SB #1 – The Warburg Effect: Break up with OXPHOS, Lactic Acid Fermentation’s bored

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A defining characteristic of cancer cells is that they are known to uptake glucose and therefore grow at a much faster rate than normal cells. This observation could lead one to believe that these cells accelerate the high energy-yielding processes of glycolysis, the citric acid cycle and most notably, oxidative phosphorylation. However, Otto Warburg made a key discovery 90 years ago when he proved that this was not the case: cancer cells instead undergo lactic acid fermentation after glycolysis and therefore avoid oxidative phosphorylation (OXPHOS), even when in the presence of abundant oxygen. As we learned in BCM441, this discovery came to be known as the ‘Warburg effect’ or aerobic glycolysis.

Since this important finding, primary literature has been filled with new clues as to how and why producing significantly less ATP is beneficial for proliferating cells. A fair amount of interest has been placed towards the two key regulating enzymes of glycolysis: pyruvate kinase (PK) and phosphofructokinase-1 (PFK-1). Proliferating cells are known to accelerate the process of glycolysis, and then choose to convert pyruvate to lactate instead of taking the OXPHOS route. Christofk et al. demonstrated that cancer cells choose to splice for the M2 isoform of PK as it is the embryonic version and therefore responds directly to growth factors. Zancan et al. connected increased expression of specifically the liver isoform of PFK-1 to a higher efficiency of glycolysis in proliferative breast cancer cells over its muscle and platelet isomers.

The paper published in November 2018 by Li et al. focuses on TAp73, an oncogene and homolog of famed tumor suppressor p53, and its connection to both PFK-1 and the pentose phosphate pathway (PPP). Prior studies indicated a correlation between TAp73 and glucose-6-phosphate dehydrogenase (G6PDH), the rate-limiting enzyme of the PPP, in that TAp73 increases expression of G6PDH and thus the production of ribose and other important macromolecules for proliferating cells to use. Through their investigation, Li et al. connect the increased expression of TAp73 to the stimulation of G6PDH and PFK-1, the latter a new, high-impact connection. The stimulation of both enzymes thus rapidly promotes glycolysis and the PPP.

First, the authors demonstrate the relationship between TAp73 and glycolysis by knocking out TAp73 in first mouse embryonic fibroblasts (MEFs) and human U2OS osteosarcoma cells and then in a variety of cancer cell lines including lung cancer and glioma cells. The findings indicate that defective TAp73 results in decreased lactate output and glucose uptake. Next, the authors prove that PFK-1 is the target glycolytic enzyme of TAp73 by contrasting the expressions of each glycolytic enzyme with and without the presence of TAp73 (Figure 1). The data show only a statistically significant difference for PFK-1, therefore confirming this connection. The authors also investigate and confirm that the liver isoform of PFK-1 (PFKL) is the target enzyme of TAp73, as there was virtually no effect on its muscle or platelet forms when TAp73 was knocked out (Figure 1). This finding correlates with the results of Zancan et al. (2010).

Figure 1. Adapted from Li et al. Graph a shows significant correlations between expression of PFKL and also G6PDH and presence of TAp73, confirming PFKL as its glycolytic target. Graph b, c and image d indicate a statistically significant relationship between the liver isoform of PFK-1 and TAp73.

Using gene cloning and chromatin immunoprecipitation (ChIP), the researchers next identify three potential response elements (RE) in PFKL and conclude that TAp73 activates RE3 of PFKL, using an induced luciferase reporter plasmid as an indicator of binding. They immediately proceed this by silencing p53 and p63, oncogenic homologs of TAp73, and tracking subsequent PFKL expression, and determine that there is no correlation between either of the homologs and PFKL expression. Both of these findings indicate TAp73’s role as a transcriptional activator for PFKL by binding to its RE3 region and eliminate p53 and p63 as relevant activators in this cascade.

After providing evidence for a link between PFKL and TAp73, the authors turn their attention towards establishing the relationship between G6PDH, PFKL and TAp73. First, they deplete cells of TAp73 and look at overexpression of G6PDH and PFKL both alone and together. They find that overexpressed G6PDH and PFKL alone in the presence of TAp73-depletion did not lead to proliferation, however when overexpressed G6PDH and PFKL were together, proliferation was successful despite the depletion of TAp73 – a unexpected result. Furthermore, they find that these cells grew at the same rate as cells with sufficient TAp73 (Figure 2). This is a new, impactful conclusion for the paper because it suggests that TAp73 enhances proliferation when it activates both G6PDH and PFKL, however it is not required for cancer cells to proliferate. In other words, TAp73 up regulation is advantageous, but not mandatory for tumor growth.

Figure 2. Adapted from Li et al. B & C show that cells with knocked-out TAp73 (-) and overexpressed G6PDH or PFKL were still able to proliferate and that cells with TAp73 (+) and overexpressed G6PDH or PFKL proliferated significantly faster than the control.

The in vivo studies the researchers perform on immune-compromised mice are also an important aspect of this paper. First, the researchers inject the mice with TAp73-expressed MEFs, TAp73-depleted MEFs, and TAp73-depleted MEFs with PFKL and find that tumors are able to grow in the presence of the TAp73-depleted MEFs supplemented with PFKL. Next, they look at the effects of increased G6PDH and PFKL on tumorigenesis by injecting the mice with colon cancer HCT116 cells that have control, PFKL-overexpression and G6PDH-overexpression both in the presence and absence of TAp73. The results are surprising in that they indicate that depleted TAp73 has little effect on tumorigenesis in the presence of the overexpressed cells and that PFKL alone is sufficient for tumor growth. The results of both of these trials correlate with the conclusion drawn above: TAp73 heightens cancer growth through upregulation of PFKL and G6PDH. By implementing research in vivo, the authors show the strong impacts of these findings for future studies.

Phosphofructokinase, PFK-1, is referred to as the ‘gatekeeper’ to glycolysis for it is an irreversible reaction that has a variety of inhibitors including citrate and ATP. If cells can limit the amount of ATP they produce by avoiding oxidative phosphorylation, then the rate of PFK-1 will be increased – a tactic that links to the ‘Warburg effect’, the pathway that cancer cells choose to follow. This paper provides evidence that a transcriptional activator, TAp73, promotes the expression of the liver isoform of PFK-1, PFKL, and G6PDH, the key enzyme of the PPP – both of which are connected to tumorigenesis. Future work could include regulating the expression of particularly PFKL and also G6PDH as potential targets for anti-proliferation drugs. In addition, perhaps looking at additional activators, besides TAp73, and their relationships to enzymes in glycolysis and lactate dehydrogenase would be beneficial to therapeutic inquiry.


Christofk, H. et al. 2008. The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature 452, 230-233.

Du, W. et al. 2013. TAp73 enhances the pentose phosphate pathway and supports cell proliferation. Nature Cell Biology 15, 991-1000.

Li, L. et al. 2018. TAp73-induced phosphofructokinase-1 transcription promotes the Warburg effect and enhances cell proliferation. Nature Communications 9, 4683.

Vander Haiden, M.G., Cantley, L.C. & Thompson, C.B. 2009. Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation. Science 324, 1029-1033.

Xie, J. et al. 2014. Beyond Warburg effect – dual metabolic nature of cancer cells. Scientific Reports 4: 4927.

Zancan P., Sola-Penna, M., Marques Furtado, C. & Da Silva, D. 2010. Differential expression of phosphofructokinase-1 isoforms correlates with the glycolytic efficiency of breast cancer cells. Molecular Genetics and Metabolism 100 (4), 372-378.

7 Replies to “SB #1 – The Warburg Effect: Break up with OXPHOS, Lactic Acid Fermentation’s bored”

  1. Exciting paper with many implications towards cancer treatment and future pathway targets. The fact that the researchers proved that even without viable TAp73, the cell reproduce so long as PFKL, and G6PDH are available is incredible, and beneficial information.
    However, perhaps there is a more efficient way to eradicate the cells. Using a stimulant maybe it could be possible to induce another pathway that produced ATP leading to inhibition of PFK-1 which would slow down cancer cell proliferation by slowing lactate production?

    1. Interesting question Paul! I can’t think of another pathway that creates a substantial amount of ATP other than OXPHOS, which is what cancer cells avoid. Perhaps there is another way to inhibit PFKL besides producing a lot of ATP, for example exposing the cells to a high concentration of citrate, another PFK inhibitor. I researched this a little, and found several articles that have studied and discussed citrate’s anti-proliferation effects.

      If you have time, read the section titled “Citrate-mediated PFK inactivation in aerobic glycolytic HCC cells enhances EGCG-induced apoptosis” of this paper:
      Citation: Li et al. 2016. In vitro and in vivo study of epigallocatechin-3-gallate-induced apoptosis in aerobic glycolytic hepatocellular carcinoma cells involving inhibition of phosphofructokinase activity. Scientific Reports 6, 28479.

      There are so many articles on this topic out there but I thought this one was interesting!

  2. I like the title of this article! The results of this paper are interesting because TAp73 presents as a potential druggable target to reduce tumorigenicity of cancer cells. It is notable that TAp73 upregulates both PFKL and G6PDH – is there a connection between these two enzymes (especially considering that when only one enzyme was upregulated, without TAp73, proliferation was reduced, but when both are overexpressed, proliferation occurs)? Also, I’m assuming PFKL is the M2 isoform?

    1. Thank you Pooja for your comment! To start, this paper did not study the details of whether PFKL and G6PDH are connected nor really address the possibility, but it appears that they very likely could be. I began research into the primary literature for a possible connection, but I can’t seem to find anything valuable. I believe that if there was a definite link between the two the authors would definitely state and analyze that! This is a project to explore in the future.

      As per your PFKL question, the M2 isoform does not exist in PFKL nor PFK-1 (I should say has been found to exist.. yet) – it exists in pyruvate kinase as we learned in BCM441. With this in kind, these authors actually analyze PKM2 (see figure 2a) as a possible target of TAp73 however do not find any significant data to show a connection.

  3. I am somewhat surprised that the authors did not bring up the idea of TAp73 as a druggable target for some cancers. They discussed that TAp73 deficiency leads to higher rates of death and developmental deficiencies in newborn mice, but this shouldn’t be an argument against targeting TAp73 since most cancer occurs in adults. Do you have an idea as to why they do not discuss this, because it seems like a partial inhibition or decrease in expression would slow tumor growth.

  4. The work of this paper is very impressive. TAp73 as a tumor suppressor induces one of the regulating steps in glycolysis. The role of PFKL is to promote the production of tumor, while G6PD reduces the expression of tumor cells. As PFK-1 is a significant regulator of glycolysis, TAp73 is an important regulator of PFK-1. What also attracts me is in the discussion they talked about the p53 and TAp73 together. p53, a well-known tumor suppressor, and TAp73, as a homolog of p53, are both regulating the glycolysis but they are doing the different jobs. I believe there are more tumor suppressors in p53 family are very valuable to study.

  5. The authors claim that TAp73 which is a structural homolog of the tumor suppressor p53 (commonly mutated in cancers) is frequently overexpressed in human tumors. They also say that this change indicates a proliferative advantage that it can confer to tumor cells. Is this the only thing that could be contributing to the ]robust growth of the cells? Further, it is interesting that the mechanisms controlling PFK-1 expression (which is essential to cell growth and is almost ubiquitous) remain largely unknown in both normal and malignant cells. What are some of the challenges associated with identifying these mechanisms?

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