Metastasis of cancer cells is a multistep process, which includes survival in ECM detachment [6]. Thus, tumor cells that demonstrate anchorage-independent growth possess a survival advantage which may promote their spread to distant sites. Detachment from ECM in non-transformed epithelial cells induces cellular changes that suppress flux of metabolites, including glucose and glutamine, into energy-producing pathways such as glycolysis and the TCA cycle which contributes to reduced viability in ECM detached conditions [4]. Compared to non-transformed cells, oncogenic transformation rescues glucose and glutamine flux into the TCA cycle in detached conditions [10]. The flux of metabolites through the TCA cycle generates the coenzymes nicotinamide adenine dinucleotide or flavin adenine dinucleotide, which are then oxidized at the electron transport chain for ATP production, collectively suggesting that enhanced flux of nutrients through the TCA cycle contributes to anchorage independence in transformed cells. Additionally, intermediates of the TCA cycle are utilized by cells to mediate biosynthetic reactions required for cell proliferation, such as amino acids and lipids, suggesting that nutrient metabolism serves additional functions beyond ATP production. The present study demonstrates that detached MCF10A-ras cells display decreased glucose metabolism through glycolysis and decreased anaplerosis from glutamine compared to attached cells. However, detached cells specifically display increased flux of pyruvate, the end product of glycolysis, to oxaloacetate through upregulated expression and activity of PC.
Previous studies identified that PC is required for breast cancer metastasis to the lung. For example, in an in vivo model of breast cancer, metastatic lesions at the lung contained high levels of PC expression and activity [15]. Additionally PC was required for breast to lung metastasis in an animal model [13]. The organotropic role of PC in breast to lung metastasis is consistent with previous literature showing a cellular requirement for PC activity in primary lung tumors [22]. Given the requirement for PC in both primary and metastatic cancer cell growth in the lung, these data may highlight PC as an attractive therapeutic target. The present work identified a novel mechanism by which detached breast cancer cells reprogram energy metabolism to increase the expression and activity of PC. Further, depletion of PC in detachment impairs cell survival, suggesting a specific molecular mechanism by which PC mediates breast to lung metastasis. Additionally, overexpression of PC, a characteristic of metastatic breast cancer [14, 16], improves detached viability of MCF10A-ras breast epithelial cells. Collectively, these results suggest that increased PC expression may promote metastasis by supporting survival in ECM detached conditions throughout the metastatic cascade.
Metabolic flexibility has recently been identified as a contributor to the successful metastasis of cancer cells [23]. As cancer cells progress through metastasis, they require adaption to changing nutrient conditions in variable microenvironmental conditions. As a compensatory mechanism, PC may play a crucial role in conferring metabolic flexibility to cancer cells. For example, in healthy BALB/c mice, the ratio of pyruvate/glutamine in the lung interstitial fluid is three times higher than in serum [15]. This varying ratio of pyruvate/glutamine may require cancer cells to switch from utilizing glutamine as a means of replenishing the TCA cycle to increasing anaplerosis through PC activity for successful colonization. Indeed, PC activity was increased in lung metastases compared to primary breast tumors [15]. In the current study, a PC switch was demonstrated in detached cells as detachment resulted in decreased glutamine incorporation into the TCA cycle and increased flux of glucose into aspartate and malate pools through PC activity. Further support for metabolic reprogramming in favor of PC over glutamine was demonstrated by the decreased mRNA abundance of PC following the addition of DM-αKG to detached cells. These results demonstrate that replenishing the TCA cycle with a metabolite of glutamine reverses the increase in PC abundance. Therefore, detached cancer cells display metabolic flexibility by increasing PC expression and activity in response to reduced glutamine metabolism.
PC’s role in aspartate biosynthesis has been well documented in cancer models. In renal cell carcinoma with succinate dehydrogenase (SDH) ablation, the addition of aspartate rescues cells from PC depletion [24]. Consistent with these results, SDHA or SDHB inhibition resulted in an increase in PC activity in both neuroendocrine and prostate cancer cells [25, 26]. In both of these models, increased PC activity following SDH inhibition resulted in the replenishment of aspartate. Thus, inhibition of the forward TCA cycle by inhibition of SDH activity increases PC-mediated TCA cycle anaplerosis. Consistent with this, the current study identified increased PC activity in detached cells indicated by enrichment of 13C6-glucose derived M + 3 labeling patterns in the intracellular pools of aspartate and malate. These results suggest that detached cells use PC activity to compensate for an inability to synthesize oxaloacetate from glutamine through the forward TCA cycle.
In summary, these findings demonstrate that detachment of MCF10A-ras cells leads to a decreased flux of glucose and glutamine into the TCA cycle compared to their attached counterparts. However, PC expression and activity is increased in detached cells, and this effect is reversed with exogenous addition of the TCA cycle intermediate DM-αKG. These results indicate that PC plays a critical role in promoting the survival of cells in matrix detached conditions and highlights a potential therapeutic target for the prevention of cancer metastasis.