Fundamental Finding Yields Insight Into Stem Cells, Cancer; Opens Door To Drug Discovery

The team found that the protein beta-arrestin2, earlier linked to a variety of inhibitory functions, also plays a critical role in activating the so-called hedgehog (Hh) signaling pathway, which plays a central role in early development and normal cell proliferation. When left unchecked, uncontrolled cell growth spurred by the hedgehog pathway can lead to the development of cancerous tumors.

The researchers report their findings in the Dec. 24, 2004, issue of Science. The work was funded by the National Institutes of Health.

"Studies have found a wide breadth of functions for beta-arrestins, but none had revealed a role for these proteins in development," said James B. Duke Professor Marc Caron, Ph.D., a researcher in the department of cell biology, the Duke Institute for Genome Sciences and Policy and senior author of the study. "The involvement of beta-arrestin2 in the hedgehog signaling pathway provides a previously unappreciated paradigm for its role in promoting growth, differentiation, and malignancies."

The finding in zebrafish could lead to new drugs that block the growth of tumors by disrupting the beta-arrestin2 protein's normal function, the researchers said. In other cases, drugs that activate beta-arrestin2 might also drive the proliferation of therapeutic stem cells, they added.

Hh proteins play a central role in cell proliferation and embryonic patterning. In humans, inhibitory mutations in the pathway result in developmental defects such as holoprosencephaly – an often fatal condition characterized by abnormal brain development and facial deformities. In contrast, mutations that spur overactivity of the pathway lead to basal cell carcinoma, the most common form of skin cancer, and medulloblastoma, an aggressive form of brain cancer. About one in five childhood brain tumors are medulloblastomas.

In their experiments, the researchers injected zebrafish embryos with a chemical that specifically blocked the function of beta-arrestin2. Humans and zebrafish, both vertebrates, share fundamental developmental pathways, the researchers said. Zebrafish embryos are an ideal model for study because their transparent embryos allow researchers to easily observe their early development.

The injected embryos, which almost completely lacked the beta-arrestin2 protein, exhibited characteristics earlier linked to defects in the Hh signaling pathway, including curved bodies, underdeveloped heads and abnormal muscle development, the researchers reported. Furthermore, injected embryos exhibited reduced activity of other genes that respond to Hh activity compared to normal embryos, suggesting that loss of beta-arrestin2 blocked their activity, the researchers found. Injection of other substances that activate the Hh pathway restored normal development in embryos lacking beta-arrestin2.

"It appears that beta-arrestin2 is a positive force in Hh signaling in living organisms," Caron said. "The current finding opens up new avenues for study of normal developmental regulation and the manner in which abnormalities in that regulation can lead to cancer. Drugs that disrupt the function of beta-arrestin2 might also offer an alternative approach to cancer therapy."

Another experimental system developed by the researchers -- which includes cells with fluorescently tagged beta-arrestin2 -- might offer a useful tool for identifying drug compounds that either disrupt or promote the protein's activity, said Gregory Fralish, Ph.D., of Duke, co-author of the study. Beta-arrestin2 normally concentrates at the periphery of cells as it binds to activated receptors nestled in the cell membrane, including a component of the Hh pathway known as Smoothened, he explained. Quantifying changes in the amount of fluorescent beta-arrestin2 at the cell surface in the presence of other compounds would therefore identify those that modify Hh pathway activity – compounds of potential therapeutic use.

In a companion paper in the same issue of Science, researchers led by Howard Hughes Medical Institute investigator and James B. Duke Professor Robert Lefkowitz, M.D., also at Duke, demonstrated that beta-arrestin2 does indeed interact with activated Smoothened in cells. That interaction promotes the internalization of activated Smoothened, a finding entirely consistent with the protein's newly recognized role in the signaling pathway, Lefkowitz said.

"The system offers the opportunity to screen for Hh pathway antagonists that might control cancer and, alternatively, for compounds that promote the pathway's activity – a function that might be of use in stimulating the growth of therapeutic stem cells," Fralish said.

Collaborators on the study include lead author Alyson Wilbanks, Margaret Kirby, Larry Barak, and Yin-Xiong Li. Collaborators on the companion study include lead author Wei Chen, Xiurong Ren, Christopher Nelson, Larry Barak, all of Duke; James Chen and Philip Beachy of HHMI and Johns Hopkins University and Frederic de Sauvage from Genentech of South San Francisco.