An increased dependence on glycolysis for ATP synthesis as opposed to the more efficient mitochondrial oxidative phosphorylation (OXPHOS) has long been established as a hallmark of carcinogenesis [12
]. This subject has received increased attention recently as researchers have more closely considered the role of mitochondrial uncoupling in cancer metabolism and carcinogenesis [34
] or conversely, the direct role that some oncogenes may serve as metabolic switches [36
]. Because previous studies [8
] of protein expression and signaling disruption in the MCF10A breast cancer progression model of cell lines have demonstrated dysregulation of metabolic proteins, these cell lines were used to interrogate the poorly understood relationship between Ras-driven oncogenesis, metabolism and cellular respiration.
The MCF10A progression series of cell lines is a useful tool to study the process of tumor formation and metastatic progression driven by the HRAS
oncogene, which is overexpressed in half of all breast cancers [37
]. To understand the disruption of cellular signaling and metabolism in this breast cancer progression model, we employed quantitative SILAC proteomic analysis to accurately determine protein expression changes at the cellular and subcellular level. The ultimate benefit of using SILAC based subcellular fractionation is that protein trafficking can be assessed because the inevitable cross-contamination from other organelles is normalized for each cell line. Therefore, when the fold-change difference between cell lines increases or decreases in a specific subcellular fraction relative to the whole cell value, it is indicative of a difference in subcellular enrichment or depletion, respectively, across the cell lines (). Using this method, translocation of proteins from one organelle to another can be appreciated, offering insight into potential mechanisms in metabolic dysfunction and metastatic progression beyond that garnered from total protein expression levels. This technique uncovered organelle specific regulation of several proteins involved in cellular respiration, and the observed dysregulation of specific respiratory pathways was consistent with metabolic analysis.
The diverse metabolic profiles of the MCF10A progression series were investigated by comparing the different cell lines for relative mitochondrial copy number, protein expression and localization, capacity for oxidative phosphorylation versus glycolysis, and steady-state ATP levels. All four cell lines had similar expression of mtDNA, while the preneoplastic cell line, T1K, had the lowest expression of mitochondrial membrane proteins, most of which were reduced or unchanged in the malignant CA1h and CA1a cells as well. One notable exception is that the mitochondrial outer membrane protein, porin 2, was up-regulated in CA1a cells whereas porins 1 and 3 were not. Mitochondrial porins 2 and 3 overexpression has been implicated in Ras-activated cancers as the functional target of the anti-cancer drug, erastin [38
], which cuts off the supply of ATP from the mitochondria to hexokinase.
The overall reduction in mitochondrial protein expression in the transformed cell lines is indicative of a reduced capacity for OXPHOS in these cells. Some of the proteins of the TCA cycle, which feed NADH to drive OXPHOS, were also impaired. The mitochondrial TCA enzymes (CS, ACO2, DLST, SDHB, FH and MDH2) were somewhat reduced but largely unchanged in transformed cells compared to 10A. demonstrates that basal oxygen consumption rates as well as total cellular capacity of OXPHOS decline with increasing tumor grade. Although reduced capacity in transformed cells compared to parental 10A cells is expected, since all three tumorigenic cell lines have lowered expression of ETC proteins compared to 10A (), the progressive impairment of OXPHOS is not reflective of the trend in ETC proteins. In fact, ETC protein expression is lowest in T1K cells and only slightly reduced in CA1a cells compared to MCF10A. The observation that CA1a has the lowest capacity for OXPHOS then suggests that electron transport is uncoupled by exogenous factors in the CA1a mitochondria. In this dataset, direct perturbation of the mitochondria is reflected in the mitochondrial enrichment of integrins, catenins and HIF1 target genes.
While mitochondrial TCA enzymes appeared to be largely unchanged, the cytosolic paralogs of these proteins were higher with respect to cancer progression. Cytosolic MDH1 and IDH1 protein expression were both higher in the cytosol of CA1a, and overall expression and cytosolic localization of the cytosolic aconitase (ACO1) was elevated in all three transformed cell lines. High levels of these cytosolic enzymes can draw intermediates from the TCA cycle toward the production of pyruvate as part of pyruvate-malate or pyruvate-isocitrate cycling in CA1h and CA1a cells. The ECAR measurements in indicate that T1K cells have the highest lactate secretion, 10A and CA1a cells are similar, and CA1h cells have the lowest lactate secretion, but are contrary to the predicted trends in glycolytic activity based on the observed OCR values and expression of phosphofructokinase. One contributing factor leading to decreased ECAR in the aggressive cancer cells relative to the preneoplastic T1K cells is the increase in LDHB (). Higher LDHB will elevate the stoichiometry of LDHB to LDHA, leading to the formation of more LDH1 and LDH2 tetramers that have a preference for conversion of lactate to pyruvate in a reverse reaction, which can then feed cellular respiration through the TCA cycle [39
Furthermore, CA1h and CA1a mitochondria were found to have increased expression of the hypoxia-inducible, tumor-associated lactate/proton symporter, monocarboxylate transporter 4 (MCT4), as well as the MCT4 cofactor, basigin, that is required for its activity [40
]. These data strongly suggest that, in the CA1h and CA1a cells, cellular lactate is pumped back into the mitochondria to be converted to pyruvate and fed into the TCA cycle. With levels of pyruvate carboxylase that are higher in CA1h compared to CA1a and the weaker capacity for OXPHOS seen in CA1a, it is reasonable that CA1h cells can more efficiently clear lactate through this process.
Our analysis provides the first evidence that some oncogenic signaling proteins are increasingly trafficked to the mitochondria in malignant cells, offering key insight into their role in the Warburg effect of carcinogenesis. The increase in mitochondrial integrins coupled with decreasing OXPHOS in concert with tumor cell grade is consistent with the recent report that siRNA knock-down of β4-integrin rescues cell senescence in aged cardiac myocytes, in part by restoring mitochondrial function [41
]. The exact role of this unique integrin signaling in cancer progression and metabolic dysfunction requires further study, but the advantage of using quantitative subcellular proteomics to elucidate these mechanisms is substantial. The increase in mitochondrial integrins coupled with decreasing OXPHOS in concert with tumor cell grade is consistent with the recent report that siRNA knock-down of β4-integrin rescues cell senescence in aged cardiac myocytes, in part by restoring mitochondrial function [41
]. The exact role of this important oncoprotein in cancer progression and metabolic dysfunction require further study, but the advantage of using quantitative subcellular proteomics to elucidate these mechanisms is substantial. Finally, the combination of proteomic and metabolic analysis applied to this model system provides a foundation for the integrated analysis of metabolic dysfunction in cancer.