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ONCOLOGY
Key Relay Protein Shapes Cancer MessageRefolding of Messenger Molecules Needed for Breast Tumor DevelopmentA tiny enzyme able to latch onto proteins and change their shape plays a critical and surprising role in promoting some of the most belligerent forms of breast cancer. Like most cancer cells, those that give rise to breast tumors are a motley bunch, some more aggressive than others. Among the fastest growing are those that produce an overabundance of the receptor Her2/Neu. Once activated, Her2/Neu sends a signal, via a series of molecular messengers, to an agent lying in wait inside the nucleus that tells the cell to enter the cell cycle. Though researchers have identified some of the middlemen, it is not clear how the message is delivered to the cell cycle gatekeeper, cyclin D1. Pin1 pins down proteins. The path to breast cancer (left) begins when Neu, Ras, and other factors signal kinases to phosphorylate the proteins c-jun, beta-catenin, and NF-kappaB (pink ovals). Once phosphorylated, the proteins are tackled by Pin1, which causes them to undergo conformational changes (pink rectangles). Now fully activated, the proteins turn on genes that result in the phosphorylation of cyclin D1. Note that phosphorylated cyclin D1 is modified and stabilized by Pin1. (Image adapted from original by Kun Ping Lu) It now appears these middlemen must be tackled and twisted into a new shape by a 163-amino-acid protein, Pin1, if they are to deliver Her2/Neu's cancer-promoting message. HMS researchers studying Her2/Neu-mutant mice found that more than 90 percent developed breast cancer, but only if they carried at least one copy of the Pin1 gene. By removing Pin1, the team prevented all but a few cancer-primed mutant mice from developing tumors. The findings by Gerburg Wulf, HMS instructor in medicine, Kun Ping Lu, HMS associate professor of medicine, both at Beth Israel Deaconess Medical Center, and colleagues appear in the July 15 EMBO Journal. What is more, the researchers were able to see the protective effects of removing Pin1 very early, even before the tumors developed. Using a novel culture assay, Wulf, Lu, and colleagues isolated and observed mammary epithelial cells from young Pin1-carrying and knockout mice and found that they could predict which cells would develop into breast tumors. MO with a TwistThe discovery comes at a time when pharmaceutical and biotechnology companies are racing to develop inhibitors of Pin1. What has made the diminutive protein such an attractive target is its unique mode of operation. To become activated and carry out their functions, proteins must receive a phosphate group, which induces the protein to change its shape, making it accessible to other proteins. Until recently, it was assumed that phosphorylation was sufficient to induce these activating conformational changes. But it turns out, to carry out their functions, certain proteins require additional modifications. Pin1 brings about these postphosphorylation changes by latching onto proline amino acids, but only those that are adjacent to phosphorylated serines or threonines. Once attached, Pin1 induces the protein to further change shape, making it more accessible to interaction with other proteins. The discovery that Pin1-induced conformational changes are necessary if Her2/Neu's middlemen are to deliver the breast cancer-inducing message suggests that Pin1 inhibitors could provide powerful therapies.Such therapies are greatly needed. Currently women carrying the Her2/Neu mutation--which accounts for 20 percent of breast cancer patients--are treated with herceptin. But the response rates are modest. "Most people get a so-so response that prolongs life a few months," said Wulf. Screening for, and inhibiting, Pin1 could tip the scales in the patients' favor. "You could envision that someone who is Her2/Neu-positive and has high Pin1 levels might benefit not only from herceptin therapy but from a combined modality treatment. Giving a Pin1 inhibitor might potentiate the effectiveness of herceptin therapy--that is one scenario," she said. Pin1's Path to CancerHints that the shape-twisting Pin1 might be playing a role in breast cancer first emerged in 1999 when Wulf, Lu, and colleagues found that human breast cancer cells harboring the Her2/Neu mutation and other genetic defects express high levels of the protein. By blocking Pin1, they were able to prevent cultured Her2/Neu-carrying cells from uncontrollably proliferating. But it was not clear if getting rid of the enzyme would stop the progression to cancer in living animals.
Having discovered the role of Pin1 in Her2/Neu-positive breast tumors, Kun Ping Lu (left), Priti Garg (center), Gerburg Wulf, and their colleagues plan to see if Pin1 plays a role in other breast cancers like those induced by BRCA1 and BRCA2 mutations. (Photo by Leah Gourley) To find out, the researchers crossbred Pin1 wildtype and knockout mice with Her2/Neu transgenic mice. The Pin1 strains were also mated with two other transgenic mice, one carrying a cancer-inducing mutation, Ras, that delivers its message to the same pro-mitotic nuclear protein, cyclin D1, as Her2/Neu; and one carrying a mutation, c-myc, that delivers its message to another pro-mitotic agent. More than 90 percent of Her2/Neu and Ras mice carrying Pin1 developed tumors. On the other hand, about 90 percent of mice lacking both copies of Pin1 remained breast cancer-free. Knocking out Pin1 in the c-myc mice had no such effect. To be sure that the protective effect was due to the absence of the Pin1 protein inside the mammary epithelial cells themselves and not from some other advantage found in the knockout strain, Wulf and her colleagues extirpated these cells from two- to three-month-old mice. Inspired by recent work on in vitro human breast cell lines by HMS professor of cell biology Joan Brugge, Wulf and her colleagues grew the transgenic mouse cells in three-dimensional cultures, using media that more accurately mimic living tissue. Cells carrying at least one copy of Pin1 displayed abnormalities not seen in the Pin1 knockouts. "The cells already had an oncogenic program," Wulf said. Lu thinks that the 3-D assay could have enormous clinical application. "We believe that this new 3-D model can be used to identify diagnostic markers for early stages of breast cancer and to screen for and test anti-breast cancer drugs," he said. He and colleagues have developed a company, Pintex Pharmaceuticals, to develop and test Pin1 inhibitors. Based on his most recent findings--and also previous work, showing that Pin1 interacts with cyclin D1 by several routes (see figure)--he is optimistic. "The promising thing about Pin1 is that it hits many oncogenic pathways. So inhibiting it could have far-reaching effects," he said. --Misia Landau |
