Unraveling
the Genetics
A Stanford Professor and Undergraduate’s
Genomics Approach.
CANCER
By Hahn Nguyen
In the 9.3 minutes it takes you to skim
through this magazine, one person
in the United States will have died
from colon cancer. According to the American Cancer
Society’s estimates, 145,000 people will be diagnosed and 56,730 people
will die from colectoral cancer in our country alone each year. Colon
cancer is currently the third most common form of cancer in the western
world. Tumors and polyps in the colon, rectum, and appendix characterize
this type of cancer. In an attempt to combat this disease, an undergraduate
student working in a cancer biology lab at Stanford University School of
Medicine has brought us one step closer to unlocking the genetics behind
colon cancer.
This lab, headed by Assistant Professor of Pathology Jonathan
Pollack, uses a genomics approach to improve our knowledge of cancer
and patient care. That is, they use DNA microarrays to discover gene
patterns in cancer cell lines that model the disease as a whole. “Our main
interest is understanding genetic instability and how it promotes cancer
development and progression,” says Pollack. “And this is our first foray
into colon cancer.” Craig Giacomini, who is now a Stanford first-year
medical student, began leading this research in his junior year as a Stanford
undergraduate. Published in Cancer Research this past October, their
recent research indicates colon cancer, in terms of genes expressed and
of
COLON
Professor Jonathan Pollack
gene pathways, is actually two and student Craig Giacomini
distinct types of disease. That prove that what we call
is, colon cancer occurs due
to two scientifically distinct
“colon cancer” is actually
disease pathways. Through two distinct diseases
analysis of cancer cell lines, and suggest a source of
their research also suggests chemoresistance in patients
a source of chemotherapy-
resistance in patients.
While not all mutations are harmful, those that increase the mutation
rate, otherwise known as mutations in the “mutator” genes, often give rise
to cancers. Mutations of this sort can be divided into two subtypes based
on the class of mutator phenotypes: microsatellite instability (MSI) and
chromosome instability, also known as microsatellite stable (MSS).
Microsatellites consist of simple sequence repeats, such as a string
of adenine nucleotides, found throughout the genome and sometimes
in genes. In MSI mutations, microsatellites are copied incorrectly. As
a result, the sequences become longer or shorter, and consequentially
mutations occur throughout the genome. In the other mutator type, MSS,
pieces of chromosomes are gained or lost in mitosis. This causes excess
or lack of expression of certain genes. The phenotypic differences in MSI
are a tendency for polyps to form on the right side of the colon and for
patients to fair better.
To further understand the role of MSI and MSS in colon cancer, Craig
Giacomini in Professor Pollack’s lab looked for similarities between the
Layout by Jason Shen Volume IV 27
Unraveling the Genetics of Colon Cancer
two mutator phenotypes from a genetic standpoint. Do the two mutator
phenotypes target two completely separate sets of genes or the same
genes in different ways? “That was the goal in looking for signatures to
distinguish between the two,” notes Pollack. The researchers working in
his lab sought to find collections of specific genes that are expressed or
not expressed which are unique to each form of the disease. This would
result in a unique “signature” or group of certain genes whose expression
significantly differ in the two causes of colon cancer.
Cancer is such a complicated disorder
with so many genetic DISRUPTIONS
occurring. Microarray technology
gives us an excellent tool to look at
something so COMPLEX
To explore the gene differences, Giacomini led research in profiling
gene expression in 18 different colon cancer cell lines. His group used
complementary-DNA microarrays to discover a statistically robust gene
expression signature. The microarray represents approximately 21,000
different genes, and allows the researchers to analyze which genes are
expressed in the cell lines.
A microarray can be thought of as a grid of DNA spots, where each
spot contains a unique DNA sequence that codes for
a certain gene. These gene-specific probes contain
complementary strand DNA, and will hybridize to a
cell line sample which expresses that same particular
It’s really great and a little
gene. With fluorescent tags, researchers can determine
to what degree any particular gene is expressed in
their sample cell line, compared to a generic cell line.
strange at first to be able to
Microarray technology essentially allows thousands
of experiments to be performed at once, as thousands look myself up on PUBMED
of genes are tested for expression levels.
In this project, Giacomini used this technology to profile the genes
expressed in the colon cancer cells. “Cancer is such a complicated disorder
with so many genetic disruptions occurring. Microarray technology gives
us an excellent tool to look at something so
complex,” Giacomini says.
The result? Of the 21,000 genes surveyed,
as few as eight differentially expressed genes
can accurately classify MSI and MSS colon
cancer. In other words, eight different genes
are expressed in higher levels in MSI and are
the best predictors that determine whether a
cancer cell is MSS or MSI derived. Professor
Pollack’s lab tested this signature on an
independent set of colon and gastric tumors
from the University of Hong Kong, which
had already been identified as MSI or MSS
mutated. Ignoring the tumors’ classifications,
the Giacomini profiled the tumors’ gene
expressions and predicted which tumors fell
into which category. Their predictions were
100% accurate for colon cancer cell lines,
85% for primary colon tumors, and 84% for
28 Stanford Scientific
primary gastric tumors (gastric tumors also have MSI and MSS mutations).
Since the gene signature is also highly accurate for gastric tumors, this
may reflect universal information about MSS and MSI mutations in other
forms of cancers.
These are not necessarily genes that cause colon cancer, but rather they
are biomarkers of the tumors being MSI or MSS. “That’s the tip of the
iceberg,” Pollack declares. “As a general conclusion, it indicates that these
two types of cancer are really distinct, not just from mutation mechanism,
but also from the genes involved in their pathogenesis.
From an expression standpoint, they are distinct and
hopefully in the future can be treated differently.”
If colon cancer were simply one type of disease,
the same genes would be mutated despite which
mechanism caused the mutation. Pollack’s lab would
have found nearly identical gene expression in MSI
and MSS colon cancer. However, that was not the
case. Analyzing microarray data, Giacomini identified
217 genes in total that are significantly different in the
two types. Inspection of just eight of these genes is
sufficient to classify whether the tumor is caused by a
MSI or MSS mutation.
Giacomini’s results are evidence that the term “colon cancer” actually
refers to two different types of cancer, in terms of gene expression and
pathways. The disease has two different mutation sources, making it, in
fact, two different diseases, separable on a molecular and genetic level.
Ideally, physicians and researchers will develop treatments that work on
each optimally. Currently, patients diagnosed with MSI colon cancer tend
to fair better overall. Stanford’s research suggests that the best treatment
targets in the different types of colon cancer are likely distinct from each
other, and that in the future, physicians should treat the two with drugs
specific to each form of the disease.
The eight genes differentially expressed
in the classifier signature could reflect the
effects of specific mutations or the sensing of
ongoing DNA mutation activity. To address
this ambiguity, the research group profiled the
genes of corrected cancer cells, cells that have
been genetically altered to stop the mutator
phenotype. If the genes responded to ongoing
mutation activity, they would no longer
be expressed in corrected cells. However,
Giacomini found the genes still expressed in
these cells, indicating that the eight predictor
genes represent the effects of prior mutations.
These genes do not sense the damage; they
are the result of the damage.
Almost half of the eight predictor genes
are metallothioneins, a family of small
proteins that protects cells against many
attacks, including chemotherapy. The eight predictor
genes are distinguishable because they are expressed
at higher levels in MSI tumors. In parallel to the lab’s
findings, it has been observed in treatments that MSI
tumors tend to be resistant to some chemotherapy.
“This immediately suggests a link between expression
of metallothionein genes resistance,” explains Pollack.
Additional tests in cultured cells can determine whether
or not metallothioneins confers chemo-resistance. “That
could improve treatment now because there are currently
some drugs that inhibit production of metallothionein,”
says Pollack. “Conceivably, we would begin to use
these drugs in combination with current chemotherapy.
“Our study shows the power of a genomics approach,”
continues Pollack. “We have a hypothesis and we can
test it by profiling expression across thousands of
genes, since we don’t know for which specific gene to
test… but by taking a genomics approach, we can learn
something about cancer that we wouldn’t otherwise be
able to see or address.”
Professor Pollack has headed this cancer research
lab for the past four years. His lab performs ongoing
research in prostate and breast cancer, using DNA
microarrays to identity and describe molecular
phenotypes of tumor cells. The lab has also recently
discovered a gene signature in leukemia that can
predict how well a patient will fair in cancer treatment.
“Although heart disease is the top cause of death in the
United States, I believe as heart treatments improve
cancer will take over as a leading cause of death,” notes
Pollack. “There’s interesting biology underlying cancer
research, and there’s a chance to make a difference in
people’s lives.”
Craig Giacomini began working in Pollack’s lab
as an undergraduate at Stanford, and had his results
accepted by premiere cancer journal Cancer Research
in the spring of his senior year. “It’s really great
and a little strange at first to be able to look myself
up on Pubmed [the National Library of Medicine’s
journal archive]. Working in Professor Pollack’s
lab gave me insight into medically-based research,
and it’s something I’d want to do in the future,” said
Giacomini. Although his main interests consist of
research, Giacomini is currently working toward his
medical degree at the Stanford School of Medicine. “I
want to stay in academic medicine,” he explains. “And
combine research with clinical practice.”
“In this research, Giacomini was 90% of the
effort,” says Pollack. “One of the great things about
Stanford, as opposed to Harvard or UC Berkeley, is
that the Undergraduate School sits right next to the
School of Medicine. There are many opportunities for
undergraduates which are beneficial in both directions,
and Giacomini’s research is certainly an example.” S
Hanh Nguyen is a freshman majoring in Biology
with a biochemistry concentration and has career
goals in disease researching. When she’s not
reading science articles, Hanh enjoys guitar,
drinking coffee, and yoga.
Studying Cancer
< A DNA microarray is a small grid on
a slide. Each spot on the grid contains
a unique DNA sequence that codes for a
certain gene. These gene-specific probes
are made up of complementary DNA—
nucleic acids that are complementary,
and will attach to, the DNA sequence
that expresses the gene of interest.
With fluorescent tags, researchers
can determine where a particular DNA
sample “sticks” to its complement, or
hybridizes, and therefore to what degree
any particular gene is expressed in their
sample. Microarray technology essentially
allows thousands of experiments to be
performed at once, since as many as
21,000 (or 42,000 with newer technology)
genes are concurrently tested for
expression levels.
Giacomini analyzes >
microarray data. “The great
thing about microarrays
is being able to look
at thousands of genes
simultaneously,” he says.
< Giacomini observes
cancer cell lines. He first
submitted his research to
Proceedings in the National
Academy of Sciences, which
unfortunately did not review
or publish his paper. “That
was quite frustrating,” says
Giacomini. “Although PNAS
was my first choice, I was
still psyched when Cancer
Research accepted my
paper.”
Microarray data collected by Prof. >
Jon Pollack and Craig Giacomini.
Each row represents a particular
gene and each column represents
a different sample cancer cell line.
“The higher the red intensity of the
square, the higher that particular
gene is expressed in that specific cell
line, compared to the average of all
the cell lines,” explains Giacomini. “If
the gene expression is low, the cor-
responding square is green.” Pollack
continues, “We derived our eight gene signature from these above genes, because
these particular genes are more highly expressed in MSI colon cancer, as seen in this
microarray data.
Volume IV 29