Essential Cell Biology 5th edition

730
HOW WE KNOW
MAKING SENSE OF THE GENES THAT ARE CRITICAL FOR CANCER
The search for genes that are critical for cancer sometimes
begins with a family that shows an inherited
predisposition to a particular form of the disease.
APC—a tumor suppressor gene that is frequently deleted
or inactivated in colorectal cancer—was tracked down
by searching for genetic defects in such families prone to
the disease. But identifying the gene is only half the battle.
The next step is determining what the normal gene
does in a normal cell—and why alterations in the gene
promote cancer.
Guilt by association
Determining what a gene—or its encoded product—
does inside a cell is not a simple task. Imagine isolating
an uncharacterized protein and being told that it acts
as a protein kinase. That information does not reveal
how the protein functions in the context of a living cell.
What proteins does the kinase phosphorylate? In which
tissues is it active? What role does it have in the growth,
development, or physiology of the organism? A great
deal of additional information is required to understand
the biological context in which the kinase acts.
Most proteins do not function in isolation: they interact
with other proteins in the cell. Thus one way to begin to
decipher a protein’s biological role is to identify its binding
partners. If an uncharacterized protein interacts with
a protein whose role in the cell is understood, the function
of the unknown protein is likely to be in some way
related. The simplest method for identifying proteins
that bind tightly to one another is co-immunoprecipitation
(see Panel 4−2, pp. 140–141). In this technique,
an antibody is used to capture and precipitate a specific
target protein from an extract prepared by breaking
open cells; if this target protein is associated tightly with
another protein, the partner protein will precipitate as
well. This is the approach that was taken to characterize
the Adenomatous Polyposis Coli gene product, APC.
Two groups of researchers used antibodies against APC
to isolate the protein from extracts prepared from cultured
human cells. The antibodies captured APC along
with a second protein. When the researchers examined
the amino acid sequence of this partner, they recognized
the protein as β-catenin.
The discovery that APC interacts with β-catenin initially
led to some wrong guesses about the role of APC in
colorectal cancer. In mammals, β-catenin was known
primarily for its role at adherens junctions, where it
serves as a linker to connect membrane-spanning
cadherin proteins to the intracellular actin cytoskeleton
(see, for example, Figure 20–23). Thus, for some
time, scientists thought that APC might be involved in
cell adhesion. But within a few years, it emerged that
β-catenin also has another, completely different function.
It is this unexpected function that turned out to be
the one that is relevant for understanding APC’s role in
cancer.
Wingless flies
Not long before the discovery that APC binds to β-catenin,
developmental biologists working on the fruit fly
Drosophila had noticed that the human β-catenin protein
is very similar in amino acid sequence to a Drosophila
protein called Armadillo. Armadillo was known to be a
key protein in a signaling pathway that has important
roles in normal development in flies. The pathway is activated
by the Wnt family of extracellular signal proteins,
the founding member of which was called Wingless,
after its mutant phenotype in flies. Wnt proteins bind to
receptors on the surface of a cell, switching on an intracellular
signaling pathway that ultimately leads to the
activation of a set of genes that influence cell growth,
division, and differentiation. Mutations in any of the proteins
in this pathway lead to developmental errors that
disrupt the basic body plan of the fly. The least devastating
mutations cause flies to develop without wings; most
mutations, however, result in the death of the embryo. In
either case, the damage is done through effects on gene
expression. This strongly suggested that Armadillo, and
hence its vertebrate homolog β-catenin, were not just
involved in cell adhesion, but somehow mediated the
control of gene expression through the Wnt signaling
pathway.
Although the Wnt signaling pathway was discovered
and studied intensively in fruit flies, it was later found
to control many aspects of development in vertebrates,
including mice and humans. Indeed, some of the proteins
in the Wnt pathway function almost interchangeably
in Drosophila and vertebrates. The direct link between
β-catenin and gene expression became clear from work
in mammalian cells. Just as APC could be used as “bait”
to catch its partner β-catenin by immunoprecipitation,
so β-catenin could be used as bait to catch the next protein
in the signaling pathway. This was found to be a
transcription regulator called LEF-1/TCF, or TCF for
short. It too was found to have a Drosophila counterpart
in the Wnt pathway, and a combination of Drosophila
genetics and mammalian cell biology revealed how the
gene control mechanism works.
Wnt transmits its signal by promoting the accumulation
of “free” β-catenin (or, in flies, Armadillo)—that is, of
β-catenin that is not locked up in cell junctions. This free
protein migrates from the cytoplasm into the nucleus.
There it binds to the TCF transcription regulator, creating
a complex that activates transcription of various
Wnt-responsive genes, including genes whose products
stimulate cell proliferation (Figure 20–54).