Leena Hilakivi-Clarke

Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland.

It has been suggested that environmental contaminants that mimic the effects of estrogen contribute to disruption of the reproductive systems of animals in the wild, and to the high incidence of hormone-related cancers and diseases in Western populations. Previous studies have shown that functionally, cadmium acts like steroidal estrogens in breast cancer cells as a result of its ability to form a high-affinity complex with the hormone binding domain of the estrogen receptor. The results of the present study show that cadmium also has potent estrogen-like activity in vivo. Exposure to cadmium increased uterine wet weight, promoted growth and development of the mammary glands and induced hormone-regulated genes in ovariectomized animals. In the uterus, the increase in wet weight was accompanied by proliferation of the endometrium and induction of progesterone receptor (PgR) and complement component C3. In the mammary gland, cadmium promoted an increase in the formation of side branches and alveolar buds and the induction of casein, whey acidic protein, PgR and C3. In utero exposure to the metal also mimicked the effects of estrogens. Female offspring experienced an earlier onset of puberty and an increase in the epithelial area and the number of terminal end buds in the mammary gland.

Antiestrogen resistance in breast cancer and the role of estrogen receptor signaling.

Antiestrogens include agents such as tamoxifen, toremifene, raloxifene, and fulvestrant. Currently, tamoxifen is the only drug approved for use in breast cancer chemoprevention, and it remains the treatment of choice for most women with hormone receptor positive, invasive breast carcinoma. While antiestrogens have been available since the early 1970s, we still do not fully understand their mechanisms of action and resistance. Essentially, two forms of antiestrogen resistance occur: de novo resistance and acquired resistance. Absence of estrogen receptor (ER) expression is the most common de novo resistance mechanism, whereas a complete loss of ER expression is not common in acquired resistance. Antiestrogen unresponsiveness appears to be the major acquired resistance phenotype, with a switch to an antiestrogen-stimulated growth being a minor phenotype. Since antiestrogens compete with estrogens for binding to ER, clinical response to antiestrogens may be affected by exogenous estrogenic exposures. Such exposures include estrogenic hormone replacement therapies and dietary and environmental exposures that directly or indirectly increase a tumors estrogenic environment. Whether antiestrogen resistance can be conferred by a switch from predominantly ERα to ERβ expression remains unanswered, but predicting response to antiestrogen therapy requires only measurement of ERα expression. The role of altered receptor coactivator or corepressor expression in antiestrogen resistance also is unclear, and understanding their roles may be confounded by their ubiquitous expression and functional redundancy. We have proposed a gene network approach to exploring the mechanistic aspects of antiestrogen resistance. Using transcriptome and proteome analyses, we have begun to identify candidate genes that comprise one component of a larger, putative gene network. These candidate genes include NFκB, interferon regulatory factor-1, nucleophosmin, and the X-box binding protein-1. The network also may involve signaling through ras and MAPK, implicating crosstalk with growth factors and cytokines. Ultimately, signaling affects the expression/function of the proliferation and/or apoptotic machineries.

Preventing excessive weight gain during pregnancy - a controlled trial in primary health care.

Objective:To investigate whether individual counselling on diet and physical activity during pregnancy can have positive effects on diet and leisure time physical activity (LTPA) and prevent excessive gestational weight gain.Design:A controlled trial.Setting:Six maternity clinics in primary health care in Finland. The clinics were selected into three intervention and three control clinics.Subjects:Of the 132 pregnant primiparas, recruited by 15 public health nurses (PHN), 105 completed the study.Interventions:The intervention included individual counselling on diet and LTPA during five routine visits to a PHN until 37 weeks’ gestation; the controls received the standard maternity care.Results:The counselling did not affect the proportion of primiparas exceeding the weight gain recommendations or total LTPA when adjusted for confounders. The adjusted proportion of high-fibre bread of the total weekly amount of bread decreased more in the control group than in the intervention group (difference 11.8%-units, 95% confidence interval (CI) 0.6–23.1, P=0.04). The adjusted intake of vegetables, fruit and berries increased by 0.8 portions/day (95% CI 0.3–1.4, P=0.004) and dietary fibre by 3.6 g/day (95% CI 1.0–6.1, P=0.007) more in the intervention group than in the control group. There were no high birth weight babies (⩾4000 g) in the intervention group, but eight (15%) of them in the control group (P=0.006).Conclusions:The counselling helped pregnant women to maintain the proportion of high-fibre bread and to increase vegetable, fruit and fibre intakes, but was unable to prevent excessive gestational weight gain.

Prepubertal exposure to zearalenone or genistein reduces mammary tumorigenesis.

SummaryPrepubertal exposure to a pharmacological dose (500 mg kg–1) of the phyto-oestrogen genistein can reduce the incidence and multiplicity of carcinogen-induced mammary tumours in rats. However, such an exposure also disrupts the function of the hypothalamic–pituitary–gonadal axis, making it unsuitable for breast cancer prevention. We studied whether prepubertal exposure to genistein at a total body dose broadly comparable to the level typical of Oriental countries, approximately 1 mg kg–1 body weight, affects mammary tumorigenesis. We also studied whether prepubertal exposure to zearalenone, a major source for phyto-oestrogens in the USA, influences breast cancer risk. Prepubertal rats were treated between postnatal days 7 and 20, with 20 μg (~ 1 mg kg–1 body weight) of either genistein or zearalenone. Zearalenone exposure significantly reduced both the incidence and multiplicity of mammary tumours induced by 7,12-dimethylbenz(a)anthracene (DMBA). Genistein exposure significantly reduced tumour multiplicity, but not tumour incidence, when compared with vehicle-treated animals. Furthermore, 60% of the tumours in the genistein group were not malignant, while all the tumours analysed for histopathology in the vehicle and zearalenone groups were adenocarcinomas. A higher number of differentiated alveolar buds, and lower number of terminal ducts, were present in the DMBA-treated mammary glands of the phyto-oestrogen exposed rats. The concentration of oestrogen receptor (ER) binding sites after the DMBA treatment was low in the mammary glands of all groups but a significantly higher proportion of the glands in the zearalenone exposed rats were ER-positive (i.e. ER levels ≥ 5 fmol mg–1 protein) than the glands of the vehicle controls. Our data suggest that a prepubertal exposure to a low dose of either zearalenone or genistein may protect the mammary gland from carcinogen-induced malignant transformation, possibly by increasing differentiation of the mammary epithelial tree.

The role of early life genistein exposures in modifying breast cancer risk

Review of the existing literature suggests that consumption of soy foods or an exposure to a soy isoflavone genistein during childhood and adolescence in women, and before puberty onset in animals, reduces later mammary cancer risk. In animal studies, an exposure that is limited to the fetal period or adult life does not appear to have the same protective effect. A meta-analysis of human studies indicates a modest reduction in pre- and postmenopausal risk when dietary intakes are assessed during adult life. These findings concur with emerging evidence indicating that timing may be vitally important in determining the effects of various dietary exposures on the susceptibility to develop breast cancer. In this review, we address the mechanisms that might mediate the effects of an early life exposure to genistein on the mammary gland. The focus is on changes in gene expression, such as those involving BRCA1 and PTEN. It will be debated whether mammary stem cells are the targets of genistein-induced alterations and also whether the alterations are epigenetic. We propose that the effects on mammary gland morphology and signalling pathways induced by pubertal exposure to genistein mimic those induced by the oestrogenic environment of early first pregnancy.

Tallness and overweight during childhood have opposing effects on breast cancer risk

Using birth and school health records we studied how weight and height during childhood affect breast cancer risk among 3447 women born during 1924–33 at the University Hospital of Helsinki, Finland. Through linkages with the National Hospital Discharge Registry and the Cause of Death Registry we identified177 women who during 1971–1995 had been admitted to hospital with breast cancer, of whom 49 had died from the disease. Of these, 135 (76%) were aged 50 years or more at the time of diagnosis, and therefore likely to have been post-menopausal. Hazard ratios for breast cancer rose with increasing weight and length at birth, though neither trend was statistically significant. At each age, from 7 to 15 years, the girls who later developed breast cancer were on average taller and had lower body mass than the other girls. Unadjusted hazard ratios rose across the range of height (P = 0.01 at age 7 years) and fell across the range of body mass index (P = 0.009 at age 7 years). In a simultaneous analysis the hazard ratio for breast cancer was 1.27 (95% CI 0.97–1.78, P = 0.08) for every kilogram increase in birth weight and 1.21 (95% CI 1.06–1.38, P = 0.004) for every kg/m2 decrease in body mass index at 7. Our findings indicate that tallness in childhood is associated with increased risk of developing breast cancer. One possible explanation is persisting high plasma concentrations of insulin-like growth factors in talll women. In contrast, we found that being overweight in childhood reduces breast cancer risk. The increased adipose tissue-derived oestrogen levels in overweight children could induce early breast differentiation and eliminate some targets for malignant transformation.

Effects of early postnatal handling on brain β-adrenoceptors and behavior in tests related to stress

The present study investigated the effects of early postnatal handling and temporary maternal isolation, between the 5th and 20th postnatal days, on various behaviors related to stress measured in adulthood in male Wistar rats. In addition, beta-adrenoceptor binding in the brain was measured. The handling consisted of daily 3-min sessions during which a pup was gently held by an investigator. The isolated rat pups were kept separated from their nursing mothers for 1 h daily. Subsequently, it was found that the time spent immobile in Porsolts swim test was shortened, and voluntary alcohol (5% v/v) consumption was reduced in the handled rats, as compared with the non-handled and isolated animals. No differences in the measure of anxiety--food consumed in a novel environment--or the time spent in social, aggressive and defensive behaviors in a resident-intruder paradigm, were noted. Neither did the density or affinity of beta-adrenoceptors in the frontal cortex or hippocampus differ significantly between the groups. The results indicate that short-lasting maternal separation does not cause sustained effects on behavior in the rat. Early postnatal handling leads to shortened immobility in the swim test and reduced voluntary alcohol consumption, suggesting that handled rats show an improved ability to cope with stress.

Psychosocial factors in the development and progression of breast cancer

The factors responsible for the genesis of breast cancer remain unclear. Emerging, although controversial, evidence suggests that factors related to life-style, such as dietary fat or alcohol intake, or exposure to various forms of stressors, are associated with mammary tumorigenesis. The possible role of life-style factors in breast cancer is important in light of the fact that mortality to this disease is increasing in most countries and that development of curative therapies for breast cancer has not been forthcoming. Thus, determining the role of life-style factors in the onset and progression of breast cancer, particularly among individuals genetically vulnerable to breast cancer or women with breast cancer in remission, is critical to prevent this disease. We will review the three main hypotheses which have been suggested to link psychosocial factors to the etiology of cancer, emphasizing data obtained through animal models. Interpretation of the existing data suggests that the number of stressful life-events does not predict vulnerability to develop breast cancer or survival from it; a certain level of stress appears to protect from malignancies. The crucial factor affecting tumor growth is the interaction among stress, an individuals personality, and available psychosocial support, and the effect of this interaction on an individuals ability to cope with stress. In addition, other risk factors for breast cancer known to be closely associated with psychosocial factors, namely dietary fat and alcohol consumption, may interact with the effects of psychosocial factors on breast cancer.

Fetal origins of breast cancer

Susceptibility to breast cancer might be pre-determined in utero. Alterations in the fetal hormonal environment, caused by either maternal diet or exposure to environmental factors with endocrine activities, can modify the epigenome, and these modifications are inherited in somatic daughter cells and maintained throughout life. These epigenetic modifications might lead to changes in mammary gland development, such as increased vulnerability of epithelial targets for malignant transformation. According to this hypothesis, on post-pubertal exposure to an initiating factor, such as a carcinogen, high levels of hormones and radiation, the mammary epithelial targets, perhaps stem cells, in terminal end buds/terminal ductal lobular units would be at an increased risk of malignant transformation. The increased susceptibility for cancer initiation might result from high levels of cell proliferation, reduced apoptosis and/or altered stromal regulation. Thus, maternal diet and environmental exposure might increase the risk of breast cancer by inducing permanent epigenetic changes in the fetus that alter the susceptibility to factors that can initiate breast cancer. Identifying the epigenetically altered target genes and their ligands might lead to strategies to prevent this disease in some women.

Glucose-regulated protein 78 controls cross-talk between apoptosis and autophagy to determine antiestrogen responsiveness.

While more than 70% of breast cancers express estrogen receptor-α (ER+), endocrine therapies targeting these receptors often fail. The molecular mechanisms that underlie treatment resistance remain unclear. We investigated the potential role of glucose-regulated protein 78 (GRP78) in mediating estrogen resistance. Human breast tumors showed increased GRP78 expression when compared with normal breast tissues. However, GRP78 expression was reduced in ER+ breast tumors compared with HER2-amplifed or triple-negative breast tumors. ER+ antiestrogen-resistant cells and ER+ tumors with an acquired resistant antiestrogen phenotype were both shown to overexpress GRP78, which was not observed in cases of de novo resistance. Knockdown of GRP78 restored antiestrogen sensitivity in resistant cells, and overexpression of GRP78 promoted resistance in sensitive cells. Mechanistically, GRP78 integrated multiple cellular signaling pathways to inhibit apoptosis and stimulate prosurvival autophagy, which was dependent on TSC2/AMPK-mediated mTOR inhibition but not on beclin-1. Inhibition of autophagy prevented GRP78-mediated endocrine resistance, whereas caspase inhibition abrogated the resensitization that resulted from GRP78 loss. Simultaneous knockdown of GRP78 and beclin-1 synergistically restored antiestrogen sensitivity in resistant cells. Together, our findings reveal a novel role for GRP78 in the integration of cellular signaling pathways including the unfolded protein response, apoptosis, and autophagy to determine cell fate in response to antiestrogen therapy.