Blog #1
Pyruvate Carboxylase and Lung Cancer Proliferation
How does the expression of pyruvate carboxylase affect non-small cell lung cancer growth and proliferation?
Re-written summaries by: Anuva Gajjar
Date Published: 12/27/2021
Original research links:
https://pubmed.ncbi.nlm.nih.gov/33931119/
https:/pubmed.ncbi.nlm.nih.gov/25607840/
Introduction
Pyruvate Carboxylase is a mitochondrial enzyme that catalyzes the reaction that transforms pyruvate into oxaloacetate. Pyruvate Carboxylase aids in anaplerosis-the process of replenishing precursors in the cell cycle. Pyruvate is an anapleroic source that requires the activity of pyruvate carboxylase. Glutamine, another precursor, requires the activity of glutaminase. Anabolic Biosynthesis, or the process of building complex molecules, requires these precursors. Pyruvate Carboxylase normally regulates the processes of gluconeogenesis (the formation of glucose), the synthesis of fatty acids, and the secretion of insulin. As a result, pyruvate carboxylase activity has primarily been studied in areas where these processes take place such as pancreatic cells, the liver, and adipose tissue. Cancer cells have to proliferate, or divide, more quickly and consequently have higher anabolic energy demands. Higher activity of PC has already been shown during adipose differentiation (differentiation of body fat cells). Scientists used transduced cells with different types of RNA, shEV and shPC55 to alter the expression of the pyruvate carboxylase gene. The process of inhibiting gene expression is called knockdown.
Methods
6 week old mice were injected with NSCLC cancer cells containing either shEV, or shPC55, mixed with Matrigel, (a model of basement membrane, which contacts with the basal layer of epithelial cells, endothelial cells, and fat and smooth muscle cells). The mice were NSG mice, which meant they were immunodeficient. For 36 days, the size of the tumors were measured by caliper, and calculated as πab/4, (a and b being the maximum and minimum measures of the dimension). After 36 days, they were injected in the tail vein with 100 μl 1.075 M [U-13C]-glucose 3 times at 15 minute intervals. After, the scientists took blood samples after the first injection and then again an hour later. The tumors were then excised (surgically cut out), weighed, and finally flash frozen in liquid nitrogen. The metabolites or the substances made when the glucose was broken down were extracted and measured, in order to determine the activity level of the pyruvate carboxylase, and its effect on tumor size.
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Results
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Cell growth was decreased by PC knockdown, with shPC55 being more effective than shPC54 (supplemental figure 4C). This supports shPC55's ability to knockdown PC to a larger extent (Supplemental Figure 4A).
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(Figure 4c) Tumor xenografts from shPC55-transduced A549 cells showed a 2-fold slower growth rate than did control shEV tumors.
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(Figure 4D) The extent of PC knockdown in the mouse xenografts was less than that in cell cultures, leading to less attenuation of PC expression (30%-60% of control) and growth inhibition.
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PC expression in the excised tumors correlated with the individual growth rates.
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PC knockdown reduced oxidation of 13C6-glucose through the Krebs cycle. Hence, entry of glycolytic 13C5-citrate (blue circles in Figure 5A).
Conclusion
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PC expression and activity may promote the spread of metastasis in the lungs
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Increased PC activity boosts citrate production, which aids cancer cell proliferation and development
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As cancer cells advance, they are exposed to higher amounts of oxidative stress, implying that the protection provided by PC expression is crucial for metastatic spread.
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Specific phases in the metastatic cascade necessitate the use of PC. The metabolic flexability required for cancer growth and metastasis is facilitated by PC's regulatory effects on energy pathways.
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PC suppression compromises anaplerotic input into the Krebs cycle, which in turn reduces the activity of the Krebs cycle.
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PC knockdown reduced tumor growth in a mouse xenograft model, hence in human NSCLC CA cells, PC knockdown could cause a decrease in CA cell proliferation.
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PC-mediated anaplerosis is necessary for tumore survival and proliferation in early stage NSCLC
Overall, it is supported that Pyruvate Carboxylase mediated anaplerosis in the growth and survival of cancer cells is critical for the metabolic flexibility necessary for metastasis. Several studies have shown a requirement for PC expression for specific steps in metastasis in vitro and vivo. In vitro: The term in vitro refers to a medical study or experiment which is done in the laboratory within the confines of a test tube or laboratory dish. In vivo: The term in vivo refers to a medical test, experiment, or procedure that is done on (or in) a living organism, such as a laboratory animal or human and its essential role in mammary to pulmonary metastasis (breast to lung cancer). PC’s central role in regulating energy metabolism may increase cellular metabolic plasticity, Metabolic plasticity is the ability of the cell to adjust its metabolism to changes in environmental conditions. Increased metabolic plasticity is a defining characteristic of cancer cells, which gives them the advantage of survival and a higher proliferative capacity, that is required to adapt to changes in nutrient availability during cancer growth and progression. PC’s role in coordinating energy metabolism is particularly relevant in the pulmonary microenvironment, as PC expression and activity may drive the growth of metastatic cells at this site. Increased PC activity promotes the synthesis of citrate, further promoting cancer cell growth and progression. Cancer cells are exposed to increasing levels of oxidative stress throughout progression, suggesting that protection conferred by PC expression is critical for metastatic progression. PC is required for specific steps of the metastatic cascade. PC’s regulatory effects on energy pathways plays a central role in providing metabolic flexibility necessary for cancer progression and metastasis.
Further Research
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How can inhibition of Pyruvate Carboxylase be used as cancer treatment?
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Does lack of Pyruvate Carboxylase decrease proliferation in other types of cancer?