Abstract 2327: Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions

Abstract

Abstract Breast cancer is the most diagnosed cancer in women, and metastasis accounts for 90% of cancer related deaths. In order to metastasize, cancer cells must survive in conditions where they are detached from the extracellular matrix (ECM). Untransformed cells decrease energy metabolism when detached from ECM, but oncogenic transformation results in rescued energy metabolism of detached cells. Expression of the anaplerotic enzyme pyruvate carboxylase (PC) is required for breast to lung metastasis, and may play a role in promoting ECM detached cell survival by supporting metabolic plasticity. PC activity results in the carboxylation of pyruvate to synthesize the TCA cycle intermediate oxaloacetate (OAA). We hypothesize that PC expression is required for cell survival in ECM detached conditions. The Harvey-ras transformed MCF10A human breast epithelial cell line (MCF10A-ras) were utilized for these studies. ECM detachment was modeled using Poly-HEMA coated plates in combination with MTT assays to measure cell viability. qPCR was used to quantify mRNA abundance of metabolic genes. Protein expression was quantified using western blot. PC activity was determined using [13C6]-glucose flux analysis by comparing 3 carbon labeled (M+3) citrate, malate, and aspartate. Results show a 94% increase in PC mRNA expression and a 90% increase in PC protein expression, in detached cells compared to attached cells. In addition PC knockdown decreases viability in ECM detached conditions significantly by 15%, while PC overexpression increases viability by 17%. Further, replenishing the TCA cycle with OAA, the product of the PC reaction, rescues PC knockdown cells by 14%. Increased PC activity in detached cells was determined by a 23% increase in M+3 malate and a 61% increase in M+3 aspartate. However, an 11% decrease in M+3 citrate was observed, suggesting that detached cells do not utilize increased PC expression to replenish the TCA cycle intermediate, citrate. Given the larger relative increase in PC activity demonstrated by M+3 aspartate, we hypothesized that PC activity is required for detached cell survival to provide substrate for aspartate synthesis. Providing aspartate (2.5 mM), a downstream product of PC, in detached cells with PC knockdown rescued cell viability with no benefit of aspartate supplementation in detached PC expressing cells. Further, the aspartate synthesizing enzyme, glutamate OAA transferase 2 (GOT2) mRNA expression was unchanged in detached cells. In summary PC expression and activity is increased in detached cells compared to attached cells, and OAA produced by the PC reaction is utilized by cells to synthesize aspartate and maintain cell viability in detached conditions. PC may be a useful target for future treatments aimed at preventing metastasis. Citation Format: Madeline P. Sheeley, Dorothy Teegarden. Pyruvate carboxylase rescues MCF10A-ras cell viability in extracellular matrix detached conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2327.