A team led by scientists at the University of Texas MD Anderson Cancer Center has identified a panel of proteins linked to poor prognosis in hormone-receptor-positive breast cancer.
The study, which the group presented this week at the American Association for Cancer Research annual meeting in Chicago, analyzed expression and phosphorylation levels of twelve major translation-regulating proteins in 190 patients with stage 1 to stage 3 HR-positive breast cancer, finding four that predicted overall survival.
According to Funda Meric-Bernstam, medical director of MD Anderson's Sheikh Khalifa Bin Zayed Al Nahyan Institute for Personalized Cancer Therapy and one of the leaders of the study, all four of the identified proteins are regulated by the PI3K/mTOR signaling system, which has emerged as a potentially key pathway in breast cancer.
"This is a very interesting pathway right now in cancer research," Meric-Bernstam said, citing in particular a 724-subject study published in the New England Journal of Medicine in February that found that adding the mTOR inhibitor everolimus – marketed by Novartis as Afinitor – to conventional endocrine therapy improved progression-free survival in postmenopausal HR-positive patients.
"In laboratory models, activation of [the PI3K/mTOR] pathway confers resistance to the endocrine therapy that we give to HR-positive breast cancer [patients]," Meric-Bernstam told ProteoMonitor. "So we think there could be a potential advantage to targeting it."
The researchers didn't set out to investigate PI3K/mTOR signaling proteins, however, she noted. Rather, they looked at proteins involved in translation, working under the hypothesis that abnormalities in translation could play a role in cancer development.
"Tumors have to grow fast and to be able to do that they need rapid protein synthesis," Meric-Bernstam said. "Additionally, there have been studies suggesting that if you have abnormalities in the translation of certain genes, that can give cancer cells a competitive advantage. So we didn't start off with the question of whether PI3K signaling is important. We started off with the question of whether translation is important."
Their results, however, "feed into the importance of the [PI3K/mTOR] pathway and how it is logical to target it for [breast cancer] therapy," she said.
In the study, the researchers found that increased phosphorylation of ribosomal protein s6 and translational initiation factor 4E-binding protein 1; increased expression of eukaryotic elongation factor 2 kinase; and decreased expression of programmed cell death protein 4 were linked to either recurrence or overall survival.
The patients in the trial were all treated solely with tamoxifen, which, Meric-Bernstam said, allowed the researchers to look specifically at proteins linked to relapse and resistance in response to endocrine therapy.
"It was a unique group of patients because none … at that time got chemotherapy – even patients who had a little bit more advanced disease," she said. "So we could ask the question purely: 'What is the chance of relapse if we treat with endocrine therapy alone, and which proteins play a role in that?'"
She noted, however, that it was impossible to say whether the identified proteins in fact conferred resistance to tamoxifen or if they simply played a role in making the tumors more aggressive in the first place.
The researchers measured the proteins and their phosphorylation levels via reverse phase protein array on an instrument from Aushon Biosystems. The technique – which uses cell lysates spotted in array formats that can then be probed with antibodies to multiple proteins of interest – proved particularly useful due to its ability to work with limited amounts of sample, Meric-Bernstam said.
"It's a very helpful discovery tool because you can use a very small amount of protein from the tumors," she said. "It's a very interesting way of looking at multiple proteins at the same time."
MD Anderson maintains a Functional Proteomics Reverse Phase Protein Array Facility that contains two Aushon microarray printers and serves researchers throughout the center.
Moving forward, Meric-Bernstam and her colleagues hope to expand the study to a larger number of patients as well as investigate patients treated with aromatase inhibitors. She said she is also involved in several ongoing clinical trials looking at different PI3K/mTOR inhibitors as well as cell culture and animal model work investigating alterations in these pathways due to treatment with inhibitors.
Additionally, her lab has recently completed a study investigating the effects of sample-collection protocols on phosphoprotein work. This has emerged of late as a significant issue for the field due to the fact that phosphorylation is a highly labile post-translational modification that can degrade if samples are not quickly preserved. Several companies, including Theranostics Health and Denator, have developed technologies aimed at improving sample collection and storage for phosphoproteomics.
In an interview with ProteoMonitor last year, Mayo Clinic research Edith Perez, who also studies breast cancer, said that sample collection questions made her skeptical of essentially all protein phosphorylation work done to date (PM 12/16/2011).
“Many people have tried to do phosphorylation work, and it is a pity that people have failed to realize that phosphorylation of proteins can change in a few minutes after surgery has been performed,” Perez said at the time. “It’s really hard to understand the validity of what has been published so far. We can all keep saying that phosphorylation is important, but we don’t really have any way to truly measure it.”
In recognition of this problem, Meric-Bernstam used immunohistochemistry and RPPA to compare protein phosphorylation in immediately preserved biopsies and less-immediately preserved surgical samples from 50 breast cancer patients. Her team is currently in the process of examining the data from this study, which, she said, will hopefully address the question of how sample collection affects phosphoproteomic research.
"There have been a couple of [previous] publications that have looked at this question, but not as thoroughly as we're doing it," she said. She noted that the RPPA work that identified the four proteins linked to HR-positive patient outcomes was done "using surgical samples" that were not preserved immediately.
"Clearly there are concerns about if [these] surgical samples have as much phosphorylation as they would if they had been collected immediately," she said. However, "that's not something we addressed in this study."
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