We know from epidemiological studies that exposure to ionizing radiation (IR) is one of the major risk factors for breast cancer especially if exposure occurs at a young age [1,2]
We performed Quantitative real time PCR (qRT-PCR) of NFkβ, which is the nodal molecule of the highest scoring molecular network obtained from Ingenuity Pathway Analysis (IPA)
Summary
Breast tissue is among the most sensitive tissues to the carcinogenic actions of ionizing radiation and epidemiological studies have linked radiation exposure to breast cancer. We undertook this study to delineate radiation-induced persistent alterations in gene expression in mouse mammary glands 2-month after radiation exposure. Despite the fact that radiation exposure causes persistent changes in gene expression, data on radiationinduced long-term alterations in gene expression in mammary tissues are of limited availability. There are no reports in the literature of long-term microarray-based studies of changes in mouse mammary gland gene expression after exposure to radiation doses relevant to therapeutic procedures. Considering the carcinogenic potential of radiation exposure to mammary glands, it is important to understand how radiation modulates long-term gene expression changes and how these changes relate to oncogenic signaling pathways known to be involved in breast cancer initiation and progression. The goals of this study were to characterize persistent transcriptomic alterations and explore how these alterations relate to biological functions in whole body exposed 6 to 8 week old female C57BL6/J mouse mammary glands 2 months after exposure to 2 Gy γ radiation
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