Ohio State’s Carlo M. Croce - A
Macro View of MicroRNA
Featured Science Watch®
19: 3-4, March/April 2008)
RNA genes of only tens or hundreds of nucleotides have
been called the biological equivalent of
dark matter— "all around
us but almost escaping detection." These genes are
found in that portion of DNA that doesn’t code
for proteins and so was commonly known as "junk
DNA," which explains why they received so little
research attention until the late 1990s. Nowadays,
however, research into these so-called microRNAs
(miRNAs) and their role in cellular function and
dysfunction—notably, in the latter case,
cancer—has blossomed into one of the hottest
fields of biology and medicine.
Leading the pursuit of miRNAs as fundamental players in the cancer process
is the Ohio State University cancer geneticist Carlo Croce, who reported in
November of 2002 that chronic lymphocytic leukemia (CLL), the most common
human leukemia, appears to be caused by the loss of two miRNA genes. These
two miRNAs target the BCL2 oncogene.
Croce’s 2002 paper reporting this discovery has now been cited more
than 300 times (see adjoining table, paper #5). More recently, a 2005
report by Croce and colleagues on this topic promptly found its place in
the Hot Papers database, garnering more than 170 citations in just over two
years (A. Cimmino, et al., PNAS, 102: 13944-9, 2005).
This is just one of 10 reports published by Croce and colleagues in the
last two years that registered as Hot Papers during 2007, a total that
earned him a spot on the annual list of "hot" authors in this issue’s
Indeed, Croce’s research on miRNAs has made him, at this writing, the
seventh most-cited author in biology in the latest update to Thompson
Science IndicatorsSM database, based on papers
published and cited over the last decade. Since 1991, Croce has published
four articles that have each garnered more than 500 citations, and over 60
papers exceeding 100 citations each.
Croce, 62, did his undergraduate and medical studies at the University of
Rome, where he graduated summa cum laude in 1969. He then moved to the
Wistar Institute in Philadelphia and pursued his research in cancer
genetics there for much of the next 20 years. In 1988, Croce became
director of the Fels Institute for Cancer Research at the Temple University
School of Medicine. Three years later, he moved to Thomas Jefferson
University to head up the Kimmel Cancer Institute/Kimmel Cancer Center. In
2004, Croce relocated to Ohio State University, Columbus, where he is now
chair of the Department of Molecular Virology, Immunology, and Medical
Genetics, and director of the Human Cancer Genetics Program and the
Institute of Genetics.
Croce spoke to Science
Watch® from his office in
When was the first miRNA gene discovered, and how
did the field evolve over its first few years?
The first microRNA was discovered by Victor Ambros at the Dartmouth Medical
School in 1993. He discovered this gene known as lin-4, which is
important for the development of the worm, C. elegans. Then, other
researchers—Gary Ruvkun at Harvard, most prominently—found
other miRNA, such as let-7. After that there was a pretty long
gap. Very few people were interested in miRNA until 1998, when it became
clear that small interfering RNA, or siRNA, was taking advantage of the
same pathway that exists for miRNA.
What do miRNAs actually do?
Cited Papers by Carlo M. Croce and
Colleagues, Published Since
(Ranked by total citations)
M. Ohta, et al.,
"The FHIT gene,
spanning the chromosome 3p14.2
fragile site acid renal
breakpoint, is abnormal in
digestive tract cancers,"
Cell, 84(4): 587-97,
S. Haldar, J. Chintapalli,
C.M. Croce, "Taxol induces
bcl-2 phosphorylation and death of
prostate cancer cells,"
Cancer Res., 56(6):
G. Sozzi, et al.,
"The FHIT gene at
3p14.2 is abnormal in lung
85(1): 17-26, 1996.
S. Haldar, A. Basu, C.M.
Croce, "Bcl2 is the
guardian of microtubule
Res., 57(2): 229-33,
G.A. Calin, et
deletions and down-regulation of
micro-RNA genes MiR15 and
MiR16 at 13q14 in chronic
PNAS, 99(24): 15524-9,
SOURCE: Thomson Scientific
They bind to the 3’ untranslated regions of RNA and cause a block of
translation and some degree of degradation. In other words, they are
regulators of gene expression. But it was only in 1998, with the discovery
of siRNA, that this became clear. siRNA uses cellular mechanisms for
silencing that also exist for miRNA. So at this time more people started to
get interested. In 2000, homologues of miRNA were found in
Drosophila. And then in 2001, several laboratories found that, in
fact, this miRNA had homologues in rats, mice, and humans.
And this was in so-called junk DNA?
Well, it was called junk. Clearly, it’s not.
When did you make the first connection between
miRNA and cancer?
That was in 2002. For a long time, we’d been taking advantage of
specific chromosomal alterations to identify and characterize oncogenes or
tumor suppressor genes that are involved in a large variety of human
cancers—from acute leukemia to solid tumors, like lung cancer. We had
characterized many of them, but then, in the early 1990s, we tried to
identify the tumor suppressor on chromosome 13 in band q14 that was related
to chronic lymphocytic leukemia. This region is deleted in about 50% of CLL
For seven years we tried to find this tumor suppressor gene and we
couldn’t do it, although we characterized all the genes in this
region that are preferentially lost. Finally, by using CLL cells carrying
the translocation and this small deletion, we were able to find that the
culprits were not protein-coding genes, but two miRNA genes, called
miR-15 and miR-16.
As it turns out, these miRNA genes are lost, or expressed at a very low
level, in CLL. We showed that they are knocked out or knocked down in 68%
of CLL cases. And this was the first link between miRNA and cancer. Then we
mapped all known miRNA genes, and found that many mapped to regions
involved in rearrangements in cancer. We then developed the first chip to
detect the global expression of miRNA in normal tissue versus cancer
Subsequently, we started making the signatures for specific human cancers,
looking at the function of various miRNAs to see if they work as oncogenes
or tumor suppressor genes. We also developed the first genetic mice model.
We deregulated a mouse miRNA gene, miR-155, in mice and observed
that those mice developed cancer. That paper was published two years ago.
The mice developed leukemia?
Yes. After we deregulated one miRNA, 100% of those mice developed an acute
Why do you think miRNA escaped detection or notice
until as late as 1993?
Because people were affected by dogma. The dogma was that RNA exists to
make proteins. So all this RNA found to be non-coding was thought to be
When you discovered that these two miRNA genes
were the culprits in CLL, was it obvious or did it take some convincing
that you hadn’t made a mistake?
When I realized that the only things that could be involved in CLL were
these two miRNAs, it was a revelation. I asked myself, how could we have
been so dumb? Again, it really was a revelation, because it indicated that
another totally different class of genes could play a major role in cancer.
How much of your laboratory time and resources are
now devoted to miRNA research?
I have a pretty large laboratory, and I’d say 70% of my people work
How would you describe the state of the miRNA
research universe circa early 2008?
The big picture is that these miRNA can certainly be used for cancer
diagnostics and prognostics. And not only for cancer—it’s
likely that many other diseases are also due to miRNA dysfunction. I think
we’ll soon see miRNA used as therapeutic drug. MiRNA can get into
cells, so we can use miRNAS that are lost in cancer as therapeutic
agents—we can put them back. And if they’re over-expressed in
cancer, we can use anti-miRNAs—small molecules—that can get
into cells and regulate them. In a year or two we will probably see the
development of miRNA-based therapies.
Are there biotech or pharmaceutical companies
already pursuing the idea of miRNA therapeutics?
Not yet, but they are certainly starting to think about it.
How many miRNA genes are there likely to be in
About 450 have been discovered so far, and there’s another 700 that
have been predicted. Some of those will turn out not to be correct.
What are the predictions based on?
And how are these predictions generated using
You look at the structure of RNA and how they’re made. Then you keep
all this in mind and look at the gene areas and the DNA and see which DNA
might possibly generate this mature RNA. That gives you the prediction,
which you then have to validate.
What makes you think miRNA might play a role in
diseases other than cancer?
We know many diseases which are genetic, as shown by twin studies and
family studies, but we can’t find the genes involved. For example,
bipolar disease and schizophrenia are genetic diseases. Twin studies and
family studies indicate that alterations in genes are the cause of these
diseases. But you can’t even map them. Nobody has found them. So this
suggests that they might be caused by alterations in miRNAs, because
that’s precisely the scenario you would see if this were the case.
And so it’s likely that one day we’ll find that these diseases
are due to genetic alterations of miRNAs or defects in the processing of
miRNAs. Alzheimer’s is another one of these cases where it’s
plausible that miRNAs are the culprit.
So the failure to identify the genes responsible
suggests to you that miRNA is the answer?
Yes. How did we find the involvement of miR-15 and miR-16
in chronic lymphocytic leukemia? We looked for the protein-coding gene and
couldn’t find it. The reason we couldn’t find it is because
this disease is caused by miRNAs—in this case, the loss of the genes.
So if we apply the same line of reasoning to these other diseases, then the
fact that a lot of very smart people looked for many, many years to find
the protein-coding genes and failed suggests that the disease is due to
alterations not in protein-coding genes, such as miRNA genes.
Are there other types of RNA that might play a
role in cancer or other diseases?
We had a paper in the September 2007 issue of Cancer Cell
reporting that another large family of non-coding RNAs, what we called
ultraconserved, non-coding RNAs, are expressed in a lot of different
tissues, and are differentially expressed in different tissues, and their
pattern of expression is altered in cancer (G.A. Calin, et al.,
Cancer Cell, 12: 215-29, 2007). The RNAs are much bigger than
miRNA, from 200 nucleotides to 1.8 kilobases.
These RNAs are conserved 100% between rats, mice, and humans, hence the
name. We find that these ultraconserved, non-coding RNAs are dysregulated
in cancer. It’s also possible that miRNAs might regulate not only
protein-coding RNAs but other non-coding RNAs as well.
Give us a prediction for where miRNA research will
take us in the next five years.
It will be a revolution. We will have drugs based on this, and a lot of
novel diagnostic and prognostic markers will be developed. The great
opportunity will be in therapy. We will understand the diseases much
better, including cancer, heart disease, and many other diseases, and
we’ll be able to use miRNA or anti-miRNA to go in and perhaps reverse
or alter the disease process.