The Mouse as a Modelfor Breast Development and Breast Cancer Research Dr. Tiffany Seagroves Laboratory of Dr. Johnson tseagrov@ biomail.ucsd.edu
I. BREAST CANCER INTRODUCTION Statistics Risk Factors Hormones
Breast Cancer Statistics • The lifetime probability of being diagnosed with breast cancer for American women is 1 in 8 ( NCI, SEER, 1997 ) AGE IS the MOSTIMPORTANT RISK FACTORMedian Age of Diagnosis isBetween 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
Breast Cancer Statistics, cont. (Y-me National Breast Cancer Foundtaion; www.y-me.org)• Most common form of cancer in women, excluding skin cancer• 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
Factors Associated with an Increased Risk of Breast Cancer [Love et al. 1996]• 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)
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 promotenormal development of the breast (to prepare for lactation) act to promote breast cancers later in life?
The Role of “Pregnancy Hormones” in Breast Development and Lactation1) 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
II. ANATOMY ANDHISTOLOGY OF THE HUMAN BREAST Whole gland TDLU unitWhole mount and H&E staining
Slice of a Whole Human Breast skin Fatty tissue Brown area= “epithelium”
Acinar 15-yr The Structuralorganization female Units (Terminal Ductal Lobular Units, TDLU) of22 yr nulli-parous 30 yr nulli-parous the Human Breast 55 yr parous in 80 yr menopause parous Taken from Cardiif and Wellings, 1999
Human TDLU Whole Mount extralobular terminal ducts acini duct lobules Cardiff website
Which is mouse, which is human? (row A vs row B?)AB Wellings and Cardiff, 1999
IV. WHY THE MOUSEMAMMARY GLAND IS APOWERFUL GENETIC TOOL Reasons Experiment approaches How to make a transgenic mouse to study development/cancer
Why Use the Mouse as a Model for Breast Cancer?• Histology is comparable to human• 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
How to Biopsy a Mouse Mammary Gland QuickTimeª and a decompressor are needed to see this picture. QuickTimeª and a decompressor are needed to see this picture.
Mammary Gland Transplantation Remove endogenous epithelium from stromal fat pad Transplant a fragment of tissue Containing epithelium from a donor OR inject purified cells Transplanted epithelium grows out Into fat pad in 6-8 weeks
How To Make a Mammary-Gland Specific Transgenic Mousemilk protein geneminimal promoter Your Favorite cDNAusually MMTV or WAP Inject into isolated mouse nucleus Check mammary gland of female progeny for increased expression of your gene
V. WHAT DO MICE HAVE TO DO WITH BREAST CANCER? Differences between breast and mouse mammary tumors How to get mice to develop tumors
General Differences Between Human Breast and Mouse Mammary Tumors SIMILARITIES DIFFERENCES• Molecular lesions causing • Most tumors mouse breast cancer in human have proven to cause breast cancer metastasize to the lung. in mice Most human metastasize to• Similar morphological the regional lymph nodes. patterns of lesions appear in • Mouse tumors have much both species less fibrosis and• Development of cancer consistent with multi-hit inflammation kinetics • *Half of human breast• Breast cancers in both species cancers are hormone- are metastatic dependent. Most mouse• Breast cancers may be tumors are hormone hormone- independent independent* Taken from Thompson and Cardiff
How to Cause a Mammary Tumor in a Mouse• 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)
VI. HUMAN BREAST PATHOLOGY Examples of Benign Diseases Tumor Grades/Types Hyperplasia vs Carcinoma
How do pathologists classify and grade breast tumors? Benign vs. hyperplastic vs. carcinoma Well- vs. poorly-differentiated Nuclear morphology- uniform or not Degree of proliferation Ductal or lobular in origin?
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• HISTOLOGIC GRADE-higher the grade, less chance for survival• 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 Hyperplasias Hyperplasia: Any increase in cell number without cytologic changes in cellular morphology MILD SEVERE highlow power power Atypical Hyperplasia: Any increase in cell number WITH cytologic changes in cellular morphology, especially nuclear morphology fine needle aspirationlow power note different staining intensity of nuclei BUT cells still attached to each other
DCIS (ductal carcinoma in situ): the “precursor” to breast cancerIt is in situ or “in place” because the cells are still bound by the extracellular matrix Solid Cribiform Papillary
Breast Carcinomas the tumor is invading the breast, it has broken through the matrix Well-differentiated Abonormal nuclei from fine needle aspiration of carcinoma still see glandular structures resembling aciniPoorly-differentiated arrows point to nuclei with different morphology. Note also cells no longer attached mass of cells, no to each other resemblance to acini
Metastasis• 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 Liver mets, (white spots)• It is more rare for rodent tumor models to exhibit metastasis, but when observed, are usually restricted to lung
VII. MOUSE MAMMARY TUMOR PATHOLOGY Benign Disease Hyperplastic Alveolar Nodules Carcinomas Comparative with Human Tumors
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) F. tumors from chemical carcinogen treatment2) 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: TGFα, neu, c-src, myc transgenic mice Classification by Cardiff, 2000
Examples of Mouse Carcinomas neu (erb-2) myc more solid more glandular ras ret-1 different ras myc neunuclear morphologies from different genes
both MMTV-induced mouse tumors left; DCIS, solid form right; neu transgeneleft; schirrhous carcinomaright; src transgeneleft; papillary carcinomaright; a protein kinase transgene
IX. BACK TO HORMONES:HOW DO WE KNOW PREGNANCY CAN BEPROTECTIVE AGAINST BREAST CANCER?
A combination of E+P treatment reduces chemical carcinogen-inducedtumorigenesis in rodents (Nandi et al. 1995, 1999)
REFERENCEShttp://mammary.nih.govhttp://pathology.ucdavis.edu/tgmice/firststop.htmlhttp://www.y-me.orghttp://www.komen.orghttp://www.nci.nih.gov/THE JOURNAL OF MAMMARY GLAND BIOLOGY AND NEOPLASIA