Author: Brendan Trafford
Hepatocellular carcinoma (HCC) is one of the deadlier cancers in the world today, with a 5-year survival rate of 57% worldwide. Although this cancer kills almost 600,000 people per year, there is only one drug on the market right now to fight this cancer. This drug is called Sorafenib and it works by interfering with protein kinase signaling in cancerous cells and non-cancerous cell’s membranes. The one major problem with this drug is that not only does it kill rapidly growing cancer cells, but it also kills rapidly growing healthy cells in the body; this can lead to many side effects such as diarrhea, hair loss, fatigue and weight loss. It can even cause more serious side effects such as severe vomiting, seizures and irregular heartbeat. In a recent review article by Nissim Hay, it was found that many cancers, including HCC, expressed the oncogene for Hexokinase 2 enzyme (this is an isozyme of hexokinase 1), which has a lower Km and higher affinity for glucose than HK1. In previous studies, it was found that HK2 was required for tumor initiation and growth and that removing the gene for HK2 could lead to a therapeutic cure for HCC. Using this knowledge, the authors attempted to determine if HK2 was critical for cancer proliferation and how removing HK2 could help kill the cancer.
The first portion (Figure 1) of this experiment involved proving that HK2 expression is a key factor for hepatocarcinogenesis. To do this, the authors took liver samples from people with dysplasia, carcinoma and cirrhosis. They found HK2 expression was significantly higher in dysplasia and carcinoma cases when compared to normal liver cells. Cirrhosis cells had no significant change in HK2 expression when a Friedman test was run. The second experiment conducted in figure 1 involved two groups of mice; one group had the gene encoding HK2 and the HK2 knock down (HK2 KD) group did not. After treating each group of mice with diethylnitrosamine (DEN) they found that the group without the HK2 cells had lower amounts of tumor incidence when compared to the group with HK2 cells. Both experiments allowed the group to confidently say there was a correlation between HK2 and cancer prevalence.
Figure 2 involved growing two groups of cells, HepG2 and Huh7 cells (both forms of cancerous liver cells), with and without doxycycline (Dox)-inducible HK2 shRNA (a gene that can be silenced used doxycycline). First, the group induced the knock down of the HK2 gene and found a substantial reduction in hexokinase activity, which showed that HK2 was the primary isozyme in liver cells. Proliferation curves were then made and showed a 50% decrease in proliferation between silenced HK2 gene cells and the non-silenced ones. The final portion of figure two involved Huh7 cells that were put into mice containing either doxycycline (Dox)-inducible HK2 shRNA cells or not. The mice were fed normal mice chow until tumors formed within the mice. After tumor formation occurred, the experimental group was transitioned into a doxycycline diet while the control group stayed on normal mice chow. The experimental group showed a 50% decrease in tumor growth in comparison to the control group. These findings reinforce the theory that HK2 plays a key role in the proliferation of HCC cells.
Figure 3 compared the proliferation of rat WT HK2, MTB (HK2 that is not bound to the mitochondrial membrane), SA (kinase dead mutant) and GCK or hexokinase 4 in Dox-inducible shRNA cells. The results showed that not only does HK2 need to be active in the cell, but it needs to be attached to the mitochondrial membrane to allow the cell to rapidly proliferate. It also showed that the higher Km hexokinase 4 could not allow the cell to proliferate at levels to that of HK2.
Figure 4 was done to understand the changes to glycolysis, glutaminolysis and the citric acid cycle when HK2 was silenced. Glucose uptake, lactate secretion and pyruvate secretion rates were all measured using radioactive C13 isotope in glucose to see how silencing HK2 changed glycolysis product formation. In this experiment, glucose uptake was reduced by 40%, alongside a reduction of lactate secretion by 40%, and pyruvate secretion of 35%. From the lactate that was excreted, almost all of it had the radioactive C13 from the glucose, meaning that almost all energy in the cell came from glycolysis. This shows that the cancer cells most important way to get energy is through glycolysis. Glutamine (measured using Glutamine C13) uptake and production were not changed during this alteration to HK2, meaning that the cell did not attempt to increase substrates into the TCA. Although the substrate concentrations of the TCA were lower, there was no noticeable difference in flux to the TCA substrates. This means the cells most likely function at a lower metabolic level. This drop in metabolic function is most likely the driver behind the lack of growth in HCC when HK2 is silenced.
The experiments in Figure 5 attempted to determine what specific points of the cell had differing metabolic fluxes, to see what the consequences of HK2 were. This was done by taking the results of the tests in figure 4 and fitting them to a metabolic network model. The only substantial metabolic flux difference to appear, besides the glycolytic flux changes, was a 2-fold increase in extracellular serine uptake that was separate from endogenously created serine from glucose derivatives. A later experiment run by the group found that depriving the cells of serine further decreased the amount of HK2 KD cells.
Figures 6 works to understand how the cell compensates for its energy loss to glycolytic energy loss from HK2 knock down. In Figure 5, they noticed that when HK2 is knocked down, the cell has a noticeable increase of O2 consumption. The proposed mechanism of thought was that the cells were increasing their coupling efficiency between glycolysis and oxidative phosphorylation. This would increase basal metabolic rate, raising O2 consumption and overall leading to higher ATP formation. The group also found that HK2 and HK2 KD cells can run at the same overall respiration rate, but HK2 KD cells always run at full capacity; whereas HK2 cells run at 50% of the basal respiration rate because they can get majority of their energy from glycolysis. They also noticed that HK2 KD cells infused with the drug metformin leads to an even higher amount of O2 consumption.
Figure 7 aims to understand how metformin and HK2 KD increases energetic cell stress. The scientists predicted that the combined effects of metformin and HK2 KD would lead to increased AMPK and then inhibit mTORC1 activity. mTORC1 is a gene for a protein that controls the synthesis of enzymes in glycolysis. Surprisingly, they found that metformin and HK2 KD inhibited mTORC1 but not using AMPK. Instead, these two changes increase the presence of a protein called REDD1, which inhibits mTORC1 through a separate pathway. To prove this the group used siRNA to degrade REDD1 in cells with metformin and HK2 KD and found that mTORC1 inhibition was diminished. By doing this the scientist were further able to decrease the survivability of cancer cells and therefore decrease tumor genesis.
The last Figure of this paper determined if there were synergistic effects between HK2 KD and Sorafenib. Two groups of mice were treated: one group had HK2 KD cells and was exposed to Sorafenib and the other had HK2 KD cells. The group found that the mice with HK2 KD and Sorafenib had 2 times the amount of tumor cell death than those with just HK2 KD.
This paper is extremely relevant to the world today. If we could find a way to inhibit just this isozyme of Hexokinase doctors could specifically kill HCC cells. The group also showed how mixing HK2 inhibition with current drugs on the market, Sorafenib, leads to substantial death of liver cancer cells.
This paper relates to our class because just last week we took a test on the inner workings of the citric acid cycle and glycolysis. I also feel this paper directly relates to the Warburg effect we discussed in class where PKM2 (another isozyme of a glycolytic enzyme) leads to the proliferation of cancer cells.
 Lee et al., “The Actual Five-Year Survival Rate of Hepatocellular Carcinoma Patients after Curative Resection.”
 DeWaal et al., “Hexokinase-2 Depletion Inhibits Glycolysis and Induces Oxidative Phosphorylation in Hepatocellular Carcinoma and Sensitizes to Metformin.”
 “Sorafenib (Nexavar) | Cancer in General | Cancer Research UK.”
 “Sorafenib MedLine Plus Drug Information.”
 Hay, “Reprogramming Glucose Metabolism in Cancer.”
 Patra et al., “Hexokinase 2 Is Required for Tumor Initiation and Maintenance and Its Systemic Deletion Is Therapeutic in Mouse Models of Cancer.”
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