Ectopic Notch1 activation alters mammary cell fate during - TSpace

117 Pages·2009·8.23 MB·English

by  Aaron Kucharczuk

Ectopic Notch1 activation alters mammary cell fate during
puberty and promotes the development of lactating
adenomas during pregnancy

by

Aaron Kucharczuk

A thesis submitted in conformity with the requirements for
the degree of Masters, Department of Molecular Genetics,
University of Toronto

©Copyright by Aaron Kucharczuk 2009
Ectopic Notch1 activation alters mammary cell fate during
puberty and promotes the development of lactating
adenomas during pregnancy

Aaron Kucharczuk

Department of Molecular Genetics
University of Toronto

Masters Degree

August 2009

Abstract

The role that each of the Notch receptors play in controlling alveolar development and cell fate
determination in the mouse mammary gland has remained unclear. By utilizing a cre-conditional
constitutively active intracellular Notch1 knock-in I define, in vivo, that ectopic Notch1 activation is
sufficient to inhibit ductal outgrowth, cause the formation of alveolar-like cell accumulations, and
promote Elf5+/ER- cell fate, at the expense of ER+ cell fate, in the mammary gland of pubescent mice.
Furthermore, ectopic Notch1 in the pregnant mammary gland is sufficient to promote the formation of
pregnancy/lactation-dependent lactating adenomas. These lactating adenomas consist of differentiated
secretory cells and normally regress during involution but progress into non-regressing tumours after
multiple pregnancies. These lactating adenomas exhibit decapitation secretions characteristic of apocrine
differentiation. Together these results suggest that Notch1 may function to promote Elf5+/ER- cell fate
and may be misregulated in pregnancy-associated masses and apocrine-carcinoma of the breast in
humans.

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Acknowledgments

I wish to thank Dr. Sean Egan’s support, guidance, patience, vision, and instruction. From Sean I
learnt important lessons on asking the right questions and he provided me the freedom to explore how to
find the answers. Keli Xu and Kelvin Wang provided tremendous technical expertise and advice for
which I will always be thankful. To the rest of the Egan lab, thanks for the many enlightening discussions
about life past and present.
I also wish to thank my parents for their constant support in so many aspects of my life. They not
only gave me life but also filled it with all the love and affection one could wish for and for that I am
forever grateful. I dedicate this thesis to them.

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Table of Contents
Abstract…………………………………………………………………………………………………......ii
Acknowledgements…………………………………………………………………………………..…….iii
Table of Contents…………………………………………………………………………………………..iv
List of Tables and Figures……………………………………………………………………………..…....v
List of abbreviations…………………………………………………………………………………..…...vi
1. Introduction……………………………………………………………………………….………..…….1
1.1 Mammary Gland Development……………………………………………………..………1
1.1.1 Puberty…………………………………………………………………………..……….1
1.1.1.1 Estrogen and Ductal Outgrowth…………………………………………..………….7
1.1.1.2 Progesterone and Ductal Branching……………………………………..……….....10
1.1.2 Pregnancy…………………………………………………………………..…………..11
1.1.2.1 Wnt/Progesterone and Alveolar Specification………………………..…………….11
1.1.2.2 Prolactin and Lactogenic Differentiation…………………………..……………….12
1.2 Notch Signaling Pathway………………………………………………..…………………13
1.2.1 Notch and Breast Cancer…………………………………………………………….....14
1.2.2 Notch and Breast Cancer Models…………………………………………………...….15
1.2.3 Notch and Mammary Gland Development……………………………………………..16
2. Objective………………………………………………………………………………………..…...…19
3. Materials and Methods...........................................................................................................................20
3.1 Breeding/Strains…………………..…………………………………………………...…….20
3.2 Genotyping…………………………………………………………………………...……...20
3.3 Wholemount………………………………………………………………………...……….21
3.4 Histology: Immunohistochemistry and Immunofluorescence ……………………………....21
3.5 Flow Cytometry……………………………………………………………………………...22
3.6 Transmission Electron Microscopy………………………………………………………….26
4. Results………………………………………………………………………………………………….29
4.1 Generation of Transgenic System for Analysis of Notch1 Function in the Developing
Mammary Gland……………………………………………………………………………..29
4.2 Pubertal Phenotype of Ectopic Notch1IC Expression ……………………………………….35
4.2.1 Notch1IC Inhibits Ductal Growth when Activated in Body Cells………………………..35
4.2.2 Notch1IC Expression Causes the Formation of Alveolar-Like Structures within
Ducts……………………………………………………………………………………..46
4.2.3 Notch1IC Cell Autonomously Promotes Specification of Hormone Receptor
Negative Cells……………………………………………………………………………58
4.3 Effects of Ectopic Notch1IC Expression during Pregnancy…………………………………65
4.3.1 Notch1IC Expression Promotes Formation of Pregnancy-Dependent Lactating
Adenomas………………………………………………………………………………..65
4.3.2 Notch1IC-Induced Adenomas Contain Highly Proliferative Luminal Secretory
Cells with Two Distinct Morphologies………………………………………………….69
4.3.3 Notch1IC-Induced Adenomas Exhibit Irregular Subcellular Structures and
Morphologies…………………………………………………………………………….81
4.4 Other Effects of Ectopic Notch1IC Expression……………………………………………...88
4.4.1 Notch1IC Induces Facial Tumours and Lymphoma…...................………………………88
5. Discussion……………………………………………………………………………………………...93
5.1 Pubertal Effect of Ectopic Notch1IC Expression…………………………………………….93
5.2 Effects of Ectopic Notch1IC Expression during Pregnancy………………………………….95
6. References……………………………………………………………………………………………...99

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List of Figures
Figure 1……………………………………………………………………………………………………4
Figure 2……………………………………………………………………………………………………6
Figure 3……………………………………………………………………………………………………9
Figure 4……………………………………………………………………………………………………28
Figure 5……………………………………………………………………………………………………32
Figure 6……………………………………………………………………………………………………37
Figure 7……………………………………………………………………………………………………39
Figure 8……………………………………………………………………………………………………41
Figure 9……………………………………………………………………………………………………43
Figure 10…………………………………………………………………………………………………..45
Figure 11…………………………………………………………………………………………………..49
Figure 12…………………………………………………………………………………………………..51
Figure 13…………………………………………………………………………………………………..53
Figure 14…………………………………………………………………………………………………..55
Figure 15…………………………………………………………………………………………………..57
Figure 16…………………………………………………………………………………………………..60
Figure 17…………………………………………………………………………………………………..62
Figure 18…………………………………………………………………………………………………..64
Figure 19………………………………………………………………………………………………67, 68
Figure 20…………………………………………………………………………………………………..72
Figure 21…………………………………………………………………………………………………..74
Figure 22…………………………………………………………………………………………………..76
Figure 23…………………………………………………………………………………………………..78
Figure 24…………………………………………………………………………………………………..80
Figure 25…………………………………………………………………………………………………..83
Figure 26…………………………………………………………………………………………………..85
Figure 27…………………………………………………………………………………………………..87
Figure 28…………………………………………………………………………………………………..90
Figure 29…………………………………………………………………………………………………..92

List of Tables
Table 1……………………………………………………………………………………………………..24
Table 2……………………………………………………………………………………………………..34

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List of Abbreviations
ADAM17 ADAM metallopeptidase domain 17
CD24 Heat stable antigen
CD29 Beta1 integrin
CD49f Alpha6 integrin
CD61 Beta3 integrin
C/EBPβ CCAAT/enhancer binding protein beta
DAB 3,3'-Diaminobenzidine
Dpc Days post coitum
eGFP Enhanced green fluorescent protein
EGFR Epidermal growth factor receptor
Elf5 E74-like factor 5
ER Estrogen receptor
FBS Fetal bovine serum
FSC Forward scatter
Gata3 GATA binding protein 3
H&E Hematoxylin and eosin
HER2 Human Epidermal growth factor Receptor 2
Hes Hairy and enhancer of split
Hey Hairy/enhancer-of-split related with YRPW motif
Id2 Inhibitor of DNA binding 2
IGF2 Insulin-like growth factor 2
IHC Immunohistochemistry
Int3 Insertional site 3
Ires Internal ribosome entry site
Jak2 Janus kinase 2
K8 Cytokeratin 8
K14 Cytokeratin 14
MMTV Mouse mammary tumour virus
MMTV LTR Mouse mammary tumour virus long terminal repeat
Notch1IC Intracellular Notch1 domain
PBS Phosphate Buffered Saline
PCNA Proliferating Cell Nuclear Antigen
PI Propidium iodide
PR Progesterone receptor
Prlr Prolactin receptor
RANKL Receptor Activator for Nuclear Factor κ B Ligand
RBPJκ Recombination signal binding protein for immunoglobulin kappa J region
shRNA Small hairpin Ribonucleic acid
SSC Side scatter
Stat5 Signal transducer and activator of transcription 5
TEB Terminal end bud
TEM Transmission electron microscopy
WAP Whey Acidic Protein

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1. Introduction
1.1 Mammary Gland Development
The murine mammary gland is a powerful system for the study of normal development and
transformation. Uniquely, mammary glands develop almost entirely post-natally, making
analysis of their growth and development from start to finish relatively easy to perform.
Furthermore, recent advances have made possible the identification and isolation of mammary
stem cells, which can be transplanted into an epithelial cell divested mammary fat pad to
generate a complete and functional mammary gland in recipient mice1,2. Indeed, the mammary
system has been used to model stem cells in human breast cancer, a disease that according to the
World Health Organization accounts for 10.4% of all cancer incidences and is the fifth most
common cause of cancer death.
1.1.1 Puberty
Development of the mammary gland occurs largely under control of the female
reproductive hormones estrogen, progesterone, and prolactin3. At birth, the mouse mammary
gland consists of a nipple and a small arborized gland in the underlying mammary fat pad.
Following birth, the gland grows isometrically with the rest of the body until puberty when a
rapid influx of hormones induces substantial growth and differentiation. Terminal end buds
(TEBs) are highly proliferative structures, enriched in mammary stem/progenitor cells, which
invade the mammary fat pad and drive ductal development4. Terminal end buds consist of a
single outer layer of undifferentiated cap cells and multiple layers of inner body cells. As the
duct grows, trailing edges of the cap cell layer differentiate into myoepithelial cells5. Body cells
closest to the cap cell layer tend to be highly proliferative, supporting forward growth of the
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duct. These cells give rise to both hormone receptor positive and hormone receptor negative
luminal cells. Body cells furthest from the cap cell layer undergo apoptosis to create a lumen in
the developing duct (Figure 1)6. Hormone receptor positive cells are cuboidal and express the
estrogen receptor (ER), the progesterone receptor (PR), and the prolactin receptor (Prlr)7. In
contrast, hormone receptor negative cells are morphologically columnar and express both Elf5,
an Ets transcription factor required for lobuloalveolar development and milk production, and
CCAAT/enhancer binding protein beta (C/EBPβ), required for ductal morphogenesis and
alveolar differentiation8,9. As TEBs invade the fat pad they bifurcate to form a branched ductal
tree-like structure. The TEBs persist and continue to move forward until the ducts have reached
the outer limits of the fat pad, at which point they regress. Distinct from TEB bifurcation, lateral
or side branching grow from sites along the developed ductal structure. These side branches are
dependent on recurrent estrous cycles and/or pregnancy and are thought to consist largely of
alveolar precursors9. During the estrous cycle, these side branches develop small alveolar buds,
which will either develop into milk producing structures if the animal becomes pregnant or will
apoptose in a non-pregnant animal (Figure 2)10.
In the past few years, thanks to advances in transplantation methodologies and flow
cytometric analyses, various mammary cell types have been identified and isolated. For
examples, flow cytometric analysis was used to identify three populations on the basis of CD24
expression: CD24Negative, CD24Low, and CD24High, which were confirmed by quantitative PCR to
be non-epithelial, myoepithelial, and luminal cells, respectively11. Interestingly, it was found
that transplantations in cleared mammary fat pads resulted in extensive mammary gland
repopulation in myoepithelial CD24Low cells, whilst luminal CD24High cells were unable to
recapitulate the mammary gland in the divested mammary fat pad11. Indeed, it has been
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Figure 1. Cellular architecture of terminal end buds and mature ducts during puberty.
An illustrated depiction of cap cells (orange) and body cells (blue) within terminal end buds of
the growing duct. The mature duct consists of myoepithelial cells (red) that differentiated from
cap cells as well as hormone receptor positive (green) and hormone receptor negative (black)
luminal cells that differentiated from body cells.

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