Mass spectrometry analysis shows the biosynthetic pathways supported by pyruvate carboxylase in highly invasive breast cancer cells

Abstract

We recently showed that the anaplerotic enzyme pyruvate carboxylase (PC) is up-regulated in human breast cancer tissue and its expression is correlated with the late stages of breast cancer and tumor size [Phannasil et al., PloS One 10, e0129848, 2015]. In the current study we showed that PC enzyme activity is much higher in the highly invasive breast cancer cell line MDA-MB-231 than in less invasive breast cancer cell lines. 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 in viability and proliferation rates suggesting the perturbation of pathways that are involved in cancer invasiveness. 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.

Metabolomics and transcriptomics profiles reveal the dysregulation of the tricarboxylic acid cycle and related mechanisms in prostate cancer

“…First, our finding showed an increased expression of ACO2 in tumor tissues. 50 Besides, the data indicated that the MYC-regulated Gln anaplerosis may contribute to fueling the TCA cycle by providing 2-KG in PCa. 48 Another important route to control the flux into TCA cycle is from pyruvate.…”

“…Using multiple stable PC knockdown cell lines and 13C 6 -glucose and 13C 5glutamine labeled mass spectrometry analysis, it was revealed that PC played important roles in the pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for cell growth and replication in invasive breast cancer. 50 Besides, the data indicated that the MYC-regulated Gln anaplerosis may contribute to fueling the TCA cycle by providing 2-KG in PCa. The observation that glutamine transporter ASCT1 (also called SLC1A4, FC 5 1.78, p 5 0.00016), GLS and glutamate dehydrogenase GLUD1, GLUD2 (Fig.…”

Translational and HIF1α-dependent metabolic reprograming underpin oncometabolome plasticity and synergy between oncogenic kinase inhibitors and biguanides

“…Aspartate can be generated from pyruvate though oxaloacetate as via the action of PC, an enzyme mediating the conversion of pyruvate to oxaloacetate (Ling and Keech, 1966). Elevated PC expression and/or activity is observed in breast cancer and non-small-cell lung cancer (NSCLC) (Phannasil et al, 2017;Sellers et al, 2015). We show that PC mRNA translation is regulated via 4E-BPs and that 4E-BP1/2 ablation leads to dramatic increase in aspartate levels and reduction in sensitivity to KI and phenformin combination.…”

Targeting Pyruvate Carboxylase by a Small Molecule Suppresses Breast Cancer Progression

“…Compared with non‐cancerous tissues, PC expression is greatly enhanced in patients with non‐small cell lung cancer (NSCLC) and invasive breast cancer (BCA). [ 9 ] In early stages of NSCLC, growth inhibition by PC knockdown is accompanied by disrupted TCA activity and biosynthesis. [ 10 ] In particular, BCA accounts for the highest frequency of PC amplification according to The Cancer Genome Atlas (TCGA).…”

“…[ 11 ] Importantly, BCA lung metastases develop a significantly higher PC‐catalyzed generation of oxaloacetate, along with an increased intracellular pyruvate level as compared to primary tumors. [ 9b,12 ] Researchers also unveiled a potential metabolic vulnerability of glutamine independent cancer cells through inhibiting PC activity. [ 6c,13 ] Thus, PC could be a promising therapeutic target for cancer progression.…”