Silencing cancer
Thijn Brummelkamp accelerates genetics research with
RNA interference screens
Say “benign” skin cancer, and you get an
entirely wrong impression. Familial cylindromatosis
is a rare disease that doesn’t kill patients,
but does subject them to horrible and painful tumors
on the head that must be removed regularly by surgery.
Curiously enough, though, there is hope that this condition
can be treated quite successfully with aspirin. There’s
a striking little detective story behind this discovery,
based on a new and powerful biological tool that Whitehead
Fellow Thijn
Brummelkamp helped to improve.
The tool is RNA interference (RNAi), and it’s
a quick and highly flexible method for letting scientists
quickly silence a single gene at a time. It also is
highly scalable, which is paying off big time in Brummelkamp’s
chosen field of cancer research.
| “You quickly can see that there are, for
example, 20 genes involved in the response of a
cell to a particular drug,” says Whitehead
Fellow Thijn Brummelkamp. |
Until recently, our advances in understanding cancer
have come with agonizing slowness, with scientists painstakingly
isolating genes one at a time and then poking around
to find how the genes work together and create a cancer-causing
network.
In contrast, Brummelkamp can rapidly set up experiments
that screen human cancer cells for thousands of suspect
genes. “That way we can quickly find new genes
that we suspect may play a role in cancer,” he
says. Testing for those genes in samples from cancer
patients then offers “a very direct approach to
finding cancer treatments.”
Sticking in hairpins
RNAi was first demonstrated in mammals around the year
2000 and is now widely used in labs around the world.
The process selectively disables gene expression by
attacking messenger RNA, the molecule responsible for
delivering the gene’s protein recipe to the cell’s
machinery.
Originally, RNA interference was created with “short
interfering RNAs,” snippets of RNA about 21 base
pairs long. Synthesized chemically, these are expensive
and shortlived. In 2002, Brummelkamp and colleagues
at the Netherlands Cancer Institute demonstrated a powerful
alternative.
The Dutch scientists had noted that the biology of
siRNAs was very similar to the biology of microRNAs,
the naturally occurring short RNA molecules that also
affect gene expression by disabling messenger RNAs.
(See “When RNA rules").
Their concept was to get DNA to make hairpin-shaped
RNAs quite similar to microRNAs, using common genetic
manipulation techniques to do so.
“Other scientists also read the journals, and
about 10 groups started to generate a system like this,”
Brummelkamp says. “We were the first; that was
luck and a bit of hard work.”
They created the hairpin RNAs by using retrovirus vectors
(specially created viruses that splice in a segment
of DNA). These vectors are easily and cheaply made,
replicated and studied with familiar DNA tools. Variations
of this technique shot across the biomedical research
world; Brummelkamp’s paper is now cited by more
than 1,100 other papers.
Pathways to progress
The research that led to the potential cure for familial
cylindromatosis began with ubiquitin, a small protein
that aids in demolishing proteins whose time is up.
Brummelkamp and his then-co-workers at the Netherlands
Cancer Institute knew that enzymes that helped to add
ubiquitin to a protein are important in cancer. They
speculated that enzymes that aid in removal of ubiquitin
also play a major role.
The scientists took 50 human enzymes involved in removing
ubiquitin, and used their RNAi toolkit to measure what
happened when the gene for each enzyme was silenced
on several signaling pathways implicated in cancers.
Activity on the NfκB pathway (which stands for
nuclear factor kappa B) shot up dramatically when a
certain enzyme was silenced.
This enzyme was known to be mutated in familial cylindromatosis—although
its function was unknown. The researchers turned to
the scientific literature and found that simple compounds
such as aspirin inhibit NfκB.
Decoding drugs
After joining Whitehead in late 2004, Brummelkamp is
now studying how cancer therapeutics work. That’s
not at all as well understood as we might hope. “We
may know one target or a few targets, but we don’t
typically know the whole biological cascade that a drug
uses,” he says.
Postdoctoral researchers Alessio Nencioni and Helen
Pickersgill are tackling this problem in tests that
silence 7,914 human genes. Studying genes on this scale,
“you quickly can see that there are, for example,
20 genes involved in the response of a cell to a particular
drug,” says Brummelkamp.
Lab work starts by assembling 23,742 hairpin-RNA retrovirus
vectors (three for each of those target genes). Each
vector’s gene-specific sequence will later act
as a molecular “barcode” for DNA microarray
analysis. The vectors are introduced into two dishes
filled with human cancer cells, where each hairpin knocks
down expression of its target gene. One dish is treated
with a drug, the other dish is not.
After leaving the cells in culture for a suitable length
of time, the researchers harvest surviving cells from
each dish, select and amplify their DNA, label the DNA
fragments according to their dish of origin, and plop
them on a standard DNA microarray. The relative abundance
of each “barcode” quickly indicates genes
that make cells more or less sensitive to the drug.
The Brummelkamp lab is now employing this strikingly
efficient approach to targeting defective mechanisms
in cancer cells. “Cancer cells have found ways
of turning off the cellular brakes or jamming on the
accelerator to grow uncontrollably,” says Brummelkamp.
“But their strengths are also hiding specific
weaknesses. We can use RNA interference to find those
weaknesses and run them off the road.”
A first such experiment involves drugs that inhibit
mdm2, a protein that adds ubiquitin to the p53 protein
and thus helps to degrade it. P53 is the king of tumor
suppression genes, which is found mutated in about half
of human cancers. In the other half of cancers, p53
is not mutated, but the pathway is not functioning well.
Researchers have speculated that in these cancers,
inhibiting mdm2 would activate p53, and it would go
ahead with its designated role of suppressing the errant
cell. If so, that might lead to powerful therapeutics
for these diseases. In a paper appearing in April in
Nature Chemical Biology, Brummelkamp and several
Dutch colleagues identified a gene that helps to explain
why Nutlin-3, a small-molecule drug that inhibits mdm2,
is surprisingly non-toxic to normal cells.
Moving toward medicine
“If you want to make a conventional drug that
inactivates a gene, you can only work with druggable
genes,” Brummelkamp notes. “But with RNAi,
you can basically inhibit every gene you want.”
Jan Carette, another postdoc in the Brummelkamp lab,
is studying ways to inhibit genes that would correct
the defect that causes most cases of cystic fibrosis.
The disease might be addressed with an RNAi-based inhaler,
the researchers speculate.
And labs around the world are delving into other potential
RNAi medical applications, studying everything from
HIV to, well, hypoallergenic cats. “That’s
a nice application of the technology,” Brummelkamp
says, smiling. “I’m allergic to cats.”
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