The Six-Carbon Target

Unlocking how cancer cells derive energy from glucose is leading to novel cancer biomarkers and therapies

BY DARRELL E. WARD

Human beings are fundamentally solar powered. The energy that drives the human body—and nearly all life on Earth—originates in the sun and arrives in the diet. Much of that energy is stored in the chemical bonds of glucose, a simple six-carbon sugar. Cells extract the energy using a series of biochemical reactions to generate ATP, an energy-storage molecule that drives most of the myriad chemical reactions that keep cells and the body running.

In fact, a typical human cell contains an estimated 1 billion molecules of ATP at any moment, and these molecules are consumed and replaced every minute or two. Cells draw energy from glucose and transfer it to ATP using a sequence of chemical pathways: the glycolytic pathway, the tricarboxylic acid pathway (TCA) and the electron transport chain. Cancer cells derive ATP from glucose, too, but somewhat differently from healthy cells. Researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James) are studying those differences for ways to improve cancer diagnosis and treatment.

After a brief overview of glucose metabolism in healthy and cancer cells, this article provides four examples of that research, each focusing on different parts of cancer-cell energy metabolism (identified by color in the diagram at the bottom of the article). Their work has led to promising new prognostic and predictive biomarkers to improve diagnosis, and to new therapeutic targets and agents to improve cancer therapy.

From Glucose to ATP


In healthy human cells, glucose metabolism is carefully controlled. It begins in the cytoplasm (blue boxes) and ends in the mitochondria (orange boxes). Transporter proteins in the cell membrane take up the six-carbon sugar from the extracellular space and then undergo a conformational change that pops the molecule into the cytoplasm. There, the sugar enters the glycolysis pathway. This 10-step gauntlet of enzymes tears the molecule in two, generating two net molecules of ATP and two molecules of pyruvate.

The Six Carbon Target 2The pyruvate enters the mitochondria, where it is converted into acetyl-CoA. The molecule spins through the tricarboxylic acid (TCA) cycle, then shoots through the electron transport chain. That process requires molecular oxygen (O2) and generates ATP through oxidative phosphorylation. All told, this energy pathway generates about 36 molecules of ATP per molecule of glucose, plus a molecule of carbon dioxide and water.

Cancer cells require abundant ATP to drive their growth and proliferation, but genetic instability, erratic conditions in the tumor microenvironment and natural selection make each mitosis an experiment in survival for each daughter cell. Glucose metabolism becomes reprogrammed, leaving the cells less reliant on oxygen and better able to survive conditions of low oxygen.

As a result, while healthy cells generate about 10 percent of their ATP from the glycolysis-lactate pathway, glycolysis rates in cancer cells can be 200 times greater than those of matched healthy cells. This metabolic shift by cancer cells is sometimes called the Warburg effect, named after Otto Warburg, who first discovered it in 1924, or “aerobic glycolysis.”

Consequently, cancer cells generate much more ATP in the cytoplasm (blue boxes above) while continuing to rely on aerobic respiration in the mitochondria (orange boxes).

The Six Carbon Target

Barring the Doors
(PURPLE)


An early event in the reprogramming of glucose metabolism is the overexpression of glucose transporter proteins, and it enables cancer cells to stoke their increased use of glucose.

OSUCCC – James researcher Ching-Shih Chen, PhD, professor of Medicinal Chemistry, of Pharmacognosy, of Internal Medicine and of Urology in The Ohio State University College of Pharmacy, is leading a research team in the design of an agent, called CG-5, that inhibits glucose uptake in cancer cells. If all goes well, CG-5 will belong to a new class of anticancer drugs called energy-restriction mimetic agents.

Chen’s work is part of a five-year grant awarded to him by the National Cancer Institute titled “Novel Energy Restriction-Mimetic Agents for Prostate Cancer Prevention” (grant CA112250).

“Energy restriction could be a powerful new strategy for treating cancer because it targets a survival mechanism used by many types of cancer,” Chen says.

Like a cork in a bottle, CG-5 blocks glucose transport proteins, preventing the energy molecule from entering the cell. Chen and his lab team have found that blocking the transporter suppresses a series of signaling pathways and results in tumor suppression.

The Six Carbon Target 3“We have learned that restricting glucose uptake can trigger a starvation-associated cell response that leads to the degradation of a series of oncogenic proteins and is often tumor suppressive,” says Samuel Kulp, DVM, PhD, a research scientist in Medicinal Chemistry and Pharmacognosy in the College of Pharmacy, who helps lead the study.

Their studies in pancreatic cells and an animal model suggest that CG-5 might be effective in pancreatic cancer and might even prevent or reverse resistance to the chemotherapy drug gemcitabine, a mainstay in pancreatic cancer treatment.

Working with Christina Wu, MD, of Emory University, the researchers have evidence that the agent blocks an important signaling pathway in colon cancer called the WNT pathway and two downstream molecules in that pathway called cyclin B1 and TCF4. “This strongly suggests that CG-5 could potentially have application in preventing colon-cancer recurrence,” Chen says.

The Six Carbon Target 4

The Lipid Connection
(ORANGE)


Along with ATP, glycolysis and the TCA cycle produce intermediates that cancer cells need to synthesize the amino acids, nucleic acids, carbohydrates and lipids required for cell growth and mitosis. Studies led by OSUCCC – James researcher Deliang Guo, PhD, assistant professor of Radiation Oncology, have teased out the links between glycolysis and lipid synthesis in glioblastoma (GBM) cells. The findings suggest new strategies for treating cancer.

In a 2011 paper published in the journal Cancer Discovery, Guo and his collaborators show that GBM cells with mutations in the epidermal growth factor receptor (EGFR), which are common in GBM, upregulate the low-density lipoprotein receptor. The findings suggested that GBM tumors require external cholesterol for tumor growth, and that an EGFR inhibitor might be useful for the treatment of GBM with mutated EGFR.

In 2015, Guo and his colleagues published a study in the journal Cancer Cell showing how glucose levels activate lipid synthesis during tumor growth. “Our findings reveal a previously unrecognized, critical role of glucose in controlling lipid synthesis during tumor development,” Guo says.

The metabolic pathway integrates glucose metabolism, lipid synthesis and oncogenic signaling. It includes a switch that deactivates the cell’s large and energetically expensive lipogenesis systems when glucose fuel levels are low.

The study shows how high glucose levels lead to the glycosylation of a protein called SCAP. The glycosylation of SCAP activates a transcription factor called SREBP-1, which travels to the nucleus and activates genes that regulate lipid biogenesis.

When glucose levels are low, SCAP glycosylation doesn’t happen: The switch is off, and lipogenesis goes dark. Use of an animal model showed that inhibiting SCAP glycosylation significantly blocks GB tumor growth. The findings provide a novel approach to blocking lipid synthesis. “This pathway for synthesis of membrane lipids is required for cell growth,” Guo says. “So these transcription factors and their regulator, SCAP, are promising targets for anticancer therapy.”

Guo and his colleagues showed that the SCAP/SREBP-1 pathway is also regulated by a microRNA called miR-29. The study was published in the journal Cell Reports. They showed that SCAP glycosylation activates both SREBP-1 and miR-29. The microRNA operates through a negative feedback loop to inhibit SREBP-1 and SCAP.

The researchers used an animal model to show that transfection of the tumor with miR-29 significantly reduced SCAP/SREBP-1 expression in tumor tissue and prolonged the overall survival of mice with human GBM tumors. Adding fatty acids or active SREBP-1 protein reversed this effect, showing that miR-29-mediated suppression of GBM growth is due to decreased lipogenesis.

In addition, the prolonged survival of mice implanted with GBM cells and transfected with miR-29 suggests that miR-29 suppression of SCAP and SREBP-1 might offer an effective treatment for other malignancies and for metabolic syndromes. “Further analysis of miR-29 distribution in normal brain tissues versus tumor tissues will be important for improving the treatment of glioblastoma,” Guo says.

Last, in a 2016 study published in the journal Clinical Cancer Research, Guo and his colleagues discovered that GBM cells store large amounts of lipid as droplets in the cytoplasm, an abnormal behavior. They also found that elevated lipid storage in GBM correlated with aggressive tumor behavior and lower patient survival.

“Normally, cells do not store lipids in the cytoplasm,” Guo says. “When the body has too much glucose, the excess is stored as lipids in adipocytes.”

The researchers also showed that inhibiting a key gene involved in lipid-droplet formation, SOAT1, suppresses lipid synthesis that is regulated by SREPB-1. This also led to GBM-cell death.

“Our findings suggest that this approach might be most effective against GBM tumors that contain large amounts of lipid droplets,” Guo says. “SOAT1 is a more viable therapeutic target than SREPB-1, and a SOAT1 inhibitor has already been studied in cardiovascular clinical trials. So the strategy of inhibiting SOAT1 as treatment for glioblastoma could be quickly tested in cancer patients.

“Our data explain some of the underlying molecular mechanisms that enable cancer cells to survive the harsh nutritional variability of the tumor microenvironment,” he says.

IDH-Regulated Metabolism
(BLUE)


Arnab Chakravarti 2Grade II gliomas are uncommon tumors that often progress to much more lethal grade III and IV tumors (also known as glioblastoma), “but how best to treat them has been an open question,” says OSUCCC – James researcher Arnab Chakravarti, MD, chair and professor of Radiation Oncology and director of the Brain Tumor Program. “Treatment for these tumors in different countries ranges from surgery alone to surgery and radiation or combined surgery, radiation and chemotherapy.” Chakravarti was the translational-research national study chair for an international trial designed to determine optimal treatment for low-grade glioma (ClinicalTrials. gov number NCT00003375).

The trial involved 251 patients with grade II astrocytoma, oligoastrocytoma and oligodendroglioma. The study had three arms: radiation (RT) only; RT plus procarbazine, lomustine (also called CCNU) and vincristine; and observation only.

The findings, published in the New England Journal of Medicine in April 2016, showed that overall survival was significantly greater in patients receiving RT plus chemotherapy; median overall survival was 13.3 years versus 7.8 years for those who received RT alone.

Furthermore, those who benefited most from the RT-chemotherapy combination were patients with tumors that had a particular mutation in a gene called IDH1. That mutation (IDH1 R132H) signaled improved survival in GBM patients. The analysis was done using preserved tissue that was available for 57 patients in the RT-only group and 56 patients in the RT-plus-chemotherapy group.

The analysis showed that, regardless of treatment, median overall survival was significantly longer among patients with the IDH1 mutation than those without it (13.1 years vs. 5.1 years, respectively). And patients with the mutation who were treated with RT plus chemotherapy had longer overall survival than those who received RT alone.

“The numbers were relatively small but quite significant, suggesting that this mutation could be a good prognostic marker in low-grade glioma,” Chakravarti says. His lab was instrumental in validating IDH1 as a prognostic biomarker during the trial. “We are actively investigating whether IDH1 could be a useful predictive biomarker, as well, to identify glioma patients who would benefit from chemotherapy plus radiation therapy.”

IDH1, or isocitrate dehydrogenase 1, is an enzyme in the TCA cycle that normally catalyzes the conversion of isocitrate to a-ketogluterate and NADP to NADPH. The product of mutated IDH1, on the other hand, is 2-hydroxygluterate (2HG). IDH1 mutations are found in about 75-80 percent of grade II tumors, about 60 percent of grade III tumors and in 5-8 percent of grade IV tumors.

“Tumors with a normal (wild type) IDH1 gene are extremely aggressive,” Chakravarti says. “Even if they look like a grade II tumor under the microscope, they behave like grade IVs.

“We are now trying to learn how IDH1 mutation increases sensitivity for radiation-chemotherapy,” Chakravarti says.

“In the cell,” he adds, “mutatedIDH1 results in lower NADPH production, which increases oxidative stress, and in high 2HG levels. One idea is that the mutation leaves the cell less stable and more prone to oxidative stress, and the cell dies more easily.”

The Six Carbon Target 6

Glutamine Diversion
(GREEN)


OSUCCC – James researcher Nicholas Denko, PhD, MD, associate professor of Radiation Oncology, led a 2014 study published in the journal (grant number CA067166) that identified how hypoxic conditions shift glutamine use in cancer cells from energy production to lipid synthesis. The change helps cancer cells survive hypoxic conditions.

“Our results are particularly exciting because glutamine metabolism is a potential target for anticancer therapy,” Denko says. The findings might offer a new strategy for inhibiting tumor growth by developing agents that reverse the hypoxia-activate pathway, making the cells again vulnerable to hypoxia, he explains. “Tumor cells require glutamine for growth, but drugs that completely block glutamine metabolism will have unwanted side effects because glutamine is also an important neurotransmitter.

“We show that we can block the growth of model tumors by making hypoxic glutamine metabolism follow the normal-oxygen pathway,” Denko adds. He notes that such a therapy should have few-if-any unwanted side effects because normal tissue is oxygenated and already using glutamine in the normal manner.

Denko and his colleagues found that hypoxic conditions activate a gene called HIF1. This led to the breakdown of an enzyme called OGDH2, which is necessary for glutamine to produce energy via the TCA cycle. Loss of the enzyme shifted glutamine use into its conversion to citrate and lipid synthesis.

But malignant cells that were forced to express a hypoxia-resistant form of OGDH2 grew significantly slower in an animal model than tumors with normal OGDH2. “This suggests that reversing this hypoxia pathway might be an effective strategy for inhibiting tumor growth,” Denko says.

In 2016, Denko led a study that provides insights into how hypoxic conditions inhibit glucosederived pyruvate from entering the TCA cycle. Hypoxia decreases mitochondrial function by diverting pyruvate into increased lactate production. Published in the journal Scientific Reports, the study investigated the mechanism that inactivates pyruvate dehydrogenase (PDH), a large mitochondrial enzyme complex that converts pyruvate to acetyl-CoA for entry into the TCA cycle.

The researchers show that an enzyme called PDHK1 phosphorylates one of three locations on the PDH enzyme complex, and this inactivates the enzyme. The metabolic reprogramming that shifts glucose away from TCA activity to lactate production was necessary for tumor growth in an animal model. In addition, an examination of tumor tissue from head and neck cancer patients showed that high PDHK1 levels or PDH phosphorylation correlated with poorer clinical outcomes.

“This study suggests that inhibiting PDHK1 might prevent the metabolic reprogramming that stimulates tumor growth,” Denko says.

Overall, the work led by these OSUCCC – James researchers demonstrates how an understanding of cells at the molecular level helps improve cancer diagnosis and treatment. And, it reveals the remarkable mechanisms living cells use to extract energy that originates in a star.

The Six Carbon Target 5

OSUCCC – James glucose metabolism research
The four examples of OSUCCC – James research described here focus on different areas of the cell’s energy metabolic pathways. (ECS – extracellular space)

Inhibiting the glucose receptor (purple)
Ching-Shih Chen, PhD, and Samuel Kulp, DVM, PhD, lead the design of the glucose uptake inhibitor called CG-5.

Glucose and lipid synthesis (orange)
Deliang Guo, PhD, led a series of studies that identified a glucose-responsive pathway that regulates lipid synthesis in glioblastoma (GBM). High intracellular glucose levels trigger glycosylation of a protein called SCAP. That activates the transcription factor SREBP-1, which activates genes that initiate lipid synthesis. Low glucose levels leave SCAP unglycosylated, and lipogenesis ceases. Other studies showed that SREBP-1 activation also upregulates microRNA-29, which, in turn, inhibits SCAP and SREBP-1 activation. Guo has also shown that activation of a gene called SOAT1 (not shown) leads to storage of cholesterol in cytoplasmic fat droplets. Presence of the droplets correlated with poorer patient survival. Guo has also found that mutated epidermal growth factor receptor (EGFR), common in GBM, upregulates the low-density lipoprotein receptor (LDLR), suggesting that an LDLR inhibitor might be useful for the treatment of GBM with mutated EGFR.

IDH1 (blue)
Arnab Chakravarti, MD, investigated the value of mutated IDH1 gene as a prognostic and predictive biomarker for GBM.

Glutamine metabolism (green)
Nicholas Denko, PhD, MD, led research that identified how hypoxic conditions shift glutamine use in cancer cells from energy production to lipid synthesis, which helps cancer cells survive hypoxic conditions, and how hypoxic conditions inhibit glucosederived pyruvate from entering the TCA cycle.

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