Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation
Katherine Sellers, Matthew P. Fox, Michael Bousamra II, Stephen P. Slone, Richard M. Higashi, Donald M. Miller, Yali Wang, Jun Yan, Mariia O. Yuneva, Rahul Deshpande, Andrew N. Lane, Teresa W.-M. Fan
Katherine Sellers, Matthew P. Fox, Michael Bousamra II, Stephen P. Slone, Richard M. Higashi, Donald M. Miller, Yali Wang, Jun Yan, Mariia O. Yuneva, Rahul Deshpande, Andrew N. Lane, Teresa W.-M. Fan
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Research Article

Pyruvate carboxylase is critical for non–small-cell lung cancer proliferation

Abstract

Anabolic biosynthesis requires precursors supplied by the Krebs cycle, which in turn requires anaplerosis to replenish precursor intermediates. The major anaplerotic sources are pyruvate and glutamine, which require the activity of pyruvate carboxylase (PC) and glutaminase 1 (GLS1), respectively. Due to their rapid proliferation, cancer cells have increased anabolic and energy demands; however, different cancer cell types exhibit differential requirements for PC- and GLS-mediated pathways for anaplerosis and cell proliferation. Here, we infused patients with early-stage non–small-cell lung cancer (NSCLC) with uniformly 13C-labeled glucose before tissue resection and determined that the cancerous tissues in these patients had enhanced PC activity. Freshly resected paired lung tissue slices cultured in 13C6-glucose or 13C5,15N2-glutamine tracers confirmed selective activation of PC over GLS in NSCLC. Compared with noncancerous tissues, PC expression was greatly enhanced in cancerous tissues, whereas GLS1 expression showed no trend. Moreover, immunohistochemical analysis of paired lung tissues showed PC overexpression in cancer cells rather than in stromal cells of tumor tissues. PC knockdown induced multinucleation, decreased cell proliferation and colony formation in human NSCLC cells, and reduced tumor growth in a mouse xenograft model. Growth inhibition was accompanied by perturbed Krebs cycle activity, inhibition of lipid and nucleotide biosynthesis, and altered glutathione homeostasis. These findings indicate that PC-mediated anaplerosis in early-stage NSCLC is required for tumor survival and proliferation.

Authors

Katherine Sellers, Matthew P. Fox, Michael Bousamra II, Stephen P. Slone, Richard M. Higashi, Donald M. Miller, Yali Wang, Jun Yan, Mariia O. Yuneva, Rahul Deshpande, Andrew N. Lane, Teresa W.-M. Fan

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Figure 6

PC suppression hinders Krebs cycle–fueled biosynthesis.

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PC suppression hinders Krebs cycle–fueled biosynthesis. (A) 13C atom–res...
(A) 13C atom–resolved pyrimidine biosynthesis from 13C6-glucose and 13C5-glutamine is depicted with a 13C5-ribose moiety (red dots) produced via the pentose phosphate pathway (PPP) and 13C1-3 uracil ring (blue dots) derived from 13C2-4-aspartate produced via the Krebs cycle or the combined action of ME and PC (blue dots). A549 cells transduced with shPC55 or shEV were incubated with 13C6-glucose or 13C5-glutamine for 24 hours. Fractional enrichment of UTP and CTP isotopologs from FT-ICR-MS analysis of polar cell extracts showed reduced enrichment of 13C6-glucose–derived 13C5-ribose (the “5” isotopolog) and 13C6-glucose– or 13C5-glutamine–derived 13C1-3-pyrimidine rings (the “6–8” or “1–3” isotopologs, highlighted by dashed green rectangles; for the “6–8” isotopologs, 5 13Cs arose from ribose and 1–3 13Cs from the ring) (10, 45). These data suggest that PC knockdown inhibits de novo pyrimidine biosynthesis from both glucose and glutamine. (B) Glucose and glutamine carbons enter fatty acids via citrate. FT-ICR-MS analysis of labeled lipids from the nonpolar cell extracts showed that PC knockdown severely inhibited the incorporation of glucose and glutamine carbons into the fatty acyl chains (even) and fatty acyl chains plus glycerol backbone (odd >3) of phosphatidylcholine lipids. However, synthesis of the 13C3-glycerol backbone (the “3” isotopolog) or its precursor 13C3-α-glycerol-3-phosphate (αG3P, m+3) from 13C6-glucose was enhanced rather than inhibited by PC knockdown. These data suggest that PC suppression specifically hinders fatty acid synthesis in A549 cells. Values represent the averages of triplicates (n = 3), with standard errors. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test, assuming unequal variances.