When pyruvate kinase is discussed, cancer is not the usual topic of conversation. This ubiquitous enzyme catalyzes the final rate-limiting step in glycolysis; transferring a phosphoryl group from phosphoenolpyruvate to ADP, generating ATP and pyruvate. The M2 isoform (PKM2) is exclusively expressed in embryonic or adult dividing cells. Before cell division, glycolysis levels are down-regulated by PKM2 dissociation into dimers and subsequent transport into the nucleus. This allows for glycolysis to be temporarily halted during cell division. After cell division, PKM2 reverts into its cytosolic, or tetrameric form for glycolysis. No harm done, right? Not always.
This unassuming enzyme plays an important role in cancer, acting as a transcriptional coactivator and protein kinase to trigger the expression a number of genes that drive tumorigenesis. Its export into the nucleus, where the damage is done, is elicited by acetylation at a specific lysine (433) by the transcriptional coactivator complex p300A. However the conversion of PKM2 back into its ATP-generating role in the cytosol was not understood. A recent study by Abhishek Bhardwaj and Sanjeev Das at the National Institute of Immunology in New Delhi, India has revealed that SIRT6, an enzyme of the sirtuin family of NAD+-dependent deacetylases, deacetylates PKM2, driving its transport out of the nucleus. In addition to solving the PKM2 nuclear export mystery, their study identifies a mechanism for the recently identified tumor suppressor properties of SIRT6 in pancreatic, liver, and colorectal cancers. SIRT6 was already known as a jack of all trades, with multiple activities on several target proteins in addition to its epigenetic function of deacetylating histones. Bhardwaj and Das have now shown that its role as a tumor suppressor is at least partly due to its deacetylation of PKM2.
To elucidate SIRT6 tumor suppressor functions, Bhardwaj and Das performed a proteomics screen and identified PKM2 as one of the interacting proteins. They confirmed that the two proteins interact directly and that the endogenous interaction was largely lost after prolonged starvation, consistent with transport of PKM2 to the cytosol. They then showed that out of the three known PKM2 acetylation sites, SIRT6 deacetylates PKM2 specifically at the Lys 433 position. Experiments using SIRT6 knockdowns and constitutively cytosolic or nuclear PKM2 confirmed that Lys 433 deacetylation by SIRT6 was controlling nuclear export. Lastly, exportin-4 was identified as the specific transporter responsible for removing deacetylated PKM2 from the nucleus. To further define the role of SIRT6 in suppressing PKM2 driven tumorigenesis, Bhardwaj and Das considered PKM2’s nuclear activities regulating transcription.
There are two known ways in which PKM2 regulates transcription once in the nucleus; by phosphorylating STAT3 and histone H3, and by acting as a coactivator with β-catenin. Bhardwaj and Das observed that both of these processes were down regulated in parallel with PKM2 nuclear export. Phosphorylation of STAT3 and histone H3 was reduced during starvation, as well as transcription of genes controlled by STAT3 and promotor binding. The interaction of PKM2 with coactivator β-catenin was also abrogated concomitantly with deacetylation by SIRT6. Taken together, these results clearly show the regulatory role of the SIRT6/PKM2 interaction in tumorigenesis.
The importance of this interaction hit home when altered malignant phenotypes were observed using SIRT6 knockdown mutants and constitutively cytosolic or nuclear PKM2 mutants. The migration potential and invasiveness of H1299 lung carcinoma cells, were greatly reduced upon ectopic expression of SIRT6. Cells with a constitutively nuclear PKM2 mutant showed lower oncogenic potential in the presence of SIRT6 than in its absence, demonstrating the general antiproliferative functions of SIRT6. Lastly, these hypotheses were tested in mice with hepatocellular carcinoma, and a phenotype of reduced tumor size was observed.
This study clearly defined the mechanism of SIRT6 tumor suppression and demonstrated the importance of PKM2 nuclear export to the phenomenon. Having a more complete picture of how PKM2’s tumorigenic activity is controlled provides both hope and caution for cancer drug developers. Potential points for therapeutic intervention include acetylation of PKM2 by p300A and phosphorylation of STAT3 and histone H3 by PKM2. At the same time, targeting SIRT enzymes for cancer, such as SIRT2 for non-small cell lung cancer, may result in off target activity with SIRT6 and be tumor promoting.
– Hannah Lucas