The Mouse as a Model for Breast Development and Breast Cancer Research

Dr. Tiffany SeagrovesLaboratory of Dr. Johnson

tseagrov@biomail.ucsd.edu

I. BREAST CANCER INTRODUCTION

Statistics

Risk Factors

Hormones

• The lifetime probability of being diagnosed with breast cancer for American women is 1 in 8 (NCI, SEER, 1997)

Breast Cancer Statistics

AGE IS the MOST

IMPORTANT RISK FACTOR

Median Age of Diagnosis is

Between 60-65 (NIH, 2000)

Recent Decrease in UK and USA Breast Cancer Mortality at Ages 50-69 Years

PETO et al. LANCET 355:1822, 2000

• Most common form of cancer in women, excluding skin cancer

Breast Cancer Statistics, cont.(Y-me National Breast Cancer Foundtaion; www.y-me.org)

• Leading overall cause of death between women of age 40-55

• ACS estimates that 192,000 American women will be diagnosed with breast cancer this year and approximately 46,000 women will die

• There are more than 2,000,000 breast cancer survivors in the U.S. today

• Age of menarche, first child, onset menopause

• Diet, level of exercise, obesity, alcohol consumption

• Presence of benign breast disease (DCIS)

• Exposure to radiation

• Family history and genetics (estimated 5% of total cases can be contributed to genetic factors, and 20-30% cases can be linked to a family history of breast cancer)

Factors Associated with an Increased Risk of Breast Cancer [Love et al. 1996]

Increase in Diganosis of Early-Stage Breast TumorsSince the 1980s

70% of breast cancers occur in women who have no identifiable risk factors.

This is why you are supposed to examine

yourself and have yearly exams.

Factors Associated with a Significant Decreased Risk of Breast Cancer (~ 30-50%)

• Completed Pregnancy by Age 20– Exposure to “pregnancy” hormones is protective if

happens early

• Removal of Both Ovaries by Age 35– Over time, exposure to “pregnancy” hormones

increases risk because ~50% of breast tumors are initially hormone-dependent

Paradox of Hormone Function in the Breast:

Why do the same hormones that promote normal

development of the breast (to prepare for lactation) act to

promote breast cancers later in life?

1) ESTROGEN AND PROGESTERONE (E+P): Produced by corpeus luteum of ovary first 6 wks or pregnancy, then taken over by placenta.

Together, E+P stimulates growth and development of secretory units and ducts in the gland. \

2) PROLACTIN (Prl):

Produced by anterior pituitary

Stimulates production of milk

The Role of “Pregnancy Hormones” in Breast Development and Lactation

II. ANATOMY AND HISTOLOGY OF THE

HUMAN BREAST

Whole gland

TDLU unit

Whole mount and H&E staining

Source: http://mammary.nih.gov

Slice of a Whole Human Breast

Brown area= “epithelium”

skin

Fatty tissue

The Structural Units (Terminal Ductal Lobular Units, TDLU) of

the Human Breast

Taken from Cardiif and Wellings, 1999

15-yr female

22 yr nulli-parous 30 yr nulli-parous

55 yr parous in menopause

80 yr parous

Acinar organization

Human TDLU Whole Mount

duct

acini

lobules

extralobular terminal ducts

Cardiff website

TDLU Histology: H&E

III. ANATOMY AND HISTOLOGY OF THE

MOUSE MAMMARY GLAND

Whole Mount

H&E

Comparative Histology with Human

The Terminal End Bud of the Mouse Mammary Gland

TEBs are highlyproliferative unitsthat form the ductal network

Stages of Mouse Mammary Development: Whole mounts

www.mammary.nih.gov

Duct

Alveoli

A-B. 6-P, “early”

C-D. 10-P, “mid”

E-F. 15-P, “mid-to-late”

G. Lactation

H. 4 days regression

Wellings and Cardiff, 1999

Which is mouse, which is human?(row A vs row B?)

A

B

IV. WHY THE MOUSE MAMMARY GLAND IS A

POWERFUL GENETIC TOOL

Reasons

Experiment approaches

How to make a transgenic mouse

to study development/cancer

• Histology is comparable to human

Why Use the Mouse as a Model for Breast Cancer?

• Can use genetics to manipulate the mammary glands

• Have multiple pairs of mammary glands that allow for multiple biopsies

• Can purify epithelial cells from the fat and culture them

Mice have 10 Mammary Glands

How to Biopsy a Mouse Mammary Gland

QuickTime™ and a decompressor

are needed to see this picture.

QuickTime™ and a decompressor

are needed to see this picture.

Remove endogenous epithelium

from stromal fat pad

Transplanted epithelium grows out Into fat pad in 6-8 weeks

Transplant a fragment of tissue Transplant a fragment of tissue Containing epithelium from a Containing epithelium from a donor OR inject purified cellsdonor OR inject purified cells

Mammary Gland Transplantation

How To Make a Mammary-Gland Specific Transgenic Mouse

milk protein gene minimal promoter Your Favorite cDNA

Inject into isolated mouse nucleus

Check mammary gland of female progeny for increased

expression of your gene

usually MMTV or WAP

V. WHAT DO MICE HAVE TO DO WITH BREAST

CANCER?

Differences between breast and mouse mammary tumors

How to get mice to develop tumors

• Most tumors mouse metastasize to the lung. Most human metastasize to the regional lymph nodes.

• Mouse tumors have much less fibrosis and inflammation

• *Half of human breast cancers are hormone-dependent. Most mouse tumors are hormone independent*

General Differences Between Human Breast and Mouse Mammary Tumors

• Molecular lesions causing breast cancer in human have proven to cause breast cancer in mice

• Similar morphological patterns of lesions appear in both species

• Development of cancer consistent with multi-hit kinetics

• Breast cancers in both species are metastatic

• Breast cancers may be hormone- independent

SIMILARITIES DIFFERENCES

Taken from Thompson and Cardiff

• Treat young mice (time most susceptible) with a chemical carcinogen

• Make a transgenic mouse that overexpresses a gene product that regulates growth

• Make a “knockout” mouse that deletes a gene that is a tumor suppressor

• Breed them several times and watch for spontaneous tumors (rare in mice, more common in rats)

How to Cause a Mammary Tumor in a Mouse

VI. HUMAN BREAST PATHOLOGY

Examples of Benign Diseases

Tumor Grades/Types

Hyperplasia vs Carcinoma

Benign vs. hyperplastic vs. carcinoma

Well- vs. poorly-differentiated

Nuclear morphology- uniform or not

Degree of proliferation

Ductal or lobular in origin?

How do pathologists classify and grade breast tumors?

Wheel of Prognosis

• HISTOLOGIC GRADE-higher the grade, less chance for survival

What factors predict outcome of treatment?

• SIZE-the larger the size, fewer patients survive

If <2 cm, 11% lymph-node negative patients will have recurrence in 5 yr; >5cm, 25-30% patients will relapse

• ER STATUS-loss of estrogen receptor tends to be negatively associated with outcome.In particular women with ER-negative tumors are no longer responsive to tamoxifen, a widely used adjuvant therapy

• PROLIFERATIVE RATE -low rate proliferation, increased chances of survival. This factor is also independent of other factors.

• Amplification of certain growth factors or receptors or loss of certain tumor suppressors (p53)-lead to decreased survival

Benign Breast Disease

1) Fibroadenoma-overgrowth of stromaMost common benign tumor of the breast; typically occurs in the 20s-30s

2) Cysts-fluid filled epithelium

may make breasts feel “lumpy”

Human Breast HyperplasiasHyperplasia: Any increase in cell number without cytologic

changes in cellular morphology

Atypical Hyperplasia: Any increase in cell number WITH cytologic changes

in cellular morphology, especially nuclear morphology

low power

MILD

low power

SEVERE

highpower

note different staining intensity of nuclei BUTcells still attached to each other

fine needle aspiration

DCIS (ductal carcinoma in situ): the “precursor” to breast cancer

It is in situ or “in place” because the cells are still bound by the extracellular matrix

Solid

Papillary

Cribiform

Breast Carcinomasthe tumor is invading the breast, it has broken through the matrix

Well-differentiated

still see glandularstructures resembling acini

Poorly-differentiated

mass of cells, no resemblance to acini

arrows point to nuclei withdifferent morphology. Note also cells no longer attached to each

other

Abonormal nuclei fromfine needle aspiration of carcinoma

• Human tumors tend to spread to regional lymph node first (why lymph nodes under arm also biopsied with tumor)

• Then tumors spread to small capillaries of the vascular network

• Breast tumors tend to metastasize to lung, liver, brain, bone

• It is more rare for rodent tumor models to exhibit metastasis, but when observed, are usually restricted to lung

Metastasis

Liver mets, (white spots)

VII. MOUSE MAMMARY TUMOR PATHOLOGY

Benign Disease

Hyperplastic Alveolar Nodules

Carcinomas

Comparative with Human Tumors

Mouse hyperplasia: the HAN

HANs and Atypical Hyperplasias

2) From GEM, usually transgenic mice

In contrast to non-GEM, several GEM transgenic mouse models develop tumors with similar pathologies to human breast tumors and more are described every year.

examples: TGF, neu, c-src, myc transgenic mice

Classification of Mouse Mammary Carcinomas:

1) From non-Genetically Engineered Mice (GEM)A. spontaneous tumors

B. tumors as a result of infection of with mouse mammary tumor virus (MMTV)

C. tumors from chemical carcinogen treatment

Classification by Cardiff, 2000

Examples of Mouse Carcinomas

ras myc neudifferent nuclear morphologiesfrom different genes

neu (erb-2)

ras

myc

ret-1

more solid more glandular

left; papillary carcinomaright; a protein kinase

transgene

both MMTV-inducedmouse tumors

left; DCIS, solid formright; neu transgene

left; schirrhous carcinomaright; src transgene

IX. BACK TO HORMONES:

HOW DO WE KNOW PREGNANCY CAN BE PROTECTIVE AGAINST BREAST CANCER?

A combination of E+P treatment reduces chemical carcinogen-induced tumorigenesis

in rodents (Nandi et al. 1995, 1999)

http://mammary.nih.govhttp://pathology.ucdavis.edu/tgmice/firststop.html http://www.y-me.orghttp://www.komen.orghttp://www.nci.nih.gov/THE JOURNAL OF MAMMARY GLAND BIOLOGY AND NEOPLASIA

REFERENCES