Calcium uptake through the mitochondrial Ca2+ uniporter (MCU) is thought to be essential in regulating cellular signaling events, energy status, and survival. Functional dissection of the uniporter is now possible through the recent identification of the genes encoding for MCU protein complex subunits. Cancer cells exhibit many aspects of mitochondrial dysfunction associated with altered mitochondrial Ca2+ levels including resistance to apoptosis, increased reactive oxygen species production and decreased oxidative metabolism. We used a publically available database to determine that breast cancer patient outcomes negatively correlated with increased MCU Ca2+ conducting pore subunit expression and decreased MICU1 regulatory subunit expression. We hypothesized breast cancer cells may therefore be sensitive to MCU channel manipulation. We used the widely studied MDA-MB-231 breast cancer cell line to investigate whether disruption or increased activation of mitochondrial Ca2+ uptake with specific siRNAs and adenoviral overexpression constructs would sensitize these cells to therapy-related stress. MDA-MB-231 cells were found to contain functional MCU channels that readily respond to cellular stimulation and elicit robust AMPK phosphorylation responses to nutrient withdrawal. Surprisingly, knockdown of MCU or MICU1 did not affect reactive oxygen species production or cause significant effects on clonogenic cell survival of MDA-MB-231 cells exposed to irradiation, chemotherapeutic agents, or nutrient deprivation. Overexpression of wild type or a dominant negative mutant MCU did not affect basal cloning efficiency or ceramide-induced cell killing. In contrast, non-cancerous breast epithelial HMEC cells showed reduced survival after MCU or MICU1 knockdown. These results support the conclusion that MDA-MB-231 breast cancer cells do not rely on MCU or MICU1 activity for survival in contrast to previous findings in cells derived from cervical, colon, and prostate cancers and suggest that not all carcinomas will be sensitive to therapies targeting mitochondrial Ca2+ uptake mechanisms.
References
[1]
Baughman JM, Perocchi F, Girgis HS, Plovanich M, Belcher-Timme CA, et al. (2011) Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476: 341–345 doi:10.1038/nature10234.
[2]
De Stefani D, Raffaello A, Teardo E, Szabò I, Rizzuto R (2011) A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature: 1–24. doi:10.1038/nature10230.
[3]
Csordás G, Golenár T, Seifert EL, Kamer KJ, Sancak Y, et al. (2013) MICU1 Controls Both the Threshold and Cooperative Activation of the Mitochondrial Ca2+ Uniporter. Cell Metab 17: 976–987 doi:10.1016/j.cmet.2013.04.020.
[4]
Mallilankaraman K, Doonan P, Cárdenas C, Chandramoorthy HC, Müller M, et al. (2012) MICU1 Is an Essential Gatekeeper for MCU-Mediated Mitochondrial Ca2+ Uptake that Regulates Cell Survival. Cell 151: 630–644 doi:10.1016/j.cell.2012.10.011.
[5]
Perocchi F, Gohil VM, Girgis HS, Bao XR, Mccombs JE, et al. (2010) MICU1 encodes a mitochondrial EF hand protein required for Ca2+ uptake. Nature 467: 291–296 doi:10.1038/nature09358.
[6]
Aykin-Burns N, Ahmad IM, Zhu Y, Oberley LW, Spitz DR (2009) Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation. Biochem J 418: 29 doi:10.1042/BJ20081258.
[7]
Fantin VR, St-Pierre J, Leder P (2006) Attenuation of LDH-A expression uncovers a link between glycolysis, mitochondrial physiology, and tumor maintenance. Cancer Cell 9: 425–434 doi:10.1016/j.ccr.2006.04.023.
[8]
Denton RM (2009) Regulation of mitochondrial dehydrogenases by calcium ions. BBA - Bioenergetics 1787: 1309–1316 doi:10.1016/j.bbabio.2009.01.005.
[9]
Glancy B, Balaban RS (2012) Role of Mitochondrial Ca2+ in the Regulation of Cellular Energetics. Biochemistry 51: 2959–2973 doi:10.1021/bi2018909.
[10]
Marchi S, Lupini L, Patergnani S, Rimessi A, Missiroli S, et al. (2012) Downregulation of the Mitochondrial Calcium Uniporter by Cancer-Related miR-25. Curr Biol. doi:10.1016/j.cub.2012.11.026.
[11]
Madden SF, Clarke C, Aherne ST, Gaule P, O'Donovan N, et al. (2013) BreastMark: an integrated approach to mining publicly available transcriptomic datasets relating to breast cancer outcome. Breast Cancer Res 15: R52 doi:10.1186/bcr3444.
[12]
Imanishi H, Hattori K, Wada R, Ishikawa K, Fukuda S, et al. (2011) Mitochondrial DNA mutations regulate metastasis of human breast cancer cells. PLoS ONE 6: e23401 doi:10.1371/journal.pone.0023401.
[13]
Owens KM, Kulawiec M, Desouki MM, Vanniarajan A, Singh KK (2011) Impaired OXPHOS Complex III in Breast Cancer. PLoS ONE 6: e23846 doi:10.1371/journal.pone.0023846.t001.
[14]
Curry MC, Peters AA, Kenny PA, Roberts-Thomson SJ, Monteith GR (2013) Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells. Biochem Biophys Res Commun. doi:10.1016/j.bbrc.2013.04.015.
[15]
Nagai T, Sawano A, Park ES, Miyawaki A (2001) Circularly permuted green fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 98: 3197–3202 doi:10.1073/pnas.051636098.
[16]
Palmer AE, Giacomello M, Kortemme T, Hires SA, Lev-Ram V, et al. (2006) Ca2+ Indicators Based on Computationally Redesigned Calmodulin-Peptide Pairs. Chem Biol 13: 521–530 doi:10.1016/j.chembiol.2006.03.007.
[17]
Spitz DR, Malcolm RR, Roberts RJ (1990) Cytotoxicity and metabolism of 4-hydroxy-2-nonenal and 2-nonenal in H2O2-resistant cell lines. Do aldehydic by-products of lipid peroxidation contribute to oxidative stress? Biochem J 267: 453–459.
[18]
Cai Z, Chattopadhyay N, Liu WJ, Chan C, Pignol J-P, et al. (2011) Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: comparison with manual counting. Int J Radiat Biol 87: 1135–1146 doi:10.3109/09553002.2011.622033.
[19]
Dahle J, Kakar M, Steen HB, Kaalhus O (2004) Automated counting of mammalian cell colonies by means of a flat bed scanner and image processing. Cytometry 60A: 182–188 doi:10.1002/cyto.a.20038.
[20]
Joiner M-LA, Koval OM, Li J, He BJ, Allamargot C, et al. (2012) CaMKII determines mitochondrial stress responses in heart. Nature. doi:10.1038/nature11444.
[21]
Lee WJ, Monteith GR, Roberts-Thomson SJ (2006) Calcium transport and signaling in the mammary gland: targets for breast cancer. Biochim Biophys Acta. doi: 10.1016/j.bbcan.2005.12.001
[22]
Denton RM, McCormack JG (1980) The role of calcium in the regulation of mitochondrial metabolism. Biochem Soc Trans 8: 266–268.
[23]
Pinton P, Ferrari D, Rapizzi E, Di Virgilio F, Pozzan T, et al. (2001) The Ca2+ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action. EMBO J 20: 2690–2701 doi:10.1093/emboj/20.11.2690.
[24]
Hadzic T, Aykin-Burns N, Zhu Y, Coleman MC, Leick K, et al. (2010) Paclitaxel combined with inhibitors of glucose and hydroperoxide metabolism enhances breast cancer cell killing via H2O2-mediated oxidative stress. Free Radic Biol Med 48: 1024–1033 doi:10.1016/j.freeradbiomed.2010.01.018.
[25]
Qiu J, Tan Y-W, Hagenston AM, Martel M-A, Kneisel N, et al. (2013) Mitochondrial calcium uniporter Mcu controls excitotoxicity and is transcriptionally repressed by neuroprotective nuclear calcium signals. Nat Commun 4: 2034 doi:10.1038/ncomms3034.
[26]
Pan X, Liu J, Nguyen T, Liu C, Sun J, et al. (2013) The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nat Cell Biol. doi:10.1038/ncb2868.
[27]
Raffaello A, De Stefani D, Sabbadin D, Teardo E, Merli G, et al. (2013) The mitochondrial calcium uniporter is a multimer that can include a dominant-negative pore-forming subunit. EMBO J. doi:10.1038/emboj.2013.157.
[28]
Mallilankaraman K, Cárdenas C, Doonan PJ, Chandramoorthy HC, Irrinki KM, et al. (2012) MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism. Nat Cell Biol. doi:10.1038/ncb2622.
[29]
Sancak Y, Markhard AL, Kitami T, Kovács-Bogdán E, Kamer KJ, et al. (2013) EMRE is an Essential Component of the Mitochondrial Calcium Uniporter Complex. Science. doi:10.1126/science.1242993.
[30]
Plovanich M, Bogorad RL, Sancak Y, Kamer KJ, Strittmatter L, et al. (2013) MICU2, a Paralog of MICU1, Resides within the Mitochondrial Uniporter Complex to Regulate Calcium Handling. PLoS ONE 8: e55785 doi:10.1371/journal.pone.0055785.
[31]
Patron M, Checchetto V, Raffaello A, Teardo E, Vecellio Reane D, et al. (2014). MICU1 and MICU2 Finely Tune the Mitochondrial Ca2+ Uniporter by Exerting Opposite Effects on MCU Activity. Mol Cell. doi:10.1016/j.molcel.2014.01.013.
