Winner of the Cornelius L. Hopper Scientific Achievement Award

1. 1 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 : nandi@berkeley.edu; nandilab@berkeley.edu Website : http://mcb.berkeley.edu/labs/nandi/

2. 2 Breast Cancer Prevention with EstrogenS. Nandi, R.C. Guzman, G. Thordarson, E. Blank & R. Lakshmanaswamy Cancer Research Laboratory & Department of Molecular & Cell Biology University of California, Berkeley

3. 3 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.

4. 4 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.

5. 5 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

6. 6 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

7. 7 MNU-Treated Rats:Protective Effect of Pregnancy Mammary Cancer RatsIncidence %Latency (Months) Young Virgin 90 - 100 3 - 5 (~ 2 months) Parous 10 - 20 6 - 9 (~ 4-5 months) Virgin 60 - 70 6 - 9 (~ 4-5 months)

8. 8 Our Presentation • Hypotheses Explaining Parity Protection • Mammary Cancer: Multistep carcinogenesis & Hypotheses Testing • Defining the Parous Rat Phenotype: Why are they protected? • Development of Hormonal Prevention Treatments: STET (short-term E treatment) & STEPT (short-term E+P treatment) • Parous vs. STET/STEPT Phenotypes: Similar or Different? • Conclusions & Lessons Learned

9. 9 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

11. 11 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

12. 12 Hypothesis # 2: Newer Versions • Sivaraman, Medina, O’Malley et al. (2001) Parous mammary cells w/ sustained p53 expression resulting in muted proliferative response to carcinogens • Wagner, Smith et al. (2002) Parous mammary gland w/ new population of stem cells unresponsive to carcinogens • 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

13. 13 II. Mammary Cancer: Multistep Carcinogenesis Can Be Used for Hypotheses Testing (Experimental Testing of TDH vs DPH)

14. 14 Testing DPH vs TDH in Parous (PA) Rats Multistep Control DPH Testing TDH Testing Virgin (2 mo) PA (5 mo) PA (5 mo) Mammary Carcinogenesis I + high P I + low P no I Breast w/ Target cells Breast w/ Target cells Breast w/o Target cells { Initiation (I) by Carcinogen & Proliferation MNU { Initiated Cells Initiated Cells No Lesions + high P Hormone Driven Promotion & Progression (P) Preneoplasias DCIS (Microscopic) Preneoplasias DCIS + high P Mammary Cancer Mammary Cancer No Lesions

15. 15 Defining the Parous Rat Phenotype exposed to MNU: Known & Unknown Characteristics? • Incidence of Mammary Cancer: Very Low • Incidence of Preneoplasia (DCIS): unknown • Effect of  Hormonal Promotion on DCIS to Mammary Cancers: unknown • Hormone levels & Hormone Receptors in Parous Rats: unknown • Our Studies follow

18. 18 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 manyMCs. • 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 micecan also be reversed w/  hormonal promotion • Thus both parous rats & mice are protected only from promotion-progression & not from initiation. They have no loss of targetcells for MC • All supporting Decreased Promotion Hypothesis

19. 19 IV. Development of Hormonal Prevention Treatments (STET/STEPT) STET : Short-term Estrogen Treatment STEPT: Short-term Estrogen + Progesterone Treatment

20. 20 Testing the Terminal Differentiation Hypothesis (Russo & Russo 1982) Lobule TEB 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.

21. 21 EXPERIMENTAL PROTOCOLS Exp 1: CONTROL (Vehicle); PPZ (Perphenazine/Trilafon); E + P (Estrogen + Progesterone) (Both PPZ & E+P cause TEB  lobule differentiation) PPZ or E + P TREATMENT MNU ……… TERMINATION AT 43 WEEKS OF AGE 7wk 9wk 12wk (Draw blood) @ 7 wks @ 12 wks – After either treatment TREATMENT: 30 mg E & 30 mg P in SUSTAINED RELEASE SILASTIC CAPSULE & PPZ (DAILY INJECTION) for three weeks

23. 23 Plasma Levels of Estradiol (E) & Progesterone (P) Estradiol Progesterone Rats(pg/ml)(ng/ml) Young Virgin 18.3  8.8 12.7  2.1 Controls PPZ Treatment 16.6  7.8 101.5  10.3 E+P Treatment 168.6  22.1 25.8  6.6 Pregnant 55-194 up to 45-56 up to Controls 630 at term 130 at peak Hypothesis: Pregnancy levels of Estradiol in the E+P treated rats may be the reason for their protection against mammary cancer

28. 28 Summary: Hormonal Prevention Studies • Treatment of nulliparous rats for 1-3 weeks, w/ pregnancy levels of estradiolw/ 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 ugEstradiol/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

29. 29 MNU EXPOSED PAROUS VS STEPT/STET RATS -- SIMILAR OR DIFFERENT? Conclusion: Parous & STET/STEPT Phenotypes Similar

33. 33 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

34. 34 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

35. 35 VI. CONCLUSIONS & LESSONS LEARNED

36. 36 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

38. 38 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

39. 39 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

40. 40 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-N-nitrosourea-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