Attacking Cancer’s Sweet Tooth May Be Effective Against Tumors

Mice with Deadly Tumors but Without Glycolytic Pathway Survive Beyond Four-month Experiment

Cancer leads to a struggle between tumor and host, but tumor cells also engage each other in a kind of Darwinian battle. As a tumor grows, cells crowd one another and may be cut off from oxygen-carrying blood vessels—a distinct disadvantage since most cells require oxygen to produce the bulk of their energy-storing ATP. In the 1920s, the future Nobel-prize winner Otto Warburg proposed that some cancer cells evolve the ability to switch over to an ancient, oxygen-free route, the glycolytic pathway. And they continue to use this pathway even when access to oxygen is restored. Though Warburg’s proposal has since been confirmed, the role played by glycolysis in cancer has been largely ignored. Few have attempted to attack specific points along the glycolytic pathway to gain a therapeutic effect.

A team of HMS researchers has done just that, with stunning effect. Valeria Fantin, Julie St-Pierre, and Philip Leder knocked down one of the pathway’s enzymes, LDHA, in a variety of fast-growing breast cancer cells, effectively shutting down glycolysis, and implanted the tumor cells in mice. Control animals carrying tumor cells with an intact glycolytic pathway did not survive beyond 10 weeks. In contrast, only two of the LDHA-deficient mice died, one at 16 weeks, another at just over 18 weeks. Eighty percent of the mice outlived the four-month experiment. The findings appear in the June Cancer Cell.

Photo by Graham Ramsay

Valeria Fantin and Phil Leder dramatically slowed the development of tumors in mice by shutting down the glycolytic pathway. This ancient avenue for producing cellular energy could provide a rich target for anticancer therapies.

“This is an exciting contribution that reveals a surprising Achilles heel in cancer cells. It also adds to our sense of opportunity for new avenues of cancer therapeutics,” said Stuart Schreiber, the Morris Loeb professor and chair of the Department of Chemistry and Chemical Biology at Harvard University. He has been studying the Warburg effect, but is not an author on the current paper.

A Switch Between Pathways
What may further excite the growing number of researchers who are studying the Warburg effect, and cancer metabolism more generally, is the way the study resolves a long-standing debate about how and why cells switch to glycolysis in the first place. Warburg speculated that cancer cells change over to glycolysis, which occurs in the cytoplasm, because the mitochondria, where oxygen-dependent ATP synthesis occurs, are defective. The mitochondria of cancer cells appear to be mostly intact, however, which led many researchers to minimize the importance of the glycolytic switch.

But cancer cell mitochondria do display an intriguing difference. Normally, these organelles turn glucose into ATP through the oxygen-dependent process of oxidative phosphorylation (OXPHOS). This results in the expulsion of protons, which lowers the mitochondria’s membrane potential. The mitochondria of cancer cells exhibit a high membrane potential, however. Researchers suspected this might be because the cells have switched to an alternative means of producing ATP, namely glycolysis, but it was not clear if the glycolytic and mitochondrial pathways were connected in a switchlike fashion.

It appears the two pathways are indeed reciprocally linked. Fantin and her colleagues found that by shutting down the glycolytic pathway (through the knockdown of LDHA), they could lower the mitochondrial membrane potential of tumor cells. Furthermore, oxygen consumption increased in the knocked-down cells, suggesting they were reverting to the mitochondrial OXPHOS pathway—a kind of Warburg effect in reverse. “The findings provide us with an insight into a mechanism that had been suspected in the last six or seven decades,” said Leder, the John Emory Andrus professor of genetics and chair of that department at HMS.

The Role of Mitochondria
Like many of his colleagues, Leder paid little attention to Warburg’s findings for much of his career, in part, because he was busy studying cancer through the lens of molecular biology. “Mitochondrial metabolism is complicated. If you don’t have to learn it to understand cancer, all the better,” he said. About six years ago, he and Fantin, then an HMS research fellow in genetics, identified a compound, F16, that preferentially targets proliferating breast cancer cells carrying the Her2-Neu mutation while leaving healthy cells untouched. It turned out, F16 was accumulating in the mitochondria of the cells, but it was not clear why.

Fantin, now a research scientist at Merck, and Leder knew that cancer cells can switch to the glycolytic pathway and that this results in a higher mitochondrial membrane potential, meaning a more negatively charged membrane. They suspected that this negative charge was attracting the positively charged F16 molecules. One way to see if this was happening was to lower the mitochondrial membrane potential. This could be done by shutting down glycolysis, thereby forcing the cells to use OXPHOS. It turns out, in 1999, a researcher had discovered that LDHA, an enzyme in the glycolytic pathway, was overexpressed in certain cancer cells.

Using small hairpin RNA, Fantin knocked down LDHA in Her2-Neu breast cancer cells. Working with St-Pierre, then an HMS research fellow in cell biology and now at the University of Montreal, she found the cells exhibited a lower mitochondrial membrane potential and were less receptive to F16. By then the researchers had become intrigued by the Warburg effect and wanted to explore how reversing the effect—blocking glycolysis—would affect tumorigenesis. They allowed the LDHA-knockdown cells to proliferate under conditions of hypoxia and normal oxygen. Deprived of oxygen, the cells were severely disadvantaged. They exhibited a hundredfold decrease in their rate of proliferative activity compared to LDHA-rich tumor cells. Even in the presence of oxygen, the knockdown cells did not proliferate quite as well as the LDHA-rich controls, suggesting that the latter might be reverting to glycolysis in the presence of oxygen.

Tumor Target
The mouse findings provide still more striking evidence that glycolysis is a crucial strategy for tumor growth. Fantin was almost incredulous at the effect that knocking out LDHA had on the mice. “At the beginning, we could not really palpate anything on these mice. After a while, we started palpating these tiny nodules,” she said. “Everything made sense when I saw the pathology slides, and I could see clearly that the mice did have small tumors.” Intriguingly, the tumors exhibited the greatest cell death at their core, where oxygen is most limited. In fact, the only cells proliferating were those located near blood vessels.

There are reasons why cells might switch to glycolysis other than intermittent hypoxia. Cancer cells are energetically expensive—they reproduce quickly and need a readily available source of ATP. Though glycolysis uses up more glucose, it is faster than the oxidative route. And it is safer for the cell. The OXPHOS pathway is a notorious producer of free radicals, which could rise to toxic levels in a high-energy producer like a cancer cell. Glycolysis does not have this side effect.

Knocking out the glycolytic pathway could deliver a big blow to tumor cells. “LDHA could be one weak point that we could attack, but maybe, if we understand exactly all the steps involved, we could devise alternative strategies to attack the same pathway,” Fantin said. What makes the prospect of antiglycolytic therapies even more attractive is their potential safety. Healthy cells meet 90 percent of their energy needs through OXPHOS. People who lack the LDHA enzyme appear to function normally, though they exhibit side effects during high-intensity, or anaerobic, exercise. “They have muscle destruction because they lack an alternative route for producing energy,” Fantin said. It is not clear whether they have a lower incidence of cancer.

Also appealing is the idea of combining antiglycolytic therapies with anti-angiogenic ones. “If you have a molecule that is very stable, you could think about delivering it first, obliterating the glycolytic pathway,” said Fantin. Angiogenesis inhibitors would wipe out blood vessels and the oxygen supply with it, leaving the cells with no way to cope. “There is definite potential to combining these things,” she said.

—Misia Landau

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