Pyruvate carboxylase supports biosynthetic pathways required for growth and survival of highly invasive breast cancer MDA-MB-231 cells

Phannasil P1, Rattanapornsompong K1, Akekawatchai C2, El-Azzouny M3, Ansari IH4, Longacre MJ4, MacDonald MJ4 and Jitrapakdee S1

  1. Department of Biochemistry Faculty of Science, Mahidol University, Bangkok, Thailand.
  2. Department of Allied Health Sciences, Thammasat University, Bangkok, Thailand.
  3. Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA.
  4. School of Medicine and Public Health, University of Wisconsin, Madison, WI, USA.

Pyruvate carboxylase (PC), an anaplerotic enzyme, plays an essential role in various cellular metabolic pathways including gluconeogenesis, lipogenesis and amino acid synthesis. PC is up-regulated in human breast cancer tissue and its expression is correlated with tumor size and aggressive phenotype. Here we investigate the role of PC in supporting cell growth and survival using gene silencing approach combining with metabolomics analysis. We generated multiple stable PC knockdown cell lines from the MDA-MB-231 cell line and used mass spectrometry with 13C6-glucose and 13C5-glutamine to discern the pathways that use PC in support of cell growth. Cells with severe PC knockdown showed a marked reduction of proliferation rate associated with apoptosis, suggesting the perturbation of biosynthetic pathways that required for cell growth. Strong PC suppression lowered glucose incorporation into downstream metabolites of oxaloacetate, the product of the PC reaction, including malate, citrate and aspartate. Levels of pyruvate, lactate, the redox partner of pyruvate, and acetyl-CoA were also lower suggesting the impairment of mitochondrial pyruvate cycles. Serine, glycine and 5-carbon sugar levels and flux of glucose into fatty acids were decreased. ATP, ADP and NAD(H) levels were unchanged indicating that PC suppression did not significantly affect mitochondrial energy production. The data indicate that the major metabolic roles of PC in invasive breast cancer are primarily anaplerosis, pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for breast cancer cell growth and replication.