BioLinks Journal Club 1 - Cancer and Metabolism

Paper - “​O-GlcNAcylation of PGK1 coordinates glycolysis and TCA cycle to promote tumor growth”

Please read the introduction to this week’s research paper below, which includes the most crucial concepts and background involved. Don’t worry if you don’t understand everything in this summary - we will break it down over the course of the week and dive into the techniques and exact experiments included in the paper . Please remember questions that come up from reading this or anything of particular interest you would like to discuss more on Monday!

How do cancerous cells completely alter their metabolic pathways to allow proliferation?​ Under normal conditions, the majority of our body cells will undergo aerobic cellular respiration in order to convert the sugars (glucose) we eat into chemical energy or ATP that can be harnessed for bodily functions. Oxygen is a key component of the respiration process. There are three main steps through which our glucose reactant will pass; ​Glycolysis​, the TCA cycle, and Oxidative Phosphorylation. Notably, while glycolysis occurs in the cell's cytoplasm and does not require oxygen, the following two steps occur in the mitochondria in the presence of O2. While the vast majority of chemical energy is produced at the end of the respiration process, small amounts are generated at earlier stages, mostly during glycolysis. Instead, cancer cells exhibit a process known as the ​Warburg Effect​. In most types of cancers, including the focus of this study, colon cancer, the Warburg Effect encompasses a shift away from oxidative phosphorylation for ATP production towards glycolysis alone, resulting in increased levels of glycolysis. Although these cancer cells are often in the presence of oxygen, this pathway is actually similar to the process through which our muscles produce lactic acid when exercising; fermentation will occur, creating lactate as a product of glucose.

But if most of the energy from respiration is derived at the oxidative phosphorylation stage, shouldn’t proliferating tumors in need of considerable energy rely on this regular pathway?​ The Warburg Effect actually has a few benefits to cancer cells. It can occur irrespective of damaged mitochondria or in the case of hypoxia in a tumor growing far from blood vessels, and allows more control of the respiration process. As tumors need considerable energy to maintain proliferation, in compensation for the lower ATP yield per glucose molecule, glucose molecules are consumed at a much faster rate, and glycolysis alone is a much shorter process so more glucose can be processed rapidly. While researchers have been aware of this phenomenon for years, this paper investigates how this metabolic shift is able to take place.

Phosphoglycerokinase 1 (​PGK1​) is an important glycolytic enzyme which is responsible for one of the ATP producing steps of glycolysis. Given the nature of the Warburg Effect and previous studies, the team hypothesized that PGK1 played an important role in the metabolic process of colon cancer cells. As metabolism is an essential process of life, this would make PGK1 presence key to cancer development. They first confirmed this by examining the expression of the PGK1 gene in WT (non-cancerous) and seven different colon cancer cell lines, finding that PGK1 levels were significantly increased in the tumor tissues. Furthermore, when testing two samples of cancerous cell line HT-29, they found that the sample with artificially depleted PGK1

levels resulted in much lower levels of cellular proliferation. Taken together, PGK1 was clearly key to colon cancer development, and likely had a role in facilitating the Warburg Effect.​ (Fig. 1 in paper)

But what was causing PGK1 to alter cancerous metabolic pathways?​ Post-translational modifications (PTMs) are highly crucial for the function of proteins, especially enzymes. O-glcNAcylation​ is a type of glycosylation, which is a PTM involving the addition of a carbohydrate group to another compound. It was found that PGK1 undergoes O-GlcNAcylation, meaning that compound O-GlcNAc is added on to PGK1 at a specific spot in its genetic sequence termed T255. Then, cells with higher than usual O-glcNAcylation levels were compared to normal controls, resulting in almost two-fold higher PGK1 expression where there were higher levels of glycosylation. This direct relationship between O-glcNAcylation and PGK1 expression suggests that the former activates PGK1. The role of O-GlcNAc at position T255 in colon cancer development was confirmed by testing rates of cellular proliferation of normal cancer cells with O-glcNAcylation (WT), and those engineered to lack this PTM (T255V). The results demonstrated that cell count dropped more than half in the mutated cancer cells, perhaps already suggesting new therapeutic approaches to tumorigenesis, at least for colon cancer. ​(fig 2-4 in paper)

To confirm the role of PGK1 O-glcNAcylation in promoting the Warburg Effect and regulating cancerous glucose metabolism, normal colon cancer cells (WT) and those mutated to lack O-glcNAcylation (T255V) were tested for abundance of various key metabolic products. The WT cells exhibited higher glucose uptake, lactate production and ATP production than the T255V, all suggesting increased proliferation (more ATP energy) and more glycolysis (higher glucose uptake and lactate production) in the O-GlcNAcylated cells. Contrarily, levels of TCA cycle intermediates were lower in the WT compared to T255V cells, supporting the idea that cancerous cells experience a truncated respiration process that does not include any process occurring after glycolysis. ​(Fig. 7 in paper)

This experiment has huge implications for cancer research. It is evidence that O-GlcNAcylation of PGK1 is a main contributor to the mechanism of the cancerous metabolic process. Their results suggest that preventing glycosylation at this one site would disrupt vital methods of ATP generation significantly, perhaps inhibiting or at least decreasing the proliferation of many cancer types. The experiment creates a new potential avenue for cancer drugs for targeting the glycosylation of PGK1 at T255. The study also sheds more light on how oxidative phosphorylation is decreased in cancerous cells and pinpoints the glycolysis stage, not later on, where the normal aerobic cellular respiration process is disrupted. This study also opens up several new avenues of follow up research. While colon cancer was chosen specifically for this

study, many other cancers exhibit the Warburg Effect, and may rely on similar glycosylation patterns for tumorigenesis.