Canadian Cancer Society Research Institute
Annual Progress Report
Name: Sean Egan Award year: 1 of 5
Institute: Hospital for Sick Children Period covers: 07/01/2009 - 06/30/2010
E-mail: segan@sickkids.ca Phone: 416-813-5267
Project title: Lfng as lung tumour suppressor gene
Funder: Canadian Cancer Society (Ontario Award #: 020490
Panel: Hematopoiesis, Stem Cells and Differentiation
Division) Lung Cancer Research Program: Research Grants - 2009
List your cross appointments (Dept., Institution):
Program in Developmental and Stem Cell Biology, The Hospital for Sick Children, Department of Molecular Genetics,
Faculty of Medicine, The University of Toronto
Co-PIs/Co-Applicants/Additional Authors:
Changes to Co-PIs/Co-Applicants/Additional Authors listed above (Name, Designation as co-PI, co-applicant
or additional author [CP, C or A], Dept., Institution):
None
*Scientific Progress Report
Provide a scientific progress report describing the research that has been performed during this grant. The
equivalent of a 2 to 3 page report is recommended.
Over the past year, my lab has been studying Notch receptor activation/signalling in distal lung development of the
mouse, performing experiments to characterize the tumour suppressor function of Lunatic Fringe and obtained transgenic
mice to establish a model of human non-small cell lung cancers with mutant activated Notch1. In addition, we have
recently written and submitted a book chapter on Notch signalling in lung development and disease.
1) Lunatic Fringe-mediated Notch signalling is required for lung alveogenesis.
During the past year, we finished up our genetic analysis of Lfng, Notch2 and Notch3 in lung development. This work
has now been published (K. Xu, E. Nieuwenhuis, B. Cohen, W. Wang, A. Canty, J. Danska, L. Coultas, J. Rossant,
M.Y.J. Wu, T.D. Piscione, A. Nagy, A. Gossler, G.G. Hicks, C.-c. Hui, M. Henkelman, L. Yu, J.G. Sled, T. Gridley,
S.E. Egan. 2010. Lunatic Fringe-mediated Notch signalling is required for lung alveogenesis. American Journal of
Physiology: Lung Cellular and Molecular Physiology 298:L45-54).
Abstract: Distal lung development occurs through coordinated induction of myofibroblasts, epithelial cells, and
capillaries. Lunatic Fringe (Lfng) is a ß1–3 N-acetylglucosamine transferase that modifies Notch receptors to facilitate
their activation by Delta-like (Dll1/4) ligands. Lfng is expressed in the distal lung during saccular development, and
deletion of this gene impairs myofibroblast differentiation and alveogenesis in this context. A similar defect was
observed in Notch2ß-geo/+:Notch3ß-geo/ß-geo compound mutant mice but not in Notch2ß-geo/+ or
Notch3ß-geo/ß-geo single mutants. Finally, to directly test for the role of Notch signalling in myofibroblast
differentiation in vivo, we used ROSA26-rtTA/+;tetO-CRE/+;RBPJκflox/flox inducible mutant mice to show that
disruption of canonical Notch signalling during late embryonic development prevents induction of smooth muscle actin
in mesenchymal cells of the distal lung. In sum, these results demonstrate that Lfng functions to enhance Notch
signalling in myofibroblast precursor cells and thereby to coordinate differentiation and mobilization of myofibroblasts
required for alveolar septation.
2) Lunatic Fringe controls Notch activation in mammary basal cells and functions as a tumour suppressor: In
preparation (confidential).
Abstract: Inappropriate Notch activation induces tumour formation in the mammary gland. Indeed, many poor
prognosis breast cancers show high-level expression of Jagged ligands and Notch receptors, as well as Notch receptor
activation. The sugar transferase Fringe proteins tightly restrict Notch activation in vivo by blocking Notch receptor
activation in response to Jagged proteins while facilitating activation by Deltas. Indeed, ectopic expression of Fringe
and deletion of Fringe in Drosophila can both lead to inappropriate Notch activation and tissue overgrowth, depending on
the cellular context. Here we report that mammalian Lunatic Fringe (Lfng), which is expressed in basal cells of the
mammary gland, functions to suppress Notch activation in this context. Lfngflox/flox;MMTV-Cre mutants show
increased mammary epithelial proliferation in virgins and in pregnant mice. This is accompanied by accumulation of
CD61+ luminal progenitor cells culminating in formation of basal-like (luminal progenitor) tumours. Thus, Lfng controls
Notch activation in basal cells of the mammary gland to inhibit proliferation of luminal progenitor and/or bipotent
progenitor cells, as well as to prevent mammary tumourigenesis.
3) Establishment of system to generate a mouse model for human NSCLC with Notch1 mutations (confidential).
The Notch1 gene has gain-of-function mutations in 10% of human NSCLC. We have obtained reagents to study
Notch1-induced lung cancer in mice. These reagents include Cre-conditional mice expressing activated Notch1 from
the Rosa26 locus. In addition, we have obtained the SPC-rtTA/tet-O-Cre system to express Cre recombinase in the
distal lungs of mice in response to doxycycline in the drinking water. During the next year, these mice will be mated
together to generate animals expressing activated Notch1 in the distal lung. These compound transgenic mice will be
tested for lung tumour formation.
4) Notch signalling in lung development and disease (Book chapter in press for “Notch signalling in Embryology and
Cancer” (Editors: Jörg Reichrath and Sandra Reichrath).
Abstract:Notch signalling plays an essential role in development and homeostasis of multiple organs including the
lungs. Dysregulation of Notch signalling has been implicated in various lung diseases including lung cancer. Here we
review functions of Notch signalling in coordinating events during lung development, such as early proximodistal fate
generation and branching, airway epithelial cell fate specification, alveogenesis, and pulmonary vascular development.
We also discuss roles of Notch in chronic obstructive pulmonary disease, progressive pulmonary fibrosis, pulmonary
arterial hypertension, asthma, and lung cancer.
*Keywords: provide a list of up to 10 keywords that are associated with this research project.
Lung, Notch, Lunatic Fringe, tumour suppressor, mouse model, cooperating genes
Have there been any significant changes to the project since the original submission (e.g. research
methodology, cancer site, etc.)?
Since submission of this application, a publication by Westhoff et al. has revealed that approximately 10% of human
Non-Small Cell Lung Cancers (NSCLCs) have activating mutations in the Notch1 gene. This has caused us to shift our
goals somewhat towards modelling this form of NSCLC in order to make a more relevant model of the human disease.
*Summary of Findings
Provide an updated summary of your findings related to this research project in simple, non-technical
language in the spaces below. Consider how you would describe your project to a Canadian Cancer Society
or Terry Fox Foundation volunteer who could then relay this information to the donor community. For tips
on good writing please see our website: www.cancer.ca/research - under Grants and Awards, Applications.
*Project summary: (2 sentences summarizing the proposal)
Notch signalling has been directly implicated in human lung cancer. We are using genetic approaches in mice to define
the function of Notch in normal development and in non-small cell cancers of the lung.
*Research findings and update: (detail in 3-5 sentences what has been accomplished in this funding year)
This year, we have reported on our findings that the Lunatic Fringe gene, a regulator of Notch receptor signalling, is
required for proper coordination of events in development of the tiny sacs within the lung that are responsible for gas
exchange. Indeed, in Lunatic Fringe mutant mice, lungs do not develop correctly as a result of reduced Notch
signalling. We have also recently published a review article on the role of Notch signalling in lung development and
cancer. Finally, we have recently determine that deletion of the Lunatic Fringe gene in mice can lead to the
development of cancer.
*Project description: (detail in 2-3 sentences how you are conducting the study)
We have been studying the development of lung and mammary tissue in mice with mutations in Notch pathway genes,
This has led to our finding that altered Notch signalling can cause cancer in both tissues. We are currently focusing on
the establishment of a mouse model for lung cancer associated with excessive Notch signaling as observed in humans.
*Impact and relevance: (detail in 2-3 sentences how your studies will contribute to the eradication or control
of cancer)
By developing mouse models of lung cancer with altered Notch signalling, we will be able to perform genetic
experiments to identify the cooperative network of gene mutations involved in lung cancer. Such information can help
guide development of novel combination therapies to treat lung cancer in humans.
*Publications:
List the publications associated with this grant during the past year, and indicate the number of
publications. Include the full citation with PubMed ID numbers for all publications. Note that the CCS Open
Access Policy requires that publication be made available within 6 months of the publication date. See our
website for more information (www.cancer.ca/research - under Policies and Administration).
*Number of publications:
1
*List of publications:
1) K. Xu, E. Nieuwenhuis, B. Cohen, W. Wang, A. Canty, J. Danska, L. Coultas, J. Rossant, M.Y.J. Wu,
T.D. Piscione, A. Nagy, A. Gossler, G.G. Hicks, C.-c. Hui, M. Henkelman, L. Yu, J.G. Sled, T. Gridley,
S.E. Egan. Lunatic Fringe-mediated Notch signaling is required for lung alveogenesis. American
Journal of Physiology: Lung Cellular and Molecular Physiology 2010 Jan;298(1):L45-56. PMID: 19897741 [PubMed -
indexed for MEDLINE]PMCID: PMC2806195 [Available on 2011/1/1]
* Presentations:
List the presentations associated with this grant during the past year, and indicate the number of
presentations.
*Number of presentations:
3
*List of presentations:
1) The Notch Meeting, Athens, Greece September 27-October 1, 2009
2) Genentech, South San Francisco, California, February 9, 2010
3) The Manitoba Institute of Cell Biology, Winnipeg, Manitoba, April 29, 2010
* Upcoming publications:
List the upcoming publications associated with this grant during the past year, and indicate the number of
upcoming publications (including in preparation, submitted or in press manuscripts).
*Number of upcoming publications:
2
*List of upcoming publications:
1) Keli Xu, N. MoghaL and S.E. Egan, 2010. Notch signaling in lung development and disease for a book on Notch
Signalling in Embryology and Cancer (Editors: Jörg Reichrath and Sandra Reichrath). In press.
2) K. Xu et al. Lunatic Fringe controls Notch activation in mammary basal cells and functions as a tumour suppressor.
In preparation for submission.
Media coverage:
Was your research the subject of a press release, newspaper article, interview, etc. in the past year?
No
Promoting the funder:
Many researchers are given the opportunity to promote the funder through public presentations,
participation in fundraising efforts, etc.
List any such activities you or your research colleagues have been involved in during this grant year. To
acknowledge funding, researchers should state "This research is funded by the [funder name] (grant
#XXXXXX)".
None, but I have accepted an invitation to speak to CCS volunteers in Sarnia on April 6, 2011.
*Are you fluent in both official languages?
No
*Are you fluent in any other languages?
No
If Yes, please list:
Honours or Awards:
List any significant honours or awards you have received during this grant year. (Do not include research
grants).
None
Speakers Bureau:
As a recipient of funds provided from charitable donations by the Canadian public, you may be called on to
make presentations to volunteers, donors or the public about cancer research.
List, in lay terms, topics on which you are able to speak (e.g. angiogenesis, breast cancer, cancer
prevention, genetic markers of cancer).
Breast Cancer, Lung Cancer, Mouse Models of Cancer
Students and Post-Doctoral Fellows Promoting the funder:
Young researchers are uniquely positioned to generate enthusiasm about cancer research. List the names of
any students or post-doctoral fellows working on this project who would be suitable for or interested in
making presentations to the public about their work. List their hometowns, as these trainees may be invited
to speak by their local Society offices, and provide contact information, if different from your own.
Name, student or fellow (S or F), hometown, email
Please provide the following information:
STUDENTS
*Number of students working on this project this year:
1
*Number of these students that are paid for, in whole or in part, from this grant?
1
*Names of students paid from the grant (last name, first name, email)
Schachter, Nathan, nathan.schachter@utoronto.ca
SUMMER STUDENTS
*Number of summer students working on this project this year:
2
*Number of these summer students that are paid for, in whole or in part, from this grant?
2
*Names of summer students paid from the grant (last name, first name, email):
Li, Sisi: sincerelyc2@hotmail.com
Ruiz, Natalia, nataliaruiz@hotmail.com
FELLOWS
*Number of fellows working on this project this year:
1
*Number of these fellows that are paid for, in whole or in part, from this grant?
0
*Names of fellows paid from the grant (last name, first name, email):
Impact
What is the single greatest impact your research has made on cancer control in this grant year? For
example, development of new knowledge, development of new methods, research cited in clinical
guidelines, research cited in public policy documents, etc.
In the past year, we have published a paper on the role of Lunatic Fringe as well as Notch2 and Notch3 in lung
development. This paper is helping to set a context for understanding how Notch receptors contribute to inappropriate
development observed in lung cancer.
Describe any new collaborations formed in the past year resulting from this project (e.g. new collaborations
with other researchers, policy makers, industry, etc.)
I have established a collaboration with Genentech who are developing antibodies against Notch receptors and ligands to
use as therapy for breast and other cancers (confidential). We will be testing these antibodies in our mouse models of
cancer.
Describe any commercialization activity in the past year resulting from this project (e.g. patents
filed/granted, invention disclosures, copyrights, licenses, etc.)
None
Describe any leveraged funding that was obtained in the past year that would not have been raised if this
project were not funded (e.g. additional funding obtained to support complementary research, industry
contracts, in-kind resources, etc.)
I have received antibodies from Genentech for our collaboration as noted above (confidential).
If this grant was subject to a decrement at the time of award, did you secure supplemental funding?
No
If Yes, please describe and include the funder, term and amount:
*Our policy prohibits grantees from accepting funding from the tobacco industry. (See our tobacco policy at
www.cancer.ca/research - under Policies and Administration).
Please indicate if you have received tobacco funding during this grant year.
No