Transcript Document 7357885

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Winner of the
Cornelius L. Hopper
Scientific Achievement Award
Best Poster for Highest Impact on Breast Cancer.
From Research to Action: Seeking Solutions
California Breast Cancer Research Program
on September 10, 2005.
Awarded to Satyabrata Nandi Laboratory of the Cancer Research Laboratory and
Molecular Cell Biology Department of the University of California, Berkeley on the
basis of research in the Nandi Laboratory in collaboration with the Talamanates Thordarson Laboratory at the University of California, Santa Cruz and the Airo Tsubura
Laboratory at Kansai Medical University, Osaka, Japan.
Contacts
E-mail : [email protected]; [email protected]
Website : http://mcb.berkeley.edu/labs/nandi/
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Breast Cancer Prevention with Estrogen
S. Nandi, R.C. Guzman, G. Thordarson, E. Blank &
R. Lakshmanaswamy
Cancer Research Laboratory &
Department of Molecular & Cell Biology
University of California, Berkeley
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Abstract
In humans, a first full term pregnancy (FFTP) before the age of 20 reduces the risk for
breast cancer by 50% versus nulliparous females. During pregnancy, women produce
huge quantities of estrogens. The physiological roles for these huge amounts of estrogen
remain mostly unknown. Parous rats and mice are also highly protected against
carcinogen-induced breast cancer. There are two competing hypotheses to explain this
parity protection: (1) the decreased promotion hypothesis (DPH) which postulates
that FFTP causes permanently decreased secretion of mammogenic hormones such as
growth hormone and prolactin necessary for promotion-progression to mammary
cancers and (2) the terminal differentiation hypothesis (TDH) which proposes that
early FFTP results in complete loss of target cells necessary for breast carcinogenesis.
Using the rat model system we have sought to: (1) study the parous phenotype in rats
and discover why they are protected from breast cancer, (2) develop short-term and safe
hormonal prevention treatments mimicking the protective effect of FFTP in nulliparous
rats without their having to go through pregnancy and (3) determine the physiological
and molecular bases of parity protection. The results of our studies with carcinogen
induced rat mammary cancer are summarized. In the parous rat phenotype, following
carcinogen exposure, there is a very high incidence of DCIS, low incidence of
breast cancer but a high incidence of breast cancer upon prolonged hormonal
promotion. They have a permanently reduced secretion of growth hormone and
prolactin, necessary for promotion-progression of DCIS to breast cancer. All these
support the DPH hypothesis.
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We have succeeded in our attempt at development of safe, short-term
treatments for breast cancer prevention in nulliparous rats without their
having to go through pregnancy. Short term (1-3 weeks) treatment with pregnancy
level of estrogen (STET) or with estrogen plus progesterone (STEPT) reduces breast
cancer incidence by greater than 80% and cancer multiplicity by greater than 90%.
STET/STEPT fully mimics the protective effect of pregnancy in nulliparous
rats and is as effective in prevention as FFTP or ovariectomy or long-term
treatment with tamoxifen. Both non-carcinogen treated control and STEPT rats
kept alive for 24 months showed no differences in body weight, ovarian and
reproduction functions, tumorigenicity or longevity. To our knowledge,
STET/STEPT treatments are the shortest, safest and potentially universally
affordable chemoprevention procedure for breast cancer. Our molecular studies
are in progress . Supported by BCRP Grant No. 8PB-0132.
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Pregnancy, Estrogen (E) & Breast Cancer
• In humans, 1st full term pregnancy (FFTP) before age 20  risk by
50% vis a vis nulliparous FF
• Women >35 yrs of age at FFTP have the highest risk
• During pregnancy, Estriol secretion in urine  by 1,000 fold &
plasma estradiol level rises from 40 – 180 pg/ml to 15,000 –
22,000 pg/ml
• The physiological roles of these huge amounts of E remain mostly
unknown
• Parous rats & mice are also highly protected against carcinogen induced breast cancers
• Rat model system has been invaluable in devising procedures for
therapy & prevention of human breast cancer
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Our Goals Using the Rat Model System
• Define the biological bases of the parous phenotype.
Why are they protected from breast cancer?
• Develop short-term & safe hormonal prevention
treatments mimicking the protective effect of FFTP in
nulliparous rats w/o their having to go through
pregnancy
• Determining the physiological & molecular bases of
parity protection
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MNU-Treated Rats:
Protective Effect of Pregnancy
Rats
Young Virgin
Mammary Cancer
Incidence %
Latency (Months)
90 - 100
3-5
10 - 20
6-9
(~ 2 months)
Parous
(~ 4-5 months)
Virgin
(~ 4-5 months)
60 - 70
6-9
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Our Presentation
I.
Hypotheses Explaining Parity Protection
II.
Mammary Cancer: Multistep carcinogenesis &
Hypotheses Testing
III.
Defining the Parous Rat Phenotype: Why are they
protected?
IV. Development of Hormonal Prevention Treatments:
STET (short-term E treatment) & STEPT (shortterm E+P treatment)
V.
Parous vs. STET/STEPT Phenotypes: Similar or
Different?
VI. Conclusions & Lessons Learned
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I. Hypothesis # 1-- Explaining the Biological Bases
of Parity Protection in Humans
Decreased Promotion Hypothesis
(DPH): Yu et al & Kwa et al. (1981);
Musey et al. (1987): Hormonal
Change: FFTP causes permanently
decreased secretion of mammogenic
hormones in parous ♀♀ necessary for
promotion-progression to mammary
cancers
Prediction:  hormonal promotion will  mammary cancer
incidence in parous rats
Contd
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Russo & Russo, Cancer Letters 90 (1995) 81-89
Differentiation Pathway
Pregnancy
or
hCG
Target
AB
Lobules
TEB
DMBA or NMU
IDP
IDCa
AdCa
Neoplastic Transformation
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Hypothesis # 2: Newer Versions
1.
Sivaraman, Medina, O’Malley et al. (2001) Parous
mammary cells w/ sustained p53 expression
resulting in muted proliferative response to
carcinogens
2.
Wagner, Smith et al. (2002) Parous mammary gland
w/ new population of stem cells unresponsive to
carcinogens
3.
Ginger, Rosen et al. (2001) & D’Cruz, Chodosh et
al. (2002) Altered gene expression causing
refractoriness to carcinogens
All supporting TDH Hypothesis using altered gene
expressions in mammary cells
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II. Mammary Cancer:
Multistep Carcinogenesis Can Be Used
for
Hypotheses Testing
(Experimental Testing of TDH vs DPH)
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Testing DPH vs TDH in Parous (PA) Rats
Multistep
Control
DPH Testing
TDH Testing
Mammary
Carcinogenesis
Virgin (2 mo)
PA (5 mo)
PA (5 mo)
I + high P
I + low P
no I
Breast w/
Target cells
Breast w/
Target cells
Breast w/o
Target cells
Initiated
Cells
Initiated
Cells
No Lesions
Initiation (I)
by
Carcinogen &
Proliferation
{
MNU
{
Hormone Driven
Promotion
&
Progression (P)
+ high P
Preneoplasias
DCIS
(Microscopic)
Mammary
Cancer
Preneoplasias
DCIS
+ high P
Mammary
Cancer
No Lesions
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III.
Defining the Parous Rat Phenotype
exposed to MNU:
Known & Unknown Characteristics?
1. Incidence of Mammary Cancer: Very Low
2. Incidence of Preneoplasia (DCIS): unknown
3. Effect of  Hormonal Promotion on DCIS to
Mammary Cancers: unknown
4. Hormone levels & Hormone Receptors in
Parous Rats: unknown
5. Our Studies follow
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Parous Phenotype (MNU exposed):
Why are they protected?
• Parous rats develop high incidence of DCIS. Thus they are fully
susceptible to initiation
• Parous rats rarely develop Mammary Cancer (MC) but upon
hormone-promotion develop many MCs.
• Parous rats have persistently  serum GH & PRL (Promotional
hormones) (Thordarson et al. 1995)
• Earlier studies by us (Swanson et al. 1994, 1995) showed that
refractoriness of parous mice can also be reversed w/  hormonal
promotion
• Thus both parous rats & mice are protected only from promotionprogression & not from initiation. They have no loss of target cells
for MC
• All supporting Decreased Promotion Hypothesis
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IV. Development of Hormonal
Prevention Treatments
(STET/STEPT)
STET : Short-term Estrogen Treatment
STEPT: Short-term Estrogen + Progesterone
Treatment
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Testing the Terminal Differentiation Hypothesis
(Russo & Russo 1982)
TEB
Lobule
AB
A
B
C
OUR WORKING HYPOTHESIS
Protective effect of FFTP can be mimicked in nulliparous rats
by any agent (hormones/drugs) that will induce pregnancy-like
full lobular development before or soon after the carcinogen (MNU)
treatment.
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EXPERIMENTAL PROTOCOLS
Exp 1: CONTROL (Vehicle); PPZ (Perphenazine/Trilafon); E + P (Estrogen + Progesterone)
(Both PPZ & E+P cause TEB  lobule differentiation)
MNU
7wk
@ 7 wks
PPZ or E + P
TREATMENT
9wk
TERMINATION
……… AT 43 WEEKS
12wk (Draw blood)
OF AGE
@ 12 wks – After either treatment
TREATMENT: 30 mg E & 30 mg P in SUSTAINED RELEASE SILASTIC CAPSULE
& PPZ (DAILY INJECTION) for three weeks
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Plasma Levels of Estradiol (E) &
Progesterone (P)
Estradiol
(pg/ml)
Progesterone
(ng/ml)
Young Virgin
Controls
18.3  8.8
12.7  2.1
PPZ Treatment
16.6  7.8
101.5  10.3
E+P Treatment
168.6  22.1
25.8  6.6
Rats
Pregnant
Controls
55-194 up to
630 at term
45-56 up to
130 at peak
Hypothesis:
Pregnancy levels of Estradiol in the E+P treated rats may be
the reason for their protection against mammary cancer
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Summary: Hormonal Prevention Studies
• Treatment of nulliparous rats for 1-3 weeks, w/ pregnancy levels of
estradiol w/ or w/o progesterone (STEPT/STET) results in:
Long-term protection against MNU-induced mammary cancer w/
> 80%  in incidence, > 90%  in multiplicity &  in average age
at latency
• Treatment requires a maximum of 1 ug Estradiol/day & is non-toxic
• STEPT & Control rats (w/o MNU), kept alive for 2 yrs, showed
no differences in weight, reproduction, tumorigenicity, & longevity
• Results demonstrate a novel physiologic role of pregnancy
level of estrogen in protection against breast cancer & a new
paradigm for possible breast cancer prevention
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V. MNU EXPOSED PAROUS VS STEPT/STET
RATS -- SIMILAR OR DIFFERENT?
Characteristic
Parous
STET/STEPT
Mammary Cancer (MC)
Low
Low
DCIS
High
High
Hormone Promotion
 MC
 MC
GH & PRL Secretion


Conclusion: Parous & STET/STEPT Phenotypes Similar
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Parous & STEPT/STET Rats are
Phenotypically Similar
(all MNU Exposed)
• All susceptible to initiation & develop DCIS
• All w/ drastically  Mammary Cancer incidence
• All w/  promotional environment:  GH,  PRL
• All develop Mammary Cancers w/  promotion
• All 3 phenotypes result from  promotional hormones
Conclusion: STET/STEPT mimics the protective effects of
early FFTP in Nulliparous Rats
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HOW DO FFTP & STET/STEPT
PROTECT AGAINST BREAST
CANCER?
• Protection primarily against promotion-progression &
not against initiation
• We speculate that continuous short-term exposure to
pregnancy level of estrogen permanently modifies
mammary gland & hypothalamus-pituitary axis causing:
Decreased pituitary secretion of mammogenic
hormones (GH & PRL) resulting in permanently
decreased promotional environment.
• We conclude that DPH & not TDH can fully explain
the parous & STET/STEPT Phenotypes
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VI. CONCLUSIONS
&
LESSONS LEARNED
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Overall Conclusions & Novelty
• Pregnancy level of estrogen is the reason for the protective
effect of pregnancy against breast cancer
• STEPT/STET fully mimics the protective effect of FFTP in
nulliparous rats w/o their having to go through pregnancy
• Shortest, safe & universally affordable chemoprevention
procedure. Treatment can be non-invasive.
• STET/STEPT: As effective in prevention as early FFTP or
ovariectomy or long-term treatment w/ tamoxifen
• No loss of ovarian or reproductive functions or longevity
• Treatment efficacy can be determined from simple blood test
of GH/PRL levels, pre & post STET/STEPT
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Estrogen: An Amazing Hormone
• Estrogen & Breast have been essential for Evolution of 4600+ sp. of
mammals
• Estrogen essential for growth & function of the normal breast
• Estrogen may or may not be directly involved in Breast Carcinogenesis:
Mechanism unclear
• Supra Physiological levels of estrogen can selectively kill ER+ Breast
Cancer Cells while promoting normal breast growth
• Very high levels of both Estrogen (E) & Progesterone (P) are secreted
during Pregnancy
• High E, unless opposed by high P, can cause uterine cancer
• Pregnancy level of Estrogen is likely to be the evolutionary mechanism
for the Protective Effect of Pregnancy against Breast Cancer
• Without this, high Estrogen production & their protective effect,
Mammals would likely have been Extinct due to the radiation induced
breast & other cancers
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Contributions & Acknowledgements
• Talamantes Lab, UCSC, Santa Cruz: Frank Talamantes, Gudmundur
Thordarson
• Tsubura Lab, Kansai Medical University, Osaka: Airo Tsubura,
Jihong Yang & Colleagues
• Agilent Technologies Inc. Palo Alto, CA
• Our Lab: Raphael Guzman, Rajkumar Lakshmanaswamy, Edward
Blank, Christopher Almario, Po-yin Anne Wong, Jason Yang, Steve
Swanson, Tinya Abrams & Satyabrata Nandi
• The California Breast Cancer Research Program & the National
Cancer Institute
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Relevant References
Swanson SM, Guzman RC, Christov K, et al (1994) Pituitary-isografted mice are highly susceptible to MNU-induced
mammary carcinogenesis irrespective of the level of alveolar differentiation. Carcinogenesis 15:1341-1346.
Swanson SM, Guzman RC, Collins G, et al (1995) Refractoriness to mammary carcinogenesis in the parous mouse is
reversible by hormonal stimulation induced by pituitary isografts. Cancer Lett 90:171-181.
Thoradarson G, Jin E, Guzman RC et al (1995) Refractoriness to mammary tumorigenesis in parous rats: is it caused by
persistent changes in the hormonal environment or permanent biochemical alterations in the mammary epithelia?
Carcinogenesis 20:2847-2853.
Abrams, TJ, Guzman RC, Swanson SM et al (1998) Changes in the parous rat mammary gland environment are involved in
parity-associated protection against mammary carcinogensis. Anticancer Res 18: 4115-4122.
Guzman, RC, Yang J, Rajkumar L, et al (1999) Hormonal prevention of breast cancer: Mimicking the protective effect of
pregnancy. Proc Natl Acad Sci USA 96: 2520-2525.
Yang J, Yoshizawa K and Nandi S, et al. (1999) Protective effects of pregnancy and lactation against N-methyl-Nnitrosourea-induced mammary carcinomas in female Lewis rats. Carcinogenesis 20: 623-628.
Rajkumar L, Guzman RC, Yang J, et al (2001) Short–term exposure to pregnancy levels of estrogen prevents mammary
carcinogenesis. Proc natl acad Sci USA 98:11755-11759.
Thoradarson, G, VanHorn K, Guzman RC et al (2001) Parous rats regain high susceptibility to chemically induced
mammary cancer after treatments with various mammotropic hormones. Carcinogenesis 22: 1027-1033.
Rajkumar L, Guzman RC, Yang J et al (2004) Prevention of mammary carcinogenesis by
short-term estrogen and progestin treatments. Breast Cancer Res 6: R31-R37.
Thordarson G, Slusher N, Leong H et al (2004) Insulin-like growth factor (IGF)-1 obliterates the pregnancy-associated
protection against mammary carcinogensis in rats: evidence that IGF-1 enahnces cancer progression through estrogen
receptor-a activation via the mitogen-activated protein kinase pathway. Breast Cancer Res 6:R423-436.
* Nandi, S, Guzman, RC, Thordarson, G et al. (2005) Estrogen can prevent breast cancer by mimicking the protective effect
of pregnancy. Hormonal Carcinogenesis (Eds. Li JJ, Li SA, Llombart-Bosch, A) pp 153-165, New York: Springer.
Rajkkumar, L, Dang, D-N, Hartnett, MD et al (2005) Microarray of estrogen-induced protection against breast cancer.
Hormonal Carcinogenesis (Eds. Li JJ, Li SA, Llombart-Bosch, A) pp 419-425, New York: Springer.
Guzman, RC, Rajkumar, L, Thordarson, G, Nandi, S. (2005) Pregnancy level of estrogen prevents mammary cancers.
Hormonal Carcinogenesis (Eds. Li JJ, Li SA, Llombart-Bosch, A) pp 426-430, New York: Springer.
* Review article of our research