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Childhood Cancer Research
STATEMENT OF RESEARCH PLAN
The overall premise of the Michael Cuccione
Pediatric Oncology Laboratory is that by increasing
our understanding of how tumour cells respond
to extra- or intracellular signals, we will gain
unique insights into the pathways that are specific
for tumour cells. Targeting of these pathways
can then be used as treatment strategies, while
minimizing effects on normal growth.
 Cancer Update from John Hopkins
It is evident that detailed analysis
of cellular signal transduction pathways is central
to understanding tumor cell biology. Moreover,
the pharmaceutical industry is making an extraordinary
effort to exploit molecular targets in signal
transduction pathways for the development of
new cancer therapeutics.
One of the difficulties
has been to decide which pathways to target.
A major research strategy in our group is the
characterization of non-random genetic alterations
in human cancers as a means to more efficiently
identify genes involved in tumorigenesis. We
believe that analysis of tumour tissue, as opposed
to model systems, is essential for initial identification
and characterization of gene products involved
in altered signal transduction in human malignancies.
Then, once the involved proteins have been identified,
model systems to further study their biology,
such as transgenic or knock-out mice, can be
invoked. For example, cloning of chromosomal
translocation breakpoints allowed us to discover
several previously unrecognized oncogenes or
tumour suppressors in solid tumours. These include
the t(21;22) associated EWS-ERG chimeric transcription
factor in Ewing tumours (Sorensen et al, Nature
Genetics, 1994), the t(12;15) associated ETV6-NTRK3
chimeric tyrosine kinase in pediatric sarcomas
(Knezevich et al, Nature Genetics, 1998) and
secretory breast carcinoma (Tognon et al, Cancer
Cell, 2002), and the novel 6q21 HECT E3 protein-ubiquitin
ligase, HACE1, in sporadic Wilms' tumour
and neuroblastoma (Anglesio et al, Hum Mol Genet,
2004).
Dr. Sandra Dunn
"One of the difficulties in treating cancer is that malignant cells are sometimes resistant to radiation and chemotherapy. Molecular biologist Sandra Dunn is researching cancers affecting women and children to find the weaknesses in cancer cells and develop new, more effective treatments. The key is identifying and eliminating what cancer cells need to survive.
Dr. Dunn's work focuses on the AKT pathway that makes tumor cells resistant to chemotherapy and radiation. Her goal is to inhibit key points along this pathway to improve survival rates and to reduce morbidity associated with cancer therapy. A protein that interacts with AKT known as YB-1 has been found in many cancers yet does not exist in healthy tissue. Dr. Dunn is looking at the possibility that some cancers are dependent on YB-1. Using new technologies (Cellomics high-content screening, small interfering RNA's and cell permeable peptides), she hopes to block the interaction of AKT and YB-1, and inhibit the ability of certain cancers to resist treatment. The ability to target a cancer cell's survival mechanisms, while allowing healthy cells to thrive could increase the effectiveness of relatively low doses of chemo- or radiation therapies. This would be especially important in childhood cancers where patients are particularly vulnerable to healthy cell injury.
Dr. Dunn's research is also critical to developing new tools to diagnose cancer relapse. Presently, there are few inexpensive and non-invasive tests that reveal the reoccurrence of cancer. However, Dr. Dunn's research has identified that the presence of a serine protease known as urokinase plasminogen activator (UPA) indicates a returned cancer. The discovery of UPA could lead to the development of a simple blood test that will reveal a relapse."
To understand how these proteins might
contribute to oncogenesis requires extensive
biochemical characterization of not only the
proteins themselves but of their protein interactors,
enzymatic substrates, or other molecules involved
in common signal transduction pathways. Thus,
analysis of signaling pathways activated or suppressed
by translocation-associated oncoproteins or tumour
suppressors forms another other major focus of
our research group.
Our strategy has been to focus initially on
specific recurrent genetic alterations in childhood
cancers. In contrast to most adult malignant
tumours, which appear to have complex genetic
etiologies, many childhood cancers show recurrent
chromosomal rearrangements including translocations.
We have found that many of these translocations
disrupt key genes regulating signal transduction
pathways. For example, some chromosomal translocations
lead to oncogenic gene fusions expressing chimeric
oncoproteins.
After we identify genes altered
by a particular genetic alteration, we quickly
shift to biochemical studies to ascertain how
the altered genes affect cellular signaling.
In this way, we have been able to more efficiently
single out those pathways that may be of relevance
to particular childhood cancers. An additional
strategy in my laboratory is to combine these
studies with gene expression profiling using
the Affymetrix platform. In fact, the gene expression
patterns of a large series of childhood solid
tumors are currently being generated through
a collaborative study with other Children's
Oncology Group institutions. We hope that this
combination of cancer biology and cancer genetic
studies will allow us to more readily elucidate
those pathways that can be uniquely targeted
in tumour cells while sparing normal cells.
Once
we identify altered gene products and signaling
pathways in childhood malignancies, we screen
for potential relevance in adult malignancies.
For example, we found that the t(12;15) associated
ETV6-NTRK3 chimeric tyrosine kinase of pediatric
sarcomas is also expressed in secretory breast
carcinoma, a variant of (Tognon et al, Cancer
Cell, 2002), This has lead to a number of studies
focused on NTRK3 signaling in breast cancer.
We have also started to screen for alterations
HECT E3 protein-ubiquitin ligase, HACE1, in adult
malignancies. Interestingly, targeted inactivation
of the Hace1 gene in mice, which results in the
development of spontaneous, late onset murine
tumors. Gamma irradiation or inactivation of
a single p53 allele on a Hace1-/- background
dramatically increases tumor frequencies. Moreover,
loss of Hace1 renders mice highly susceptible
to lung cancer in response to alkylating agents.
These studies confirm the tumor suppressor activity
of Hace1, and suggest a role for this protein
in cell stress.
A further strategy we have recently implemented
for pathway dissection and analysis is to combine
phenotypic screens at the cell level with libraries
of reagents that target individual genes or combinations
that target several components of a pathway.
For cell level phenotypic analysis we have built
up a high-content screening core which consists
of a GE Incell 1000 analyser coupled to a liquid
handling station, a thermo plate handling arm
and a plate incubator hotel.
The ability to couple
fluorescent tagging of proteins (whether by reporters
or antibodies) with sub-cellular localization
and morphology has proven to be a powerful phenotypic
analysis tool. The high content screening platform
(INcell) allows rapid fluorescent or brightfield
imaging of multiwell plates (up to 384), followed
by automated analysis of fluorescent/morphological
phenotypes, for example to measure cell receptor
internalisation, nuclear blebbing, cytoskeletal
changes, cell division, apoptosis. The liquid
handling stations and plate oven hotel allow
for reagents to be added or the contents of medium
in wells to be changed and for incubations to
occur over a long period of time, with plates
being moved to the INcell reader periodically
for phenotypic assessment. The core is building
up a modest library of small molecules and a
genome wide library of RNAi and shRNA molecules
as probe reagents to conduct genome wide screens
for phenotypes at cell level.
The ultimate goal
of such studies to use this information to develop
novel molecular-based strategies for the treatment
of human malignant diseases.
The following research documents are available
for your knowledge.
A
Census of Human Cancer Genes [PDF format,
174KB]
A central aim of cancer research has been to identify
the mutated genes that are causally implicated
in oncogenesis ('cancer genes').
After two decades of searching, how many have
been identified and how do they compare to the
complete gene set that has been revealed by the
human genome sequence?
Read
Complete Article -->
Endless
Cycling [PDF format, 441KB]
Stem cells and cancer cells share certain properties,
such as plasticity and selfrenewal,
which indicates that they might have common cellular
machineries.Tsai and McKay now report in Genes
& Development a nucleolar mechanism that regulates
cell-cycle progression in stem cells and cancer
cells..
Read
Complete Article -->
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Michael's Story 
