Conventional biological wisdom holds that living cells interact
with their environment through an elaborate network of chemical
signals. As a result many therapies for the treatment of cancer and
other diseases in which cell behavior goes awry focus on drugs that
block or disrupt harmful chemical signals. Now, a new road for
future therapies may have been opened with scientific evidence for
a never seen before way in which cells can also sense and respond
to physical forces.
A team of researchers with the Lawrence Berkeley National
Laboratory (Berkeley Lab) and the University of California (UC)
Berkeley has shown that the biochemical activity of a cellular
protein system, which plays a key role in cancer metastasis, can be
altered by the application of a direct physical force. This
discovery sheds important new light on how the protein signaling
complex known as EphA2/ephrin-A1 contributes to the initiation,
growth and progression of cancerous cells, and also suggests how
the activity of cancer cells can be affected by surrounding
tissue.
"This first evidence that the EphA2/ephrin-A1 receptor-ligand
complex, which was previously thought to be strictly a chemical
sensor, can actually sense mechanical properties as well," says
chemist Jay Groves, who led this research. "This coupling of
mechanical and chemical signaling, which could never have been seen
with classical biological methods, helps explain some of the
biological mysteries concerning the onset and progression of
cancer."
Groves holds a joint appointment with Berkeley Lab's Physical
Biosciences Division and UC Berkeley's Chemistry Department. He is
also a Howard Hughes Medical Institute (HHMI) investigator. With
members of his research group Khalid Salaita and Pradeep Nair, plus
Rebecca Petit, he has co-authored a paper on this research that was
published in the March 12, 2010 issue of the journal
Science. The paper is titled, "Restriction of Receptor
Movement Alters Cellular Response: Physical Force Sensing by
EphA2."
Other co-authors were Joe Gray, Richard Neve and Debopriya Das
of Berkeley Lab's Life Sciences Division.
Cancer and EphA2/ephrin-A1
The term "metastasis" comes from the Greek word for
"displacement," and it is used to describe the process whereby
cancer cells detach from a tumor, enter the bloodstream and spread
to other tissues throughout the body. For example, cancerous breast
cells can spread to a lung and form a new breast cancer tumor
there. Central to metastasis is the EphA2/ephrin-A1 receptor-ligand
complex.
EphA2 is a member of the receptor tyrosine kinase (RTK) family
of enzymes that are key regulators of cellular processes. The
over-expression of EphA2 has been linked to a number of human
cancers, including melanoma, lung, colon and prostate, but is
especially prominent in breast cancer. Some 40-percent of all
breast cancer patients show an over-abundance of EphA2, with the
highest levels found in the most aggressive cancer cells. Ephrin-A1
is a signaling protein that is tethered to the surface of a cell's
outer membrane. It binds to EphA2 in a neighboring cell like a key
fitted into a lock. When ephrin-A1 binds with EphA2, the newly
bound complexes become activated and gather in a cluster.
"The host cell will then literally give the clusters a
distinctive tug, applying a force that pulls the clusters across
the surface of the cell to a centralized location," Grove says.
"What we found is that by applying an opposing force, we could
alter the cell's biochemical activity. When we applied a big
opposing force we were able to convert able to convert highly
invasive cells into well-behaved cells. This shows that in addition
to chemically sensing the presence of ephrin-A1, the cells also
sense the mechanical properties of the local environment in which
ephrin-A1 is displayed."
Spatial Mutation
Observations have indicated that mammalian cells are sensitive
to the physical aspects of their environment, such as the texture
or geometry of the surrounding tissue. However, evidence that
physical forces impact freely-moving signaling molecules (as
opposed to focal adhesion molecules) in the membranes of cells has
been lacking because the cell membrane is an environment that has
always been difficult to characterize and manipulate. Groves and
his research group have found a way to overcome this obstacle with
the development of unique synthetic membranes constructed out of
lipids and assembled onto a substrate of solid silica that enables
them to directly control cellular signaling activities.
"We call this approach the 'spatial mutation' strategy because
molecules in a cell can be spatially re-arranged without altering
the cell in any other way," Groves says. "We first used this
strategy in 2005 to study T cell signaling in the immune
system."
In this latest study, Groves and his colleagues worked with
mammary epithelial cells from a library of 26 model human breast
cancer cell lines that have been well-characterized by co-author
Gray and his research groups at Berkeley Lab and UC San
Francisco.
Says co-author Nair, "Gray's research has demonstrated that this
library substantially reproduces the genomic abnormalities and drug
responsiveness of primary breast cancer tumor cells from patients,
and constitutes the most comprehensive system for the study of the
various aberrations responsible for human breast cancer."
To test the sensitivity of the EphA2/ephrin-A1 signaling complex
to mechanical forces, Groves and his group patterned their silica
substrates with chromium metal lines that were 10 nanometers in
height and 100 nanometers wide. These metal lines acted as
diffusion barriers that impeded the lateral mobility of the
EphA2/ephrin-A1 complexes in the synthetic membrane. The movement
and spatial organization of the complexes were subsequently tracked
through a combination of Total Internal Reflection Fluorescence
(TIRF), reflection interference and epifluorescence imaging
techniques.
"Without the barriers, the clusters of EphA2/ephrin-A1 signaling
complexes were transported to the center of the
cell–supported membrane junction, but with the barriers in
place, there was an accumulation of clusters at the barrier
boundaries," Groves says. "This resulted in a spatial
reorganization that altered the cell's biochemical behavior."
Quantitative analysis of these changes to the spatial
organization of the EphA2/ephrin-A1 signaling complexes across the
library of breast cancer cell lines revealed a strong correlation
with the potential for metastasis. Since the patterned metal lines
in the silica substrate are analogous to the stiffness, texture and
other elastic and mechanical properties of tissue, as well as to
internal structures within the cell membrane, the results of this
study point to intriguing new possibilities for breast and other
cancer therapies.
"It's possible that the force-sensing process itself could
provide a target for therapeutic intervention," says Groves. "We're
also excited about finding targets for which there may be drugs
that have already been developed but are now being used to treat
diseases other than cancer. Given the sensitivity to mechanical
forces displayed by the EphA2/ephrin-A1 signaling complexes, it is
possible these existing drugs could be redirected to the treatment
of cancer."
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