Protective Effect Of Early Pregnancy Research Articles

Dairy animals and breast cancer: reflections on a long-term study from the 1970s that was never done.

In this short commentary I recall a long-term experiment that was sketched out to determine if the low incidence of mammary cancer in dairy animals reflects a low incidence in these species generally or is the result of a protective effect of early pregnancy and long lactations. Although that experiment was never done, I discuss these questions in the light of developing knowledge on the incidence of cancer in ruminants generally and in the mammary gland in particular.

Abstract 645: Pregnancy inhibits mammary carcinogenesis by altering the mammary epithelial and stromal interactions

Abstract A full-term pregnancy before the age of 20 is the only known natural phenomenon that can drastically reduce the risk of breast cancer in women of all ethnic backgrounds worldwide. This universal protective effect of early pregnancy is clearly of major consideration in devising prevention strategies against breast cancer. It is well known that parous subjects have an altered systemic hormonal milieu with a significant decrease in circulating GH and PRL. Determining how these hormonal alterations impact the mammary gland of early parous rats is expected to improve our understanding of the dynamics of mammary carcinogenesis. The inguinal mammary fat pad was cleared of the host mammary gland in 3-4-week-old rats. One group of rats were exposed to N-Methyl-N-Nitrosourea (MNU) at 7 weeks of age, and another group of rats were not exposed to MNU. A subset of rats from each of these experimental groups were mated at 9 weeks and allowed to go through a full-term pregnancy. All animals were terminated 6 weeks post-weaning. Mammary epithelial cells (MECs) were isolated using the collagenase cell dissociation procedure. The isolated MECs (5 × 105) were transplanted into the gland-free fat pads of respective hosts. In brief, we studied the following groups to understand the influence of early parity on epithelial-stromal interactions involved in mammary carcinogenesis: 1) carcinogen treated MECs from Age-matched nulliparous (AMNP) rats were transplanted into untreated parous rats; 2) carcinogen treated MECs from parous rats were transplanted into untreated AMNP rats; 3) untreated MECs from AMNP rats were transplanted into carcinogen treated parous rats and 4) untreated MECs from parous rats were transplanted into carcinogen treated AMNP rats. All experimental animals were palpated weekly for the presence of mammary cancers. The cancerous nature of the palpable tumors was confirmed by histopathological analysis. Digital images of immunocytochemistry result was analyzed using Image Pro Plus software. Carcinogenesis experiments were terminated nine months after carcinogen administration. Incidence, multiplicity, and latency of mammary cancers was determined for each group and analyzed for significance by the appropriate statistical test. Our data demonstrated that transplantation of MECs from AMNP rats into parous hosts did not develop into palpable tumors while MECs from parous rats when transplanted to AMNP hosts developed mammary tumors. Analysis of the mammary gland wholemounts and molecular markers clearly indicated that parity alters key signaling mechanisms and inhibits progression of mammary lesions. These findings suggests that the mammary epithelial-stromal interactions in the parous rats is altered making it resistant to mammary carcinogenesis. Citation Format: Ramadevi Subramani Reddy, Adriana Galvez, Diego A. Pedroza, Animesh Chatterjee, Courtney Perry, Elizabeth Penner, Rajkumar Lakshmanaswamy. Pregnancy inhibits mammary carcinogenesis by altering the mammary epithelial and stromal interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 645.

Abstract 350: Pregnancy at early age is associated with a reduction of progesterone-responsive cells and epithelial Wnt signaling in human breast tissue

Abstract Background: Pregnancy at early age is the most significant modifiable factor which consistently decreases lifetime breast cancer risk. However, the underlying mechanisms haven’t been conclusively identified. Studies in mice suggest a reduction in progesterone-receptor (PR) sensitive epithelial cells as well as a downregulation of the Wnt signaling pathway as being one of the main mechanisms for the protective effect of early pregnancy. The aim of our study was to validate these findings in humans. Methods: We collected benign breast tissue of 123 women who had been stratified according to age at first pregnancy and the occurrence of subsequent breast cancer, and performed immunohistochemistry for PR, Wnt4 and the Wnt-target Versican. Results: The number of PR positive epithelial cells was significantly lower in the group of women with early pregnancy and no subsequent breast cancer compared to the group of nulliparous women with subsequent invasive breast cancer (p=0.017). In women with early pregnancy, expression of Versican and Wnt4 was significantly lower compared to nulliparous women (p=0.0064 and p=0.0156 respectively), and Versican expression was also significant lower compared to women with late pregnancy (p<0.0001). Discussion: Our results confirm prior observations in mice and suggest a role of downregulation of epithelial Wnt signaling in the protective effect of early pregnancy in humans. This results in a decreased proliferation of stem/progenitor cells; therefore, the Wnt signaling pathway may represent a potential target for breast cancer prevention in humans. Citation Format: Robert Mechera, Simone Münst, Silvio Däster, Salvatore Piscuoglio, Charlotte Ng, Fabienne Meier-Abt, Walter P. Weber, Savas D. Soysal. Pregnancy at early age is associated with a reduction of progesterone-responsive cells and epithelial Wnt signaling in human breast tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 350. doi:10.1158/1538-7445.AM2017-350

Pregnancy at early age is associated with a reduction of progesterone-responsive cells and epithelial Wnt signaling in human breast tissue.

BackgroundPregnancy at early age is the most significant modifiable factor which consistently decreases lifetime breast cancer risk. However, the underlying mechanisms haven't been conclusively identified. Studies in mice suggest a reduction in progesterone-receptor (PR) sensitive epithelial cells as well as a downregulation of the Wnt signaling pathway as being one of the main mechanisms for the protective effect of early pregnancy. The aim of our study was to validate these findings in humans.MethodsWe collected benign breast tissue of 125 women who had been stratified according to age at first pregnancy and the occurrence of subsequent breast cancer, and performed immunohistochemistry for PR, Wnt4 and the Wnt-target Versican.ResultsThe number of PR positive epithelial cells was significantly lower in the group of women with early pregnancy and no subsequent breast cancer compared to the group of nulliparous women with subsequent invasive breast cancer (p = 0.0135). In women with early pregnancy, expression of Versican and Wnt4 was significantly lower compared to nulliparous women (p = 0.0036 and p = 0.0241 respectively), and Versican expression was also significant lower compared to women with late pregnancy (p < 0.0001).DiscussionOur results confirm prior observations in mice and suggest a role of downregulation of epithelial Wnt signaling in the protective effect of early pregnancy in humans. This results in a decreased proliferation of stem/progenitor cells; therefore, the Wnt signaling pathway may represent a potential target for breast cancer prevention in humans.

Parity and Short-Term Estradiol Treatment Utilizes Similar Cellular Mechanisms to Confer Protection Against Breast Cancer

Background: Protective effect of early pregnancy and short-term estrogen treatment (STET), against breast cancer is well established. The underlying mechanisms are not well understood. In this study, we compared the mammary gland cellular microenvironment influenced/induced by parity and STET alongside age-matched controls. Methods: Parous, STET, and control rats were injected with N-methyl-N-nitrosourea at 15 weeks and monitored for the development of mammary cancer. A subset of 4 rats were killed five weeks post carcinogen treatment and mammary gland samples were isolated and subjected to molecular analysis. Results: Our results demonstrated a reduction in cell survival, extracellular matrix associated proliferation, hormonal and growth factor receptor pathways in the experimental groups compared to control rats. Moreover, concomitant reductions in the EMT markers along with cell migration regulators were also observed in parous and STET groups. Hormonal receptor such as GHR, PR, ERα and growth factor receptors IGFR, EGFR and erbB2 were down regulated in the treatment groups. Further analysis revealed that parity and STET drastically reduced the expression, activation of JAK2 and nuclear localization of STATs. Conclusion: Parity and STET by targeting major cell signaling pathways involved in cell survival, cell migration and cell death reduces the mammary tumor promoting environment.

Abstract P1-08-01: Short-Term Exposure to Pregnancy Levels of Estrogen Alters Key Molecular Pathways Involved in Breast Cancer Development

Abstract Breast cancer is the most common cancer in women worldwide and attempts at prevention of breast cancer are important areas of clinical and experimental investigations. Interestingly, a full-term pregnancy before the age of twenty is the only natural phenomenon known that can drastically reduce the risk of breast cancer in women. The universal protective effect of early pregnancy is clearly a major consideration in devising breast cancer prevention strategies. Rats that have been exposed to carcinogens before or after undergoing a full-term pregnancy are protected from mammary carcinogenesis. Hormonal prevention strategies have used exogenous hormonal treatment to mimic the protective effect of early full-term pregnancy against breast cancer. We have demonstrated that short-term treatment with pregnancy levels of estradiol with or without progesterone is highly effective in conferring protection against mammary carcinogenesis in rats. In order to understand the molecular mechanism of the protective effect of short-term pregnancy levels of estradiol treatment we have performed pathway focused microarrays. Nine week old female Lewis rats were divided into three groups, each consisting of 15 rats and receiving one of the following treatments: (i) control, (ii)10 microgram estradiol (non-protective dose), and (iii) 200 microgram estradiol (protective dose). Each treatment was given in the form of silastic capsule and continued for the length of gestation (3 weeks) in rats. At the end of the treatment, the silastic capsule was removed. The rats were then terminated, 8 and 16 weeks after removal of the hormone treatment. Mammary glands were removed, immediately snap frozen in liquid nitrogen and stored at −80°C. Total RNA and protein were extracted and used for gene and protein expression analyses. Our data indicate that several proteins and genes which are involved in apoptosis, DNA repair and growth inhibition (Bax, Bclaf1, Cidea, Fas, Tnfsf10) are upregulated in the mammary glands of rats that received pregnancy levels of estradiol treatment compared to the rats that received non-pregnancy levels of estradiol treatment and untreated controls. By contrast, there were consistent decreasing levels of proteins and genes involved in growth regulation, angiogenesis, anti-apoptosis and cell cycle regulation (Dad1 Faim, Naip2, Xiap) in the mammary glands of pregnancy level estradiol treated rats when compared to the non pregnancy level estradiol treated rats and untreated controls. These alterations were similar at both the time points after withdrawal of treatments indicating that these changes are constitutive. In conclusion, short-term treatment with pregnancy levels of estradiol resulted in persistent down-regulation of growth promotion, cell cycle, anti-apoptosis, angiogenesis and oncogenesis. On the other hand, the same protective treatment persistently enhanced growth inhibition, apoptosis and DNA repair. The data obtained could be helpful to understand the mechanisms involved in short-term pregnancy level estradiol-induced protection and will also facilitate the identification of biomarkers associated with protection. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-08-01.

Abstract P2-06-14: Utilizing Human Breast Tissue and Blood to Discern Novel Protective Properties of Early Pregnancy in Reducing Breast Cancer Risk

Abstract Introduction: The incidence of breast cancer, the most common cancer in women, is very high in Western Countries and is increasing rapidly in the Third World. Yet, despite better screening, earlier diagnosis, and the development of new therapies, a cure for metastatic breast cancers remains an elusive goal. Since it is difficult to defeat breast cancer after it has occurred, finding new strategies to delay or prevent the development of breast cancer has been an important area for clinical and experimental investigations. Multinational studies have suggested that women who undergo a full-term pregnancy before the age of 20, with or without lactation, have about one half the risk of developing breast cancer compared to women who undergo their first pregnancy after the age of 35 or women who have never undergone a full term pregnancy. Thus, pregnancy at an early age protects against breast cancer development. This universal protective effect of pregnancy serves as a major clue and starting point in the search for new experimental strategies and novel biomarkers related to the prevention of breast cancer. Methods: Women were recruited who had never been diagnosed with breast cancer and fit in one of the following categories; 1) had their first child ≥25 years of age, 2) had their first child ≥35 years of age, and 3) a control group of women the same age as those in groups 1 and 2 but who have never given birth were recruited and provided a sample of breast tissue and blood simultaneously. These groups were categorized as early parous, late parous, or nulliparous and their RNA was isolated from tissue and whole blood. Human whole genome microarray analysis (Agilent) was performed to identify the genes that were persistently altered in expression due to early pregnancy. Results: The study of molecular alterations in breast cancer genes has succeeded in identifying some genetic mechanism of breast cancer development. We attempted to elucidate early pregnancy-related changes in genes in breast tissue and blood associated with resistance to breast cancer. Over 2200 genes were up-regulated 2 folds or more in the blood and breast of early parous women than late parous and nulliparous women. Approximately 5000 genes were down-regulated 2 folds or more in the blood and breast of early parous women compared to late parous and nulliparous women. Conclusion: These studies focused on the analysis of human breast tissue and blood to gain a deeper understanding of changes in parity associated with the protective effect of early pregnancy in women. From these altered gene expressions we have identified a gene signature for early parity. The persistently altered genes will be used as novel biomarkers to predict breast cancer risk and develop strategies to protect the breast from cancer development. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-14.

Activation of Stat5 and induction of a pregnancy‐like mammary gland differentiation by eicosapentaenoic and docosapentaenoic omega‐3 fatty acids

The protective effect of early pregnancy against breast cancer can be attributed to the transition from undifferentiated cells in the nulliparous to the differentiated mature cells during pregnancy. Considerable evidence suggests strongly that the n-3 polyunsaturated fatty acid (PUFA) content of adipose breast tissue is inversely associated with an increased risk of breast cancer. Here, we report that there was a decrease in the n-6/n-3 PUFA ratio and a significant increase in concentration of n-3 PUFA docosapentaenoic acid and eicosapentaenoic acid in the pregnant gland. The functional role of n-3 PUFAs on differentiation was supported by the studies in the fat-1 transgenic mouse, which converts endogenous n-6 to n-3 PUFAs. Alternation of the n-6/n-3 ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid, in the mammary gland of fat-1 mouse resulted in development of lobulo-alveolar-like structure and milk protein beta-casein expression, mimicking the differentiated state of the pregnant gland. Docosapentaenoic acid and eicosapentaenoic acid activated the Jak2/Stat5 signaling pathway and induced a functional differentiation with production of beta-casein. Expression of brain type fatty acid binding protein brain type fatty acid binding protein in virgin transgenic mice also resulted in a reduced ratio of n-6/n-3 PUFA, a robust increase in docosapentaenoic acid accumulation, and mammary differentiation. These data indicate a role of mammary derived growth inhibitor related gene for preferential accumulation of n-3 docosapentaenoic acid and eicosapentaenoic acid in the differentiated gland during pregnancy. Thus, alternation of n-6/n-3 fatty acid compositional ratio in favor of n-3 PUFA, and particularly docosapentaenoic acid and eicosapentaenoic acid, is one of the underlying mechanisms of pregnancy-induced mammary differentiation.

Tissue involution and the acute phase response.

After their roles in reproduction are completed, the mass of the uterus and the mammary gland decrease rapidly by the process of involution that involves an ordered series of events including apoptosis, neutrophil entry, the release of degradative enzymes, and phagocytosis of cellular debris. The acute phase proteins are produced by the liver and other tissues in response to inflammation or a toxic challenge. Uterocalin (SIP24/24p3) is one of these proteins. During involution, the mammary gland and uterus express high levels of uterocalin that reach an average of 0.2-0.5% of the total extractable protein at its peak. Uterocalin and its orthologues have been demonstrated in vitro to (1). bind certain fatty acids and (2). specifically induce apoptosis in neutrophils and other leukocytes. The period of uterocalin expression during involution is consistent with the hypothesis that one of its physiological roles is to induce apoptosis of invading neutrophils and delay the entry of neutrophils into the tissue until the second phase of involution. Interestingly, it has been shown that uterocalin expression remains higher in primiparous gland than in virgin glands after the pregnant glands have completely involuted. This observation and the known protective effect of early pregnancy on later development of breast cancer suggest that the ability of uterocalin to induce apoptosis in neutrophils might also decrease oxidative and carcinogenic activity in the gland and result in a lower mutation rate and thus a lower probability of cancer in the primiparous gland.

HORMONAL PROFILES IN WOMEN WITH BREAST CANCER

The literature findings on endogenous hormonal profiles in women with breast cancer are reviewed in detail. It is concluded that four sets of findings are valid: (1) diminished adrenal androgen production, probably genetic, in women with premenopausal breast cancer; (2) ovarian dysfunction (luteal inadequacy plus increased testosterone production) in breast cancer at all ages; (3) increased 16 alpha-hydroxylation of estradiol in breast cancer at all ages; and (4) evidence that prolactin is a permissive risk factor for breast cancer, and that the pregnancy-induced decrease in prolactin levels may account for the protective effect of early pregnancy against breast cancer.

Epidemiologic features of breast cancer in Slovenia, 1965–1967

As part of an international collaborative investigation, the epidemiologic characteristics of breast cancer in Slovenia have been studied. An attempt was made to interview all cases of the disease newly diagnosed during a period of 39 months. For comparison purposes, hospital patients with other diseases were interviewed. A total of 806 eligible breast cancer cases were ascertained and 772 were interviewed. Two thousand three hundred and thirty-two control patients were selected and 2308 interviewed. The incidence of ascertained cases was quite similar to that obtained from the data of the Cancer Registry of Slovenia, the rates being approximately twice as high as those in Japan and other Asian populations, but substantially lower than those in North America and Northern Europe. In regard to both the overall level of incidence and the characteristic shape of the age-incidence curve, the data from Slovenia were similar to those from Athens, Creece — the other Southern European center in the international study. There were no significant differences between cases and controls in socio-economic status, number of fetal deaths and frequency or duration of lactation. Even though some patients reported unusually prolonged lactation experience these were equally frequent among the cases as among the controls. Women first pregnant under the age of 25 had approximately 70% of the breast cancer risk of those whose first pregnancy was delayed until age 30 or older. While this trend is in the same direction as that seen in some other centers, the protective effect of early pregnancy seems substantially less in Slovenia than in the other areas studied. There was a significant association between early menarche and increased breast cancer risk. Median ages at menarche were quite late both in the cases and in the controls, similar in fact to those reported in the counterpart study in Japan. There was also an indication that late menopause was associated with increased breast cancer risk. The breast cancer patients were taller and heavier than the controls.