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Searching for Early Signs of Pancreatic Cancer
these autoantibodies can help in diagnosis because detecting circulating cancer-specific antibodies is easier than detecting circulating cancer-specific proteins.” Novelli’s team began with protein extracts from PDAC and normal pancreatic tissue. A 2DE gel showed >1000 spots in the resulting map, and a Western blot zeroed in on R-enolase. Sepa-
Pancreatic cancer might be the most vicious of all cancers. It’s difficult to detect, tumors progress and spread very rapidly, and medical intervention is often unsuccessful. Pancreatic ductal adenocarcinoma (PDAC), for example, has just a 4% survival rate after five years. But now in JPR (DOI 10.1021/ pr100213b), an interdisciplinary research team demonstrates new progress in the ongoing search for a useful pancreatic cancer biomarker. The enzyme R-enolase is up-regulated when pancreatic tumors are present, and the international group headed by Francesco Novelli of the University of Turin (Italy) carefully analyzed an autoantibody response to R-enolase that is associated with New molecular flares. Antibodies to two isoforms of onset of the disease. They conclude R-enolase (marked by arrows) represent potential that human R-enolase is phosphodiagnostic biomarkers for pancreatic cancer. rylated at a specific serine residue in the protein when tumors are present, causing the immune ration by weight in one dimension response. showed R-enolase near 47 kDa, and furTo detect cancer early, it’s critical to ther separation by isoelectric point in recognize any potential molecular the second dimension distinguished four “flares” that might be set off when isoforms of R-enolase in normal tissue healthy cells first turn malignant. Once and six isoforms in PDAC. The two isopancreatic cancer is indicated, the only forms of R-enolase associated with marker in the blood commonly used PDAC (termed ENOA1 and ENOA2) are clinically is a glycoprotein, termed more acidic than the four isoforms CA19-9. This glycoprotein is a large found in both normal and PDAC tissues. (>1000 kDa) mucin, and an increase in The research team also worked with its concentration also often accompa>250 patients, which included subnies a variety of other ailments, includgroups of healthy subjects, PDAC paing several types of cancer as well as tients, and individuals with possibly non-oncological conditions such as confounding ailments, such as autoimcirrhosis, pancreatitis, and obstructions mune disease, pancreatitis, or other of the bile duct. Assays for CA19-9 are forms of cancer. Blood samples from thus not useful for detecting pancreatic individuals were subjected to similar cancersthey create too many poten2DE and Western blot analysis. tially devastating false negatives and Akhilesh Pandey of Johns Hopkins false positives. As a “flare”, the glycoUniversity lauds the value of the approtein CA19-9 is just not specific proach. “We recently published a comenough. pendium of potential biomarkers for The enzyme that Novelli’s group expancreatic cancer,” he says, referring amined, R-enolase, offers much more to a paper last year in PLoS Med. (DOI: promise because it triggers an immune 10.1371/journal.pmed.1000046). “This response. “These are very important group and others have shown that results,” says Yasuhiro Kuramitsu of R-enolase is a prime candidate, and Yamaguchi University (Japan). “So far, characterizing these autoantibodies is many autoantibodies have been rean important step forward.” ported, and a combination assay with
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Journal of Proteome Research • Vol. 10, No. 1, 2011
Novelli’s team further used a combination of other techniques, including immuno-based methods, LC/MS/MS protein analysis, and “on-blot” phosphatase treatments. They concluded that ENOA1 and ENOA2sthe R-enolases associated with PDAC and not healthy individualssare specifically phosphorylated at serine 419. Wei Chen of Duke University offers enthusiasm about the results but evinces concern about some of the details of the analysis. “A straightforward molecular biological experiment would be a mutagenesis [of serine 419] to an alanine,” he says. “That would be the best way to remove doubt” about the role of phosphorylation at that site. Pandey agrees that further studies are warranted. “You might try almost the converse of a mutagenesis,” he suggests. “Construct a phosphorylated version of R-enolase protein as a phosphopeptide and check the immune response.” “Site-directed mutagenesis aimed to inhibit phosphorylation at serine 419 would clarify the role of this post-transcriptional modification,” Novelli agrees. “We are also conducting further proteomics studies to clarify whether the R-enolase phosphorylation is a specific feature of pancreatic cancer, if it might be shared with other types of cancer, and if it occurs in normal pancreatic cells,” he adds. The researchers also report that by combining a scoring system for the autoantibody response for R-enolase with the CA19-9 assay, they achieved 95% accuracy in sorting pancreatic cancer patients from the rest of the pool. But “with the patient samples, it’s hard to determine how they should be scored,” cautions Chen. “It’s a good early step, but they knew the answers at the outset, in a sense.” The large team includes research groups from several major hospitals and universities in Italy, “as well as a critical contribution from the Center for Proteomics and Molecular Medicine at George Mason University,” says Novelli. “Our experience shows that a multidisciplinary approach that links clinical and basic research can lead to progress in translational medicinesand particularly in pancreatic cancer.” —Steven Powell
10.1021/pr1005845
© 2011 American Chemical Society
