DURHAM, N.C. – New research by investigators at Duke
University Medical Center has provided insight into a
fundamental cellular control mechanism that governs tissue
regeneration, stem cell renewal and cancer growth. In humans,
malfunctions in the pathway have been implicated in skin and
brain cancers, as well as certain developmental defects,
according to the researchers.

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.