Role of Metabolic Pathways in Cancer

Using siRNA knockdowns to verify antibody specificity

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Metabolic reprogramming has emerged as an important indicator of cancer cells [1]. Even in the presence of oxygen, cancer cells tend to metabolize glucose into pyruvate and then lactate by aerobic glycolysis (also known as the Warburg effect) rather than by the more efficient oxidative phosphorylation pathway [2]. This observation was instrumental in linking metabolic disorders to cancer. In an elegant review by Pavlova and Thompson [3], cancer-associated metabolic characteristics are classified as: (1) deregulated uptake of glucose and amino acids, (2) use of opportunistic modes of nutrient acquisition, (3) use of glycolysis or tricarboxylic acid (TCA) cycle intermediates for biosynthesis and NADPH production, (4) increased demand for nitrogen, (5) alterations in metabolite-driven gene regulation, or (6) metabolic interactions with the microenvironment. Of these metabolic characteristics, the deregulation of glucose and glutamine metabolism has been studied extensively, and the key proteins in these pathways are being explored for potential therapeutic interventions [4,5]. Here we detail several Invitrogen antibodies that are proving useful for the study of cancer metabolism and highlight the strategies we employ to ensure antibody specificity.

Antibodies for studying glucose metabolism

Hypoxia has been found to enhance both lactate production and tumor progression through the activation of hypoxia-inducible transcription factor 1-alpha (HIF1α), glucose transporter 1 (GLUT1), hexokinase 2 (HK2), pyruvate kinase M2 (PKM2), pyruvate dehydrogenase kinase 1 (PDK1), enolase 1 (ENO1), and lactate dehydrogenase A (LDHA). PKM2 is upregulated in most cancer cells, leading to the redirection of metabolic pathways towards lactate production [6]. Thermo Fisher Scientific supplies antibodies against many of these metabolic markers, and the specificity of these antibodies has been validated using siRNA-mediated knockdown to reduce target expression. Figure 1 shows the specificity of the Invitrogen anti-LDHA and anti-PKM2 antibodies on western blots using various cell lines and in the presence of siRNA-mediated knockdown.

Epigenetic control of glucose metabolism

Epigenetic control of glucose metabolism is primarily exerted though the sirtuin family of NAD+-dependent deacetylases (SIRT1–7). In particular, SIRT6 is observed to upregulate glucose metabolism through induction of GLUT1, phosphofructokinase 1 (PFK1), aldolase C (ALDOC), PDK1, and LDHA. The mitochondrial deacetylase SIRT3 acts as a tumor suppressor; deactivating SIRT3 leads to disordered mitochondrial metabolism [7]. Figures 2A and 2B show the specificity of the Invitrogen anti-SIRT3 antibody on western blots using various cell lines and in the presence of siRNA-mediated SIRT3 knockdown.

Overlap of oncogenes and glucose metabolism

Likewise, oncogenes and tumor suppressors are linked to the regulation of glucose metabolism in cancer cells [8]. The expression of TP53- inducible glycolysis and apoptosis regulator (TIGAR) is upregulated by the wild-type tumor suppressor p53, resulting in the inhibition of fructose 2,6-bisphosphate production and redirection of glucose metabolism towards a pentose phosphate pathway [9]. Figure 2C shows the specificity of the Invitrogen anti-TIGAR antibody on western blots using extracts from MCF7 cells without and with doxorubicin treatment, which causes an increase in TIGAR expression.

Antibodies for studying glutamine metabolism

Glutamine is a nonessential amino acid that provides carbon for the TCA cycle and lipid biosynthesis, and elevated glutamine levels have been observed in many cancer cells. Glutaminolysis aided by glutaminases (GLS) leads to the production of various catabolites, including glutamate, which is subsequently converted to α-ketoglutarate by glutamate dehydrogenases (GLUD). Oncogenic molecules such as c-Myc and K-Ras can activate GLS1 and glutamic-oxaloacetic transaminases (GOT), whereas K-ras inhibits GLUD1 in proliferating cells, and therefore these glutamine metabolism enzymes have emerged as potential therapeutic targets for cancer [10]. Figure 3 shows the specificity testing of Invitrogen anti-GLS and anti-GLUD antibodies by immunofluorescence and western blot analysis, respectively, using siRNA-mediated knockdown of the target expression.

Explore our metabolic marker antibodies for cancer research

In addition to the antibodies for glucose metabolism described above, Thermo Fisher Scientific offers an extensive collection of specific, application-tested antibodies directed against various TCA cycle intermediates and lipid and fatty acid metabolism markers that play a role in tumorigenesis and disease progression.

References

1. Hanahan D, Weinberg RA (2011) Cell 144:646– 674. 
2. Warburg O, Wind F, Negelein E (1927) J Gen Physiol 8:519–530. 
3. Pavlova NN, Thompson CB (2016) Cell Metab 23:27–47. 
4. Vander Heiden MG, DeBerardinis RJ (2017) Cell 168:657–669. 
5. Martinez-Outschoorn UE, Peiris-Pagés M, Pestell RG et al. (2017) Nat Rev Clin Oncol 14:11–31. 
6. Kato Y, Maeda T, Suzuki A et al. (2018) Jpn Dent Sci Rev 54:8–21. 
7. Hirschey MD, Shimazu T, Goetzman E et al. (2010) Nature 464:121–125. 
8. Kroemer G, Pouyssegur J (2008) Cancer Cell 13:472–482. 
9. Bensaad K, Tsuruta A, Selak MA et al. (2006) Cell 126:107–120. 
10. Robinson MM, McBryant SJ, Tsukamoto T et al. (2007) Biochem J 406:407–414.