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Ubiquitous Mitochondrial Creatine Kinase (CKMT1) Drives Malignant Creatine Metabolite Profiles in Human Breast Cancer Cell Lines1Johns Hopkins University School of Medicine, Baltimore, MD, United States, 2F.M. Kirby Research Center for Functional Brain Imaging, Kennedy Krieger Institute, Baltimore, MD, United States
Synopsis
Ubiquitous Mitochondrial Creatine Kinase (CKMT1) is a mitochondrial membrane protein that catalyzes the reversible conversion of creatine (Cr) to phosphocreatine (PCr). This study shows that in two triple-negative breast cancer cell lines, CKMT1-overexpression resulted in significant increases in intracellular Cr and PCr and conferred increases in cell viability while decreasing migration, invasion and adhesion. Since CKMT1 has been identified as a prognostic indicator in several cancers, it may play a role in oncogenesis and/or cancer progression. Thus, CKMT1 holds promise as a potential diagnostic marker and/or treatment target whose expression can be monitored using magnetic resonance spectroscopy-detected Cr and PCr levels.
Introduction
Breast cancers display altered creatine metabolism, resulting in metabolite profiles with low levels of creatine (Cr)[1] and phosphocreatine (PCr)[1,2]. Despite these changes being well established in the literature[3], the molecular drivers of malignant creatine metabolite profiles, and the potential relevance of these drivers to oncogenic processes are not well understood. We have previously identified a strong correlation between these malignant creatine profiles and the mRNA expression of ubiquitous mitochondrial creatine kinase (CKMT1)[4], which is downregulated in metastatic breast cell lines and which catalyzes the reversible interconversion of Cr to PCr[5]. In this study, we sought to identify a causal relationship between the expression of CKMT1 and Cr and PCr levels in breast cancer. Here, high-resolution (HR) 1H MRS was used to identify changes in levels of Cr and PCr in breast cancer cell extracts following overexpression of CKMT1. In addition, we identified significant changes to cells’ functional cancer characteristics, including migration, invasion, adhesion, and viability.Methods
Two triple-negative breast cell lines (MDA-MB-231 and SUM159) were transduced with the pLX304 lentiviral vector to enable significant overexpression of CKMT1. Immunoblotting and SYBR Green-based quantitative RT-PCR were used to confirm CKMT1 overexpression relative to cells transfected with empty-vector.
Metabolites were extracted from empty vector-expressing (EV) and CKMT1-overexpressing (OV) MDA-MB-231 and SUM159 cells using dual-phase extraction (methanol:chloroform:water = 1:1:1). HR 1H MRS of the aqueous extract fraction was measured on a Bruker 750 MR spectrometer, and Cr and PCr concentrations were quantified using TopSpin software.
To assess the functional cancer characteristics of these cells, transwell migration and invasion (with Matrigel) assays were performed using media containing 3% FBS as chemo-attractant. Additionally, assays of cell adhesion to Matrigel were conducted, as was a WST-1 assay of cell viability.
Results
HR 1H MRS analysis revealed that CKMT1-overexpressing MDA-MB-231 cells displayed significantly higher levels of Cr and PCr relative to their respective empty-vector controls (Figure 1A-D). In addition, CKMT1-overexpression conferred significant increases in the ratio of metabolites associated with glycolysis (lactate, alanine) to those associated with oxidative energy metabolism (aspartate, glutamate, succinate, fumarate) (Figure 2A,B). These metabolic changes were accompanied by increases in the mRNA expression of enzymes associated with glycolysis and anaerobic energy generation, including hexokinase 2 (HK2), phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) and B (LDHB). Finally, CKMT1 overexpression conferred significant decreases in cell migration, invasion, and adhesion in both cell lines, and increased cell viability, as detected by a WST-1 assay (Figure 3A,B).Discussion
Consistent with our previous studies of creatine metabolism in breast cancer, which identified a correlation between creatine metabolite levels and CKMT1 expression[4], this study clearly demonstrates that CKMT1 overexpression results in increased levels of Cr and PCr. This indicates that CKMT1 participates in driving malignant creatine metabolite profiles in breast cancer cell lines. In addition, CKMT1 overexpression conferred significant increases in the cellular ratio of lactate and alanine to aspartate, glutamate, succinate, and fumarate, suggesting that CKMT1 is supportive of glycolytic energy metabolism in these cells. While we previously identified an association among metastatic potential, Cr-metabolite levels, and CKMT1 expression[4], this study demonstrates for the first time that CKMT1 overexpression results in decreased migration and invasion in breast cancer cell lines. Moreover, CKMT1 overexpression conferred increases in cell viability, consistent with prior studies that have identified CKMT1 as a potential indicator of worsened prognosis in several cancers[6]. Thus, it is possible that CKMT1 can, in the future, serve as a potential diagnostic marker and/or treatment target in breast cancer, and Cr metabolites can function as a surrogate marker of CKMT1 expression.Conclusions
CKMT1 participates in driving the levels of Cr metabolites in breast cancer, and its overexpression results in decreased cell migration, invasion, and adhesion, as well as increased cell viability. Therefore, CKMT1 may be an interesting marker or target that can be monitored using MRS.Acknowledgements
We thank all members of the Division of Cancer Imaging Research in The Russell H. Morgan Department of Radiology and Radiological Science for their help and support.References
1 Cao, M. D., Lamichhane, S., Lundgren, S., Bofin, A., Fjøsne, H., Giseødegård, G. F., Bathen, T. F. (2014). Metabolic Characterization of Triple Negative Breast Cancer. BMC Cancer. 14(941)
2 Chan, K. W. Y., Jiang, L., Cheng, M., Wijnen, J. P., Liu, G., Huang, P., van Zijl, P. C. M., McMahon, M. T., Glunde, K. (2016) CEST-MRI Detects Metabolite Levels Altered by Breast Cancer Cell Aggressiveness and Chemotherapy Response. NMR Biomed., 10(29): 806-816.
3 Wijnen, J. P. Wybe, J. M. V., Luttje, M., Korteweg, M., Luijten, P. R., Klomp, D. Quantitative 31P Magnetic Resonance Spectroscopy of the Human Breast at 7T. Magnetic Resonance in Medicine.68(2):339-348.
4 Ayyappan, V., Cheng, M., Cai, R., Tressler, C., Sonkar, K., McMahon, M.T., Glunde, K. (2018). “Ubiquitous Mitochondrial Creatine Kinase (CKMT1) Expression Correlates with MRS-Detectable Creatine Metabolic Profiles in Human Breast Cancer.” Proceedings of the 2018 Joint Annual Meeting ISMRM-EMRB.
5 Wyss, M., Kaddurah-Daouk, R. (2000). Creatine and Creatinine Metabolism. Physiological Review, 80(3):1107-1213.
6 Wijnen, J. P. Wybe, J. M. V., Luttje, M., Korteweg, M., Luijten, P. R., Klomp, D. Quantitative 31P Magnetic Resonance Spectroscopy of the Human Breast at 7T. Magnetic Resonance in Medicine.68(2):339-348.
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