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PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

ABSTRACT: PURPOSE:Radiotherapy (RT) constitutes an important part of breast cancer treatment. However, triple negative breast cancers (TNBC) exhibit remarkable resistance to most therapies, including RT. Developing new ways to radiosensitize TNBC cells could result in improved patient outcomes. The M2 isoform of pyruvate kinase (PK-M2) is believed to be responsible for the re-wiring of cancer cell metabolism after oxidative stress. The aim of the study was to determine the effect of ionizing radiation (IR) on PK-M2-mediated metabolic changes in TNBC cells, and their survival. In addition, we determine the effect of PK-M2 activators on breast cancer stem cells, a radioresistant subpopulation of breast cancer stem cells. METHODS:Glucose uptake, lactate production, and glutamine consumption were assessed. The cellular localization of PK-M2 was evaluated by western blot and confocal microscopy. The small molecule activator of PK-M2, TEPP46, was used to promote its pyruvate kinase function. Finally, effects on cancer stem cell were evaluated via sphere forming capacity. RESULTS:Exposure of TNBC cells to IR increased their glucose uptake and lactate production. As expected, PK-M2 expression levels also increased, especially in the nucleus, although overall pyruvate kinase activity was decreased. PK-M2 nuclear localization was shown to be associated with breast cancer stem cells, and activation of PK-M2 by TEPP46 depleted this population. CONCLUSIONS:Radiotherapy can induce metabolic changes in TNBC cells, and these changes seem to be mediated, at least in part by PK-M2. Importantly, our results show that activators of PK-M2 can deplete breast cancer stem cells in vitro. This study supports the idea of combining PK-M2 activators with radiation to enhance the effect of radiotherapy in resistant cancers, such as TNBC.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6790295 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

Zhang Le L Bailleul Justine J Yazal Taha T Dong Kevin K Sung David D Dao Amy A Gosa Laura L Nathanson David D Bhat Kruttika K Duhachek-Muggy Sara S Alli Claudia C Dratver Milana Bochkur MB Pajonk Frank F Vlashi Erina E

Breast cancer research and treatment 20190801 1

<h4>Purpose</h4>Radiotherapy (RT) constitutes an important part of breast cancer treatment. However, triple negative breast cancers (TNBC) exhibit remarkable resistance to most therapies, including RT. Developing new ways to radiosensitize TNBC cells could result in improved patient outcomes. The M2 isoform of pyruvate kinase (PK-M2) is believed to be responsible for the re-wiring of cancer cell metabolism after oxidative stress. The aim of the study was to determine the effect of ionizing radia ...[more]

PMID: 31372790

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Project description:BackgroundTriple negative breast cancer (TNBC) poses a serious clinical challenge as it is an aggressive form of the disease that lacks estrogen receptor, progesterone receptor, and ERBB2 (formerly HER2) gene amplification, which limits the treatment options. The Warburg phenotype of upregulated glycolysis in the presence of oxygen has been shown to be prevalent in TNBC. Elevated glycolysis satisfies the energy requirements of cancer cells, contributes to resistance to treatment by maintaining redox homeostasis and generating nucleotide precursors required for cell proliferation and DNA repair. Expression of the monocarboxylate transporter 1 (MCT1), which is responsible for the bidirectional transport of lactate, correlates with an aggressive phenotype and poor outcome in several cancer types, including breast cancer. In this study, 3-bromopyruvate (3BP), a lactate/pyruvate analog, was used to selectively target TNBC cells that express MCT1.MethodsThe cytotoxicity of 3BP was tested in MTT assays using human TNBC cell lines: BT20 (MCT1+/MCT4-), MDA-MB-23 (MCT1-/MCT4+), and BT20 in which MCT1 was knocked down (siMCT1-BT20). The metabolite profile of 3BP-treated and 3BP-untreated cells was investigated using LC-MS/MS. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BT20 and MDA-MB-231 cells treated with 3BP were measured using a Seahorse XF96 extracellular flux analyzer. The impact of ionizing radiation on cell survival, alone or in combination with 3BP pre-treatment, was evaluated using clonogenic assays.ResultsMetabolomic analyses showed that 3BP causes inhibition of glycolysis, disturbance of redox homeostasis, decreased nucleotide synthesis, and was accompanied by a reduction in medium acidification. In addition, 3BP potentiated the cytotoxic effect of ionizing radiation, a treatment that is frequently used in the management of TNBC.ConclusionsOverall, MCT1-mediated metabolic perturbation in combination with radiotherapy is shown to be a promising strategy for the treatment of glycolytic tumors such as TNBC, overcoming the selectivity challenges of targeting glycolysis with glucose analogs.

Project description: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 ? 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.

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PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

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PK-M2-mediated metabolic changes in breast cancer cells induced by ionizing radiation.

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