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Duke Team IDs Warburg Effect and Linked Proteins as Possible Breast Cancer Predictor, Drug Target


By Molika Ashford

Following their identification of several protein-signaling pathways active in breast tissue of women at risk for cancer, Duke researchers have now found that the precancerous cells of these women show evidence of the Warburg effect — increased glucose intake and insulin signaling that is a hallmark of late-stage, aggressive disease.

This pattern was something the group hypothesized as a likely result of the protein activation patterns they had identified in earlier studies. Because the Warburg effect is a known hallmark of established aggressive triple-negative breast cancer, the researchers, led by Victoria Seewaldt, director of the prevention program at the Duke University Comprehensive Cancer Center, consider it likely that measuring the effect and its associated signaling proteins in precancerous tissue could be a method of early diagnosis or monitoring. In addition, the finding also opens the possibility that the diabetes drug metformin could be used as a preventative treatment.

Seewaldt told ProteoMonitor that her team is now planning a trial of metformin as a way of blocking the Warburg effect and hopefully stalling the progression of pre-cancerous cells into cancer. The trial design may also allow the group to add treatments targeting additional pathways that they identified in their earlier proteomic analysis.

The approach shows promise for early detection of triple-negative breast cancer — a particularly aggressive form of the disease with a high mortality rate. "We want to find out what's going wrong in the beginning, because for many of these really aggressive cancers, no matter what you do to treat them, they just don't go away and too many women die," Seewaldt said.

In previous research, the Duke team had collaborated with George Mason University researchers Lance Liotta and Emanuel Petricoin, using reverse phase protein microarrays to quantify the activation of several proteins in the breast tissue of women considered to be at an elevated cancer risk based on their family history. The signatures the group found were implicated in either the AKT/mTOR/PI3K/cSrc, EFTF/MEK/ERK or HER2/bcl-2 pathways, they reported last year (ProteoMonitor 11/12/2010).

Seewaldt presented the new Warburg effect data earlier this month at the fourth AACR Conference on the Science of Cancer Health Disparities. Collaborating with the same researchers at George Mason, the Duke team examined samples from two independent sets of 39 and 38 high-risk women.

"One of our questions was, 'Can we start to detect some of the aggressive properties of cancer, like the Warburg effect, in pre-cancerous cells?' Do they take on these characteristics before they get to a cancer?" Seewaldt said.

"One thing that was striking [in our earlier research] was the activation of this AKT pathway. AKT is involved in insulin signaling, but it's also involved in how fast cells take up glucose, and that's how we got to the Warburg effect," she said. "The protein signature is like a puzzle with all the pieces in the box, and we had started to put some of the pieces together. Then we began to realize that glucose uptake would be predicted here, which is why we did these studies."

In their proteomic analysis of the 39 and 38 subjects, the group again found activation in AKT/mTOR, which is associated with the Warburg effect as well as the insulin signaling proteins pACC and IRS1, and IL6/Stat3/vimentin, involved in epithelia-to-mesenchymal transition.

The researchers also measured glucose uptake in atypical mammary epithelial cells from the high-risk women, as well as in ER-negative breast cancer cells using a fluorescently tagged glucose molecule. Both cell groups showed accumulation, indicating the Warburg effect at work both in established cancer and in precancerous tissue.

"What it looks like from this is that glucose uptake is really important because it’s a biologic test of whether the pathways are actually active or not," Seewaldt said. "You can get a protein array, but it may not have any bearing in real biology, so it was very nice to see that we could validate our predictions."

About half of the patients in the two sets were African American; a subgroup Seewaldt said sees a higher rate of these difficult-to-treat cancers. With this in mind, the team's work has centered on establishing a partnership with the Durham, NC, community through a local clinic and associated social services to seek the insight and cooperation of women most at risk for aggressive cancer to participate in the team's proteomic studies and in planning future trials of potential treatments.

Finding evidence of the Warburg effect in precancerous women, Seewaldt said, is important for two reasons. From a public health standpoint, it suggests that glucose uptake could be used as a way to track cells in order to detect the change from pre-cancer to cancer much earlier.

Additionally, from a prevention standpoint, the connection between glucose, insulin, and cancer somewhat blurs the lines between cancer and diabetes and heart disease, which has implications for how to preventatively treat African American women at a high risk of developing breast cancer who also have a higher risk of diabetes, Seewaldt said.

"When a woman becomes pre-diabetic, she gets high glucose and insulin, and we're finding these precancerous cells are very receptive to both … So anyone with pre-diabetes and precancerous cells, every time they eat, they are stimulating these cells."

"We can't look at body parts in people and do 'body part medicine,'" she argued. "We can't give people breast cancer prevention that worsens heart disease and diabetes," like the drug tamoxifen, which Seewaldt said can cause cardiovascular problems and weight gain.

"Women are not just breasts," she said.

A Four-Dollar Drug

The link between glucose intake and cancer led Seewaldt and her colleagues to identify the diabetes drug metformin as a potential cancer preventive agent. "Metformin is a really cheap drug, about four bucks a month. And it's very safe and has few side effects. It's also a drug that targets these very [pathways]," she said.

After the group's first proteomic analyses in 2010, the team had initially planned to work with a kinase inhibitor targeting one of the proteins implicated, but feedback from the Durham community members was that an expensive new drug was not going to cut it.

"We respected their view on this, because you're looking at a $50,000 drug, and I think the community viewpoint was correct, that we really needed a four-dollar drug – something that everybody could use whether they had insurance or not."

Metformin fits nicely into this requirement, as do some other potential treatments the group is considering, including vitamin D, which may impact the IL6 pathway, and good old-fashioned exercise.

Seewaldt said Duke is participating in a trial that to examine how vitamin D impacts IL6, and if that goes well, they could potentially add the supplement, as well as other potential treatments, into the metformin trial they are planning.

"The nice thing about the trial design with the protein markers is you can add things in one at a time and see, does metformin work all by itself or do you have to add something else in?" she said.

"It looks like with AKT and IL6 we are going the have to shut down both," she added. "And I think that feeds into more modern thinking about cancer signaling networks. We used to think we had to be very focused and target one molecule. But it becomes more and more evident that we have to shut down a bunch of different proteins that function as a network."

Another drug the group had hoped to try, a selective serotonin reuptake inhibitor, also met resistance from community partners, according to Seewaldt.

She said the group has a paper that will be published shortly evaluating the effect of the SSRI Prozac (fluoxetine) on the ERK/MEK pathway. Those results were "really, really nice," she said, but "if you're going to have a partnership, you have to have a partnership," and the team has tabled plans for an SSRI trial in community partner subjects.

For the metformin trial, she said that the group is "inching toward approval."

Meanwhile, the researchers are following a group of high-risk women from the Durham community — 350 by fine-needle aspirate samples and another 400 or so via MRI — to see whether the protein signatures they identified, some of which play a part in the Warburg effect, show patterns of expression linked to prognosis.

"We haven't tested the signature prospectively with enough statistical power, yet," she said. "But the fact that these things are what you see in triple negative aggressive breast cancer and we also see them pre-cancer, that gives us a lot of hope that these are useful."

The approach could also help interpret "confusing" MRIs. "They don't have a cancer in there but they have all this stuff and you'd like to biopsy the worst spot. This could help you figure out what the worst spot is," she said.

"We look at biopsies — at what they are at one point in time — but we don’t think about what these things can become because we don't have the tools," she said. "We hope this gives us the tools to do that. So we don't have to sit around waiting and doing MRIs every 30 seconds."

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