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Var det icke en försmak av ogråtna tårar som liksom en eld på din tunga brann.

när över vägar du aldrig gått en blodröd sol försvann?

Edith Södergran

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List of papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

. Hallberg G, Persson I, Naessén T, Magnusson C. Effects of pre- and postmenopausal use of exogenous hormones on receptor content in normal human breast tissue: A randomized study. Gynecological Endocrinology, 2008; 24(8):475-80

. Hallberg G, Andersson E, Naessén T, Ekman-Ordeberg G. The expression

of syndecan -1, syndecan-4 and decorin in the healthy human breast tissue during the menstrual cycle. Reprod Biol Endocrinol 2010 Apr 16;8:35

III. Hallberg G, Andersson E, Naessén T, Ekman-Ordeberg G. The effect of

short-term oral contraceptive use on the expression of syndecan-1, syndecan-4, decorin and the androgen receptor in breast tissue from women of fertile age. Submitted for publication

IV. Hallberg G, Lundström E, Andersson E, Ekman-Ordeberg G/ Naessén T.

Mammographic breast density and the expression of androgen receptor, caspase 3, Ki67 and proteoglycans in pre-menopausal women. In manuscript

Reprints were made with permission from the respective publishers.

Contents

List of papers .....................................................................................................5

List of abbreviations..........................................................................................9

Introduction .....................................................................................................11 Breast Development and Anatomy .............................................................12

Development and anatomy of the breast ................................................... 12 Risk factors for breast cancer......................................................................15 Mammographic breast density....................................................................17

Methods of evaluating density in the human breast by mammography... 18 Proliferation and apoptosis .........................................................................19

Proliferation................................................................................................ 19 Apoptosis.................................................................................................... 19 Proliferation/apoptosis ............................................................................... 20 Methods of assessing proliferation and apoptosis..................................... 20 Proliferation – Ki67 ................................................................................... 20 Methods of measuring apoptosis ............................................................... 20 Caspase....................................................................................................... 21

Sex steroid receptors and the breast............................................................21 Estrogen receptors...................................................................................... 22 Estrogen receptor content and the risk of breast cancer............................ 22 Progesterone and the PR ............................................................................ 23 Androgen and the Androgen Receptor (AR) ............................................ 23

Extracellular matrix ....................................................................................24 Interactions between epithelium and stroma...............................................25 Proteoglycans..............................................................................................26

Heparan sulphate proteoglycan (HSPG) ................................................... 26 Syndecans................................................................................................... 26 Syndecan-1 ................................................................................................. 27 Syndecan 4 ................................................................................................. 28 Small leucine-rich repeat proteoglycan (SLRP)........................................ 28 Decorin ....................................................................................................... 28

Proteoglycans and breast cancer .................................................................29 Inflammation and the mammary gland: involution ....................................29

Aims of the studies ..........................................................................................31 General aim.................................................................................................31 Specific aims...............................................................................................31

Materials and methods.....................................................................................32

Study design................................................................................................32 Participating women ...................................................................................32 Randomization and interventions ...............................................................33 Exogenous hormones given in the study ....................................................34 Sampling of breast tissue ............................................................................34

Core Needle biopsy.................................................................................... 34 Breast reduction plastic surgery................................................................. 34

Mammography and mammographic assessments.......................................35 Tissue preparation and the enzyme immunoassay (EIA) ...........................35 Immunohistochemistry (IHC).....................................................................35 Figure 5.......................................................................................................37 Tissue homogenization and extraction of RNA..........................................37 Reverse Transcription (RT) ........................................................................37 Real-Time PCR...........................................................................................38

Calculations for real-time RT-PCR ........................................................... 38 Serum analysis ............................................................................................38 Statistical analysis.......................................................................................39

Study I ........................................................................................................ 39 Study II ....................................................................................................... 39 Study III...................................................................................................... 39 Study IV ..................................................................................................... 39

Ethics ..........................................................................................................40

Results .............................................................................................................41 Participating women in study I – IV and flow-chart...................................41 Findings ......................................................................................................43

Study I ........................................................................................................ 43 Study II ....................................................................................................... 43 Study III...................................................................................................... 44 Difference in gene expression between the follicular phase and after oral contraceptives (OC) visualized in box-plots. ............................ 45 Study IV ..................................................................................................... 47

Discussion .......................................................................................................48 General discussion ......................................................................................48 Study limitations.........................................................................................53 Future perspectives .....................................................................................54

General conclusions ........................................................................................55

Sammanfattning på svenska ............................................................................56

Acknowledgements .........................................................................................58

References .......................................................................................................60

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List of abbreviations

BD mammographic breast density BMI body mass index cDNA complementary deoxyribonucleic acid E2 estradiol ECM extracellular matrix EGF epidermal growth factor EIA enzyme immunoassay ER estrogen receptor EPT estrogen progestogen therapy ET estrogen therapy FGF fibroblast growth factor FSH follicle-stimulating hormone GAG glucosaminoglycans HRT hormone replacement therapy HT hormone therapy HSPG heparan sulphate proteoglycan IHC immunohistochemistry MMP matrix metalloproteinase MPA medroxyprogesterone acetate mRNA messenger ribonucleic acid OC oral contraceptives PG proteoglycan PR progesterone receptor RR relative risk rs Spearman rank correlation coefficient RT-PCR reverse-transcription polymerase chain reaction SHBG sex hormone-binding globulin SLRP small leucine-rich repeat proteoglycan

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Introduction

Breast cancer is the most common invasive cancer among women, with a higher incidence in North America, Western Europe and Australia than in Africa and south-east Asia (1). However, with the worldwide changes in economic development and reproductive patterns, and with more global western life-style changes, the incidence is now increasing rapidly in formerly low-incidence countries (2). In Sweden, 7380 women were diagnosed with breast cancer in 2009, representing 29 % of all cancers in Swedish women (National Board of Health and Welfare). The relative 5-year survival rate is 86% and the relative 10-year survival rate is 76% (NBHW). Death from breast cancer comprises only 3 % of all deaths from cancer in Sweden. The age-specific incidence of breast cancer has changed; previously, there was a plateau at 45-59 years but, by the year 2005, the incidence had continued to increase up to the age of 70.

The overall incidence of breast cancer has increased in Sweden by 1.2% annually during the last 20 years but has slowed in the last 10 years, with an annual increase of 0.8% (National Board of Health and Welfare). After the first Women´s Health Initiative (WHI) report in 2002, there was a sharp decline in both the use of postmenopausal hormone therapy (HT) and the incidence of breast cancer among 50- to 59-year-old postmenopausal women in western countries (3). According to Lambe et al., this is an indication that hormone replacement therapy (HRT) is a “driver of population rates of breast cancer” (4). Postmenopausal HRT also has positive effects, however, with reduced risk of fractures (5), colon cancer (6), coronary calcifications (7), and coronary heart events (8), and a 40% drop in total mortality during HRT compared to during placebo treatment (9). The biological effects of different endogenous and exogenous hormones on the female breast may be explained by interactions between the glandular and stromal breast tissue. The tissue microenvironment has an impact on the initiation and maintenance of estrogen and progesterone responsiveness as well. The extracellular matrix (ECM) consists of collagens, proteoglycans (PGs), glycoproteins and fibroblasts (10, 11). The epithelium is embedded in the stroma, which makes up more than 80% of the breast volume (12). Breast stroma consists of fat tissue, interlobular dense connective tissue, intralobular loose connective tissue, and blood vessels. The focus of the thesis is to describe the expression of some receptors and markers acting and interacting in the glandular as well as the stromal tissue.

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Breast Development and Anatomy

Development and anatomy of the breast The human breast is continuously developing from intrauterine life to senescence, and is the only organ that is not fully developed at birth. Development occurs mainly in two stages: the developmental and differentiation phases (13, 14). The developmental stage includes the first stages of gland morphogenesis from nipple epithelium to lobule formation. Fifteen percent of human breast tissue in young premenopausal women consists of epithelium; this is gradually replaced by fat tissue later in life, and only 5% of the breast contains epithelium in postmenopausal women (15). The breast stroma is composed of collagen, fibroblasts, endothelial cells, adipocytes, and a macro-molecular network of PGs, and accounts for more than 80% of the breast volume (16). The development and differentiation of the breast are completed by the end of the first pregnancy. The lobuli can be classified as types 1 to 4, where lobule 1 is less differentiated and is present in the immature breast before menarche, see Figure 1. In fact, lobule 1 has the highest proliferative index and a higher concentration of estrogen receptors (ERs) and number of blood vessels per lobular structure than the other types (17, 18). In lobuli types 1 and 2, the epithelial cells are stem cells with a high proliferative capacity and are therefore highly susceptible to malignant transformation. The different lobule types are associated with different types of tumors in the breast (19). Studies of human breast cancer pathogenesis have indicated that the origin of atypical ductal hyperplasia is lobule type 1 tissue. Atypical ductal hyperplasia can evolve to invasive ductal carcinoma. Lobule type 2 is associated with atypical lobular hyperplasia and lobular cancer, while fibroadenomas have been postulated to originate in lobule type 3. Lobule type 3 tissue is representative of the pregnant breast; this type contains 80-100 ducts per lobule. Finally, lobule type 4 is only found during lactation; this type contains maximal parenchymal differentiation (19).

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Figure 1. Differentiation of lobuli in the human breast. Increased differentiation from lobuli 1 to lobuli 3. (Adapted from Russo)

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The mammary gland parenchyma arises from a single epithelial ectodermal bud. The intrauterine development of the breast goes through ten stages. The breast tissue of a newborn baby has eosinophilic cytoplasm and the breast undergoes typical apocrine secretion, which subsides within 3-4 weeks of birth (17). During puberty, both glandular breast tissue and the surrounding stromal tissue grow. Small primary and secondary ducts grow dichotomously to form club-shaped terminal end-buds (TEBs) or terminal ductal lobular units (TDLUs), which give rise to new branches: alveolar buds. Alveolar buds cluster around a terminal duct to form type 1 lobuli. Lobule formation occurs around 1-2 years after the onset of the first menstrual period, but full differentiation is only attained by full term pregnancy (20). Hormones significantly influence breast development, but their effects on parenchymal fluctuations during the menstrual cycle have not yet been fully elucidated. However, the balance of proliferation and apoptosis in the menstrual cycle never really returns to the “starting point” of the preceding cycle; mammary development proceeds with new structures budding in each cycle, until the age of 35 (21).

The ductal system is lined with luminal epithelial cells surrounded by myoepithelial cells. The myoepithelial cells are in contact with the basement membrane. Most human breast tumors are derived from the TDLUs and have the characteristics of luminal epithelial cells. Estradiol (E2) stimulates ductal elongation and progesterone receptor expression induces lobuloalveolar development. In the breast, ER and the progesterone receptor are expressed in the same cells; however, the cells that proliferate are adjacent to these cells (22). In the early stages of breast tumor development, the inverse relationship between the increased expression of ER and proliferation is disturbed (23). Increased proliferation is directly proportional with ER and PR expression levels in the lobular structures; they are at a maximum in the undifferentiated lobuli type 1 and then decrease in lobuli types 2 and 3 (24). The highest percentage of proliferating cells (measured using Ki67 levels) was found in lobuli type 1. In lobuli type 2, this percentage was reduced three-fold and, in lobuli type 3, it was reduced ten-fold. The proliferating cells are found in the epithelium, and only very occasionally in the myoepithelial cells or the intra- and interlobular stroma (14). During pregnancy, development of the female breast occurs in two phases. The first is ductal lengthening and branching at the end of the ductules to change lobuli 2 to lobuli 3. After this, the secretory phase starts: secretory acini differentiate to lobuli 4. During involution, there is regression to lobuli 3 and, because involution continues at 40-50 years of age, lobuli 2 and 1 will also be formed. In nulliparous women, the lobuli will therefore mostly be type 1. By the end of the fifth decade of life, both parous and nulliparous women will have breasts containing some type 1 lobuli, but their susceptibility to carcinogenesis could be different due to parity (20). Microarray techniques have indicated that lobuli types 1 and 3 have significantly different gene expression profiles (25). Russo et al. have

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characterized the appearance of type 1 stem cells in undifferentiated lobuli type 1. Type 1 stem cells are thought to differentiate to type 2 stem cells during early parity. In a new pregnancy, type 2 stem cells become better differentiated, better able to metabolize carcinogens and repair the induced DNA, and less susceptible to carcinogenesis (26). If the shift from stem cell type 1 to stem cell type 2 is not completed, it is thought that breast cancer may be more likely, even if the woman has an early first pregnancy (26). Pregnancy or pregnancy plus lactation cause permanent changes that affect the risk of developing cancer later in life (14).

Risk factors for breast cancer Epidemiological research has revealed many risk factors for and life-style related associations with breast cancer (27, 28). Lifestyle factors, socioeconomic conditions, age and family history all influence the risk of developing breast cancer (29, 30). Environmental factors (31, 32), including dietary factors such as the phytoestrogens, are possible complementary or independent risk factors. There is no family history of the disease in eight of nine women with BC, 5-10 % of BCs are connected with hereditary factors; BRCA-1 and BRCA-2 are two of the susceptible genes (33).

Moderate alcohol intake is associated with an increased risk of breast cancer in both pre- and postmenopausal women (34). The mechanisms by which alcohol can increase the risk of BC are probably related to low folate intake (35) and an increase in bioavailable estrogens. Studies relating the risk of breast cncer with smoking suggest that women who are early smokers, and who smoke for a long time before the first full-term pregnancy have an elevated risk (36). Postmenopausal women who have never smoked may also have an increased risk through passive smoking (37). Butler et al. found that active but not passive smoking had an inverse association with breast density, suggesting that the antiestrogenic (and not the carcinogenic) effects of smoking are reflected by breast cancer (38).

High mammographic BD has been identified as an independent risk factor for development of breast cancer. Women with high BD have an increased risk, with an odds ratio (OR) ranging from 2-6 (39-42). Benign breast disease or atypical hyperplasia increases the risk of breast cancer to between two and five times the average, and a benign breast lump increases the risk with an RR of 1.5-3.0.

Lobular involution is inversely associated with BC risk (43). This association is also present among older women with atypical hyperplasia or a strong family history of BC. When it is combined with BD, lobular involution is associated with an even greater risk of BC (43), although lobular involution and mammographic BD are each independently associated with BC risk. The combination of dense breasts and no lobular involution was associated with a

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higher BC risk than non-dense breasts and complete involution in one study (43).

The occurrence of BC is highly associated with reproductive factors such as early menarche, late menopause, nulliparity and lack of breast-feeding (28, 44). The association between breast cancer and the presence of female sex steroid hormones is convincing. Menarche before the age of 12 is associated with a relative risk (RR) of BC of 1.5. Having the first full term pregnancy before the age of 30 reduces the risk of breast cancer, while having the first full term pregnancy when older than 30 increases the risk. The completion of a pregnancy, however, does not necessarily directly protect against breast cancer; in fact, parous women under 25 years of age have an increased risk that persists for 10-15 years after the birth, although their lifetime risk is reduced (45). The incidence of breast cancer is increased with maternal age >30 years, and the risk remains increased for up to 30-40 years (45) For every child born, the lifetime risk that the mother will develop breast cancer is reduced by 7% (27). Each year of increased maternal age at first birth results in an increased risk of 3-5% for the lifetime risk of breast cancer. After the age of 35, a full-term pregnancy is associated with an even further increased risk of breast cancer (46), with a RR of 3.0 for women having their first delivery after 40 years of age (27). The increased risk and poor prognosis of breast cancer associated with a recent pregnancy is thought to be related to involution of the breast mimicking inflammation and immune suppression, with activated fibroblasts, ECM deposition, and elevated matrix metalloproteinase (MMP) levels, resembling a pro-tumorigenic environment (47). The protective effect of breastfeeding lies in its duration, where every 12 months of breastfeeding reduces the risk. However, lactation is only protective up to 10 years postpartum and has no effect on the lifetime risk (48).

Postmenopausal hormone therapy (HT) with estrogen-progestagen combined therapy (EPT) is an established risk factor, with a duration-dependent risk that is higher during continuous combined therapy than with estrogen treatment only (ET) (49, 50). The results of the extensive studies on hormones can be summarized as follows. Oral contraceptive (OC) use is associated with a slightly increased risk of breast cancer, which is normalized 5-10 years after discontinuation (51). Current users of OCs have a modestly increased RR of 1.2 (27), whereas the risk in postmenopausal women depends on the HRT regimen. For current users of ET, the RR is 0.47-0.59 (52, 53), whereas for current users of EPT the RR is 1.2 -1.7. The WHI, a large randomized controlled study, showed an increased risk of breast cancer associated with EPT [hazard ratio (HR) = 1.24] but not with ET (HR = 0.77) (6). The importance of the type of progestin used in HT has also been emphasized since studies on cell lines indicate that testosterone-like but not progesterone-like progestins may stimulate proliferation (54). The use of bilateral oophorectomy as a way of achieving remission of breast cancer in pre-menopausal women is convincing (55, 56).

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BMI has an impact on cancer risk. In premenopausal women, a high BMI is protective (RR = 0.7) (57); the opposite is noted in postmenopausal women (RR = 2.0), where a high BMI with more adipose tissue, a source of extra-gonadal estrogen, is associated with increased breast cancer risk (2, 58, 59). In postmenopausal women, obesity can increase the amount of bioavailable estrogens as a result of increased synthesis from precursors and decreased production of sex hormone-binding globulin (SHBG). Birth weight and growth during childhood and adolescence also have an impact on the risk of breast cancer (59).

Mammographic breast density High mammographic BD is associated with late first birth, lack of history of breastfeeding and use of HRT and low BD is associated with age, low parity and low BMI (60). The density of the breast is associated with the circulating levels of endogenous steroid hormones, peptide hormones, growth factors and binding proteins (42) and varies with age, parity, height, menopausal status and weight (39), as well as with the relative proportions of connective, epithelial and fatty tissues (61). BD changes in inverse proportion to obesity and age: the more obese and older the woman, the lower the density (62). BD is associated with epithelial cell proliferation and increased stromal proteoglycans (42, 63).

Hereditary factors influence the percentage BD (see the Methods of evaluating density in the human breast by mammography section for the definition of percentage BD) by about 63% (64). Breast tissue is somewhat radiographically dense in the follicular phase of the menstrual cycle (65). Different HTs have different effects on BD; the breast density where a more pronounced greater density is more likely to be associated with combined EPT than with ET (66, 67). The density increases soon after exogenous exposure and is fully developed within a few months (40, 68). Conner et al. found few differences between 19 nor-steroids and 17-OH progesterone compounds, while tibolone with its more androgenic profile had less influence on the density (69). During long-term follow up, mammographic BD tends not to change extensively (69). The addition of testosterone to conventional menopausal hormones does not substantially affect the mammographic BD (70).

More dense regions on mammograms appear to have more stroma and a preponderance of lobuli 1 (71). Harvey et al. found increased fibrous stroma and type 1 lobuli associated with increasing BD in women using HT, independent of estrogen and progesterone up-regulation, indicating that increased BD is mediated through paracrine effects (72). In nonusers of HT, increased BD was associated with a greater numbers of ducts but not with fibrous stroma or lobuli. However, in all users, after adjusting for HT use and

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age, increased BD was associated with fibrous stroma and a greater number of type 1 lobuli. Further, increased Ki 67 in ducts and in type 1 lobuli was associated with increased BD among both users and non–users of HT (72). An increase in fibrous stroma (i.e. an increase in fibroblasts) could directly increase the of aromatase present in the fibroblasts (73). Increased levels of aromatase and increased estrogen synthesis could then stimulate the cells to proliferate or make the stromal cells produce growth factors such as insulin-like growth factor (IGF)-1 or proteoglycans which, via paracrine effects, could then stimulate epithelial cell proliferation (72).

In a multiethnic population, women with higher BD had an increased risk of ER+ PR+ tumors, but not of ER- PR- tumors (74). The results did not differ with ethnicity, menopausal status, parity or HRT use. Percent BD was not a predictor of ER-PR- tumors, although it is a stronger predictor of risk than absolute density (60, 75).

Methods of evaluating density in the human breast by mammography John Wolfe developed a combined qualitative and quantitative method to describe the proportions of parenchyma, fat and fibroglandular tissue in the breast (76). Both Wolfe and the BI-RADS (the Breast Imaging Reporting and Data System, a four category system used mainly in the United States) use visual classifications that are associated with limitations in that an increase in density of at least 20-25% is required for an upgrade of one class of density (63).

There are several different visual percentage scales of mammographic BD. In 2003, BD categories were assigned using the following quantitative criteria: < 25%, 25-49%, 50-75% and > 75% (American College of Radiology 2003). Another visual scale used by Lundström et al. (Karolinska Institute) requires a 20% change in density for each class: 0-20%, 21-40%, 41 - 60%, 61-80% and 81-100% (66). Quantitative assessment of BD is achieved using a computer-assisted planimetry method (77). By lining the breast parenchyma area, the percentage BD was calculated by dividing the number of pixels of dense breast tissue by the total number of pixels in the breast area and multiplying by 100 (78). In our study (paper IV) we used a digitized computer-based assessment (70). In the analysis we used mean breast density which was the mean of left and right breast values and breast density class (< 20% vs. >40%).

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Proliferation and apoptosis

Proliferation The endometrium of the uterus and the epithelium in the breast undergo proliferation during the menstrual cycle; however, the hormonal regulation of breast tissue is different from that in the endometrium. In the breast, only a small fraction (1-10%) of the cells proliferate compared with 80-100% of the glandular cells in the endometrium during the follicular phase of the menstrual cycle (24). Proliferation takes place in an interaction between the epithelium and the stroma in breast tissue. Through the basement membrane, there is a complex process of degradation, synthesis, inflammatory cell influx and remodeling of the extracellular matrix (ECM) (79). During normal cell division, approximately one in a million breast cells undergoes spontaneous mutation (80). Hormonal regulation of proliferation in the normal breast and hormonal risk factors for development of breast cancer have been a controversial issue since in vitro and in vivo studies have yielded diverging results. In the in vitro studies, the cultured breast epithelial cells lack the normal surrounding blood vessels, fat tissue, stroma and myoepithelial cells, which disconnects them from the paracrine and hormonal influence found under in vivo conditions. On the other hand, when cells are collected in vivo by fine needle biopsy, the aspiration could preferentially collect proliferative cells that are detached from the matrix, and therefore cause a bias in the analysis (81).

While there is no controversy concerning the mitogenic effects of estrogen, progesterone has been reported to increase, decrease or have no effect at all on mitotic activity and proliferation in the breast epithelium (82-85). The debate on the actions of progestin on breast tissue continues despite recent data confirming minor cell proliferation from estrogen alone in the follicular phase of the normal menstrual cycle, and increased proliferation after ovulation, under the combined influence of estrogen and progesterone (68, 86). Progesterone counteracs the effects of estrogen in the endometrium; however, in the breast, progesterone enhances the proliferative effect of estrogen (85). Proliferation is the result of teamwork between estrogen, progesterone, prolactin, corticosteroids, growth factors and their receptors, and also involves interactions with fat tissue, connective tissue and the epithelium (87).

Apoptosis Apoptosis or programmed cell death is an active, physiological mode of cell death. Apoptosis is involved in tissue remodeling during embryogenesis as well as the removal of pre-malignant cells exposed to mutagens or ionizing radiation (88). The problem with analyzing apoptosis in in-vivo studies is that

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apoptosis it occurs only during part of the cell cycle, compared with proliferation, which occurs during the whole cell cycle except phase G0.

Proliferation/apoptosis In normal breast tissue, proliferation and apoptosis are influenced by several factors; these include the phase of the menstrual cycle, the woman's chronological age, use of OCs and recent parity (89). Earlier studies of mitosis and apoptosis in the breast during the menstrual cycle have suggested cyclical variations, with a mitotic peak at day 25 and an apoptotic peak at day 28 of the menstrual cycle (82). Regulation of proliferation occurs via three main mechanisms: receptor-mediated (90), autocrine/paracrine loop (91) and negative feedback (92). Navarrete et al. found that the extent of apoptosis did not differ significantly between the follicular and luteal phases of the menstrual cycle, reaching a maximum on day 24 with a smaller peak on day 3 of the cycle. Proliferative activity appears mainly to depend on hormonal fluctuations, whereas apoptotic activity is probably regulated by hormonal and non-hormonal factors (87). In women using HT, progestogen exposure significantly affects the balance between proliferation and apoptosis. The discontinuation of progestogen could, in fact, be a trigger for initiation of the apoptotic pathway (93).

Methods of assessing proliferation and apoptosis

Proliferation – Ki67 The murine monoclonal antibody Ki-67 reacts with a human DNA binding protein that is present in proliferating cells but absent in quiescent cells (94). The Ki-67 antigen is expressed in G1, S, G2 and mitosis but not in G0 in the cell cycle. In immunohistochemistry (IHC), the specific polyclonal MIB-1 antibody is used against the nuclear Ki67 antigen (95). The target antigen is present throughout the cell cycle in proliferating cells. MIB-1 and Ki-67 are the best proliferation markers to use in IHC (96).

Methods of measuring apoptosis The gold standard for measuring apoptosis is light microscopy and the counting of apoptotic cells. Other methods are electrophoresis, flow cytometry and laser scan cytometry. IHC using specific antibodies can recognize DNA strand breaks (TUNEL assay), plasma membrane phospholipids, or endonuclease activating enzymes such as cleaved caspase-3.

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Caspase Caspases, or cysteine-aspartic proteases are enzymes with important roles in cell survival and cell death. They are classified into two groups: the initiator caspases (caspases -2, -8, -9 and -10) and the executioner caspases (caspases -3, -6 and -7). In a complex activation process, the initiator caspases both activate and are a substrate for the executioner caspases. There are two major pathways for the actions of the caspases: the receptor pathway and the mitochondrial pathway.

Figure 2. The mammary gland experience different developmental stages during lifetime. Estrogen and progesterone will play an important role in the change of the structure of the mammary gland. (adapted from Russo & Russo 1986)

Sex steroid receptors and the breast Steroid hormones are small lipophilic compounds that can be classified as estrogens, progestins, androgens, mineralocorticoids or glucocorticoids. Each of these binds to its intracellular receptor to elicit the specific effects, but can also act in non-genomic pathways.

The steroid hormones all belong to the nuclear receptor family and arose relatively early during evolution. They are believed to have originated from a

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common ancestral gene by multiple duplication events; they all share a common three-domain structure. The N-terminal allows strong transactivation functions with many receptors, the central domain binds to DNA and the C-terminal domain binds to the ligand. The nuclear receptor family controls a wide array of processes such as differentiation, development, reproduction homeostasis, and metabolism (97). The receptor-mediated activity requires gene transcription and protein synthesis, with the effects visible in hours or days. In contrast, interactions through the second messenger systems or the plasma membrane receptor are extremely rapid (98).

Estrogen receptors The first ER, named ER , was cloned in 1986 (99). This was followed ten years later by a smaller protein receptor named ERß, first discovered in a rodent model by Kuiper (100). ERß is widespread in many organs of the body, such as the prostate epithelium, urogenital tracts, ovarian follicles, lungs, intestinal epithelium, and certain ER - deficient brain regions and muscle (101).

In normal mammary tissue, only a small fraction of epithelial cells express the ER (22). The concentration of ER is low (0-10 fmol/mg protein) in mammary tissue compared to the concentration in the human uterus (200-300 fmol/mg protein) (102). ERß expression is high in quiescent epithelialum tissue but low in proliferating breast tissue (103) with an elevated ER /ERß ratio in breast cancer tissue. Downregulation of ER during hormonal treatment has been described (104-106) and different progestagens have divergent effects on the two estrogen receptors (107).

Estrogen receptor content and the risk of breast cancer The binding of estrogens to the ER stimulates cell proliferation which, increases the risk of spontaneous mutations and neoplastic transformation (80). Alternatively, direct DNA damage by genotoxic estrogen metabolites could result in point mutations and eventually carcinogenesis (108). In normal breast tissue, a very small percentage of the epithelial cells are receptor-positive (5-15%) and 1-3 % are proliferating during the menstrual cycle (22, 109). The cells that express ER are not those that are proliferating but they are in close proximity (110). This has lead to the conclusion that estrogen stimulates proliferation indirectly through paracrine or juxtacrine factors secreted by ER-positive cells (111). In breast cancer tissue, up to 90% of the epithelial cells can express ER, which suggest a disorganized separation between steroid receptor expression and proliferation (110, 112). Levels of ER in normal breast tissue are low in female populations with a low incidence of breast cancer such as those in Japan (113), while women in Australia have high levels of ER and a high incidence of breast cancer

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(Lawson). It has therefore been proposed that higher ER expression in the normal breast epithelium could increase the breast cancer risk (114, 115). Recently, however, Lagiou et al. found an inverse association between the expression of ER/PR and breast cancer risk in a large case-control study using IHC to compare women with benign breast disease and women with breast cancer. This was statistically significant for the postmenopausal women, but there was no evidence for an association with breast cancer risk for the expression of estrogen alpha or progesterone receptor among the premenopausal women (116).

Progesterone and the PR Progesterone is secreted from the corpus luteum in the ovary during the luteal phase of the menstrual cycle. Two protein isoforms of the PR are known: PRA and PRB. In the breast, PRs are induced by ER activation, whereas ERs are down-regulated by progesterone (117, 118). PRA and PRB are similarly expressed in normal breast tissue, and heterogeneously expressed in atypical hyperplasia and cancer. In a study on breast tissue from the cynomologus macaque, Isaksson et al. found a higher PRA/PRB ratio with conjugated equine estrogens (CEE) combined with medroxyprogesterone acetate (MPA) from that with CEE treatment alone (119). One known action of progesterone in the breast is to induce lobular development (120).

Androgen and the Androgen Receptor (AR) Testosterone is synthesized by the ovaries and the adrenals in nearly equal amounts (121). The mammary gland is capable of synthesizing testosterone as well, since all the necessary enzymes needed have been reported in the normal breast tissue. Testosterone levels vary hourly and in response to stress, exercise, diet and diurnal rhythm. Androgens are converted by aromatization to estrone or E2. Of the circulating testosterone, 75% is bound to SHBG and the rest to albumin. Only the free testosterone (1-2%) will be able to pass the cell membrane, bind to the receptor and exert an effect on the target organs (121). Under estrogen-deprived conditions, testosterone is aromatized to E2 and stimulates growth via the ERα. However, if estrogens are present, androgens can have the opposite effect, inhibiting the growth stimulatory effect of estrogen (122). Postmenopausal women with high endogenous testosterone levels have an increased breast cancer risk (Endogenous Hormone and Breast Cancer Coll group 2002). Androgens exert inhibitory effects on ER- and progesterone receptor PR-negative breast cancer cell lines that express the androgen receptor (123).

The AR is a ligand-activated nuclear transcription factor. The AR is co-localized with ER and PR in the epithelial cells, but not in the stroma or myopeithelial cells. The co-expression of ER and AR in the epithelial cells

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may indicate the effect on the proliferation to be integrated in the epithelial cells. Increased collagen synthesis and pro-inflammatory effects are mediated by the AR (124). The AR can be intracellular or membrane-bound (mAR). The mAR, which triggers non-genomic effects, has been found in human breast cancer cells involved in the activation of apoptosis (125). Some of the genes involved in the androgen-estrogen conversion pathway are associated with breast cancer risk (126). In this group of genes, the mAR has been found not only in hormone-dependent tumors but also in colon tumors. Activation through albumin conjugates induced pro-apoptotic responses in vitro and also reduction of tumor incidence in vivo (127).

Extracellular matrix The ECM has been extensively studied by Bissell and Barcellos-Hoff (128). ECM has been described as “an interconnected meshwork of secreted proteins that interact with cells to form a functional unit” (79). The relationship between the ECM and the epithelial cells is reciprocal and dynamic; the ECM instructs and supports the cells and the same cells build, shape and reshape the ECM (79), se Figure 3.

The ECM exists in several biochemical and structural forms, is the sum of numerous cell types and their secreted products, and has an important role in influencing immune cell behavior (10). In the tumor microenvironment, the synthesis of macromolecules (e.g. proteoglycans) is affected by the cancer cells and the tumor ECM can counteract or facilitate the growth of the tumors (11). This dual activity has an intrinsic tissue specificity involving cell signaling, growth, cell survival, cell adhesion, migration and angiogenesis. One important mechanism is the shedding of the syndecans (proteoglycan) which facilitates the motility of both the cancer and the endothelial cells by protecting important enzymes and promoting proliferation and migration on the stromal cells. These macromolecules in the ECM could probably therefore serve as biomarkers for tumor progression and patient survival, as well as for novel cancer therapy in the future (11).

The contents of the ECM are produced by the sparsely-distributed fibroblasts or myofibroblasts. There are endothelial cells surrounding vessels and inflammatory cells. The ECM of the normal mammary gland is a mixture of collagens, fibrillar glycoproteins and proteoglycans shifting in composition during pre-adult development, estrous cycles, pregnancy and involution (129).

The ECM can be divided into three structures: the basal lamina, the intra- and interlobular stroma, and the fibrous connective tissue (79). The basal membrane delimits the ECM from the epithelial layer. Only 20 % of the luminal epithelial cells are in direct contact with the basal membrane. The rest are adjacent to the myoepithelial cells (130). Collagen I and III are the dominating molecules for strength (131, 132). Hyaluronan is important for the

25

water content of the ECM and the transportation of cells, and is active in the inflammatory response (133). In the normal breast tissue, hyaluronic acid (HA) is present in the ECM of the intralobular regions. Integrins are other proteins that are also in the ECM; these include cell-surface adhesive proteins, growth factors, fibronectin, inflammatory cytokines, and the important protein-degrading enzymes, MMPs and their tissue inhibitors (TIMPs).

Interactions between epithelium and stroma Interactions between the epithelium and the stroma are very important in the normal mammary gland and during neoplastic transformation. Steroid action is partly mediated via binding to specific intranuclear receptors. However, most cells that proliferate in response to estrogens do not contain ERs. Experimental data suggest that estrogenic stimulation and epithelial growth in the mammary gland is a paracrine event mediated via stromal cells (16, 134).

Fibroblast

TGF-

PF4IL4

Collagenfibrils

Integrin

Hyaluronan

TGF-TGF-

IL-8

Syndecan

Hyaluronan

Biglycan

GlypicanFibronectin

Decorin

Versican

FGF

Elastic fibers

Fibroblast

TGF-

PF4IL4

Collagenfibrils

Integrin

Hyaluronan

TGF-TGF-

IL-8

Syndecan

Hyaluronan

Biglycan

GlypicanFibronectin

Decorin

Versican

FGF

Elastic fibers

Extracellular matrix

Figure 3. Extracellular matrix. The space between the stroma and the epithelium with secreted proteins, proteoglycans, collagens that interact with surrounding cells forming a functional unit.

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Proteoglycans The proteoglycans are involved in the inflammatory processes required for normal functioning of the mammary glands (135, 136). Proteoglycans are multireceptor molecules that promote cellular signaling (137-139), and are divided into three main families: the larger hyalectans, the small leucine-rich repeat proteoglycans (SLRPs) and the heparan sulphate proteoglycans (HSPG) (140). Proteoglycans consist of a core protein and one or more glycosaminoglycan (GAG) side chains. GAGs are linear, negatively charged polysaccharides comprised of repeating disaccharide units of uronic acid (or galactose) and an N-substituted hexosamine (glucosamine or galactosamine).

Proteoglycans are also classified according to their location: extracellularly secreted (matrix), cell surface-associated and intracellular proteoglycans (11). Extracellular proteoglycans include the hyalectans, versicans, aggrecans, brevicans, and SLRPs such as decorin, and lumican. The basement membrane proteoglycans include perlecan, agrin and collagen. The cell surface proteoglycans include two subfamilies: the syndecans and the glypicans (11). Many different cell surface and matrix proteoglycan core proteins are expressed in the mammary gland and in mammary cells in culture. The level of expression of these core proteins, the structure of their GAG chains and their degradation are regulated by many of the effectors that control the development and functions of the mammary gland (138). Regulatory proteins of the mammary gland that bind GAG chains include many growth factors and morphogens [fibroblast growth factor (FGF), hepatocyte growth factor (HGF), matrix proteins, collagen, fibronectin, laminin, enzymes, lipoprotein lipase, and microbial surface proteins]. A single proteoglycan can bind multiple proteins and can act as a multireceptor promoting the integration of cellular signals. The SLRP and the HSPG carry a few GAG chains, while the hyalectans can carry up to a hundred.

Heparan sulphate proteoglycan (HSPG) HSPGs are found in most mammalian cells and are essential for normal tissue growth and development. HSPGs include the cell-surface HSPGs syndecan and glypican and the basement membrane HSPG perlecan.

Syndecans The syndecan family has 4 members (syndecan-1, syndecan-2, syndecan-3 and syndecan-4) encoded by four different genes. The word syndecan is derived from the Greek syndein, which means “to bind together” and one function of the syndecans is to mediate cell-cell interactions. The core protein ranges from 20 to 48kD (141). Each member has its own unique tissue distribution. The core proteins have a characteristically variable ectodomain

27

that carries the GAG chains, a trans-membrane domain that passes through the cell membrane, and a cytoplasmic tail. The syndecans bind extracellular proteins and form signaling complexes with receptors, immobilize proteins at the cell surface, and function as co-receptors for many soluble growth factors [e.g. FGF, HGF, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF)].

All syndecans can be shed from the cell surface by proteolytic cleavage near the plasma membrane. The soluble ectodomains can function through shedding as paracrine or autocrine competitors and effectors (141, 142). MMPs can shed the syndecans, but only TIMP-3 can inhibit and block the shedding of syndecan-1 and -4.

Syndecan-1 Syndecan-1, the most studied member of the syndecans, was originally isolated from a mouse mammary epithelial cell line (143, 144) and was identified as a regulated type-1 trans-membrane protein that binds to the ECM components surrounding epithelial cells (145). Syndecan-1 is expressed in epithelia and plasma cells and various developing epithelia such as epidermis and the mammary epithelium (146). Loss or overexpression of syndecan-1 in carcinoma cells has been associated with malignant progression (147, 148). Syndecan-1 is upregulated in human breast cancer and this correlates with poor prognosis (149). Leivonen et al. found that epithelial syndecan-1 expression in breast cancer was associated with ER-negative status while stromal syndecan-1 expression was associated with ER-positive breast cancer. Loss of epithelial syndecan-1 is associated with more favorable BC and concomitant expression in both stroma and epithelium is associated with an unfavorable prognosis (150). Wu et al. demonstrated that the gene expression of syndecan-1 is estrogen dependent (151). In vivo immunocytochemistry has located syndecan-1 in the epithelial cell ducts and at the terminal end buds in mouse breast tissue (152), and it is expressed in the stroma during the induction of epithelia (153, 154). Syndecan- 1 has been shown to modulate growth factor signaling in cell migration, proliferation and adhesion (148, 149). The extracellular syndecan-1 domain can be shed from the cell surface into the extracellular fluid by ectodomain shedding. However, serum syndecan-1 concentrations in healthy individuals are low (155). Shedding of syndecan 1 and syndecan - 4 is regulated by thrombin and EGF family members (156), and MMPs are also involved in the process. The regulated shedding of syndecan-1 (and syndecan-4) suggests a physiological role (re-epithelialization) for the soluble ectodomains (138). Increased syndecan-1 levels leads to increased shedding of syndecan, which inhibits the growth of cancer cell lines through effects on growth factors (153).

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Syndecan 4 Syndecan-4, also named amphiglycan, is the only HSPG located in the cell matrix adhesion sites known as focal adhesions where, along with the integrins, it has an important role (141) (overexpression of syndecan-4 increases focal adhesion formation). Syndecan-4 is found in fibroblasts, and epithelial and smooth muscle cells (157, 158). Experiments with knock-out mice indicate that there are no obvious developmental defects associated with a lack of syndecan - 4 (159). Syndecan-4 is the second most abundant HSPG produced by breast carcinoma cells and, according to Mundhenke, is expressed at high levels in normal human mammary epithelium (160). Syndecan-4 mediates breast cancer cell adhesion, forms complexes with growth factors (FGF-2), and with fibronectin initiates intracellular signaling.

Small leucine-rich repeat proteoglycan (SLRP) The fourteen SLRPs that have been identified are characterized by a protein core with leucine-rich repeats, N-terminal cysteine clusters with C-terminal repeats, and at least one GAG chain (161, 162). They have been classified into 5 groups according to their GAG chains, cysteine rich sequences at the N-terminal ends, and number of exons in the genes. The SLRPs can influence proliferation, differentiation, survival, adhesion migration, and inflammatory responses (163), and can interact with cytokines, ligands and surface receptors. The leucine-rich repeats are particularly relevant for protein-protein interactions (161). They bind to collagen fibrils through the core protein and are located around the tumor microenvironment (11). SLRPS can signal through EGF receptors (EGFR), HGF receptors IGF-1 receptors, and Toll-like receptors.

Decorin Decorin, which is abundant in connective tissue ECM, has a molecular weight of 36kD. Decorin affects a number of biological processes including inflammatory processes, wound healing and angiogenesis through effects on endothelial cell migration, and suppression of endogenous production of the vascular endothelial growth factor (164). It is thought that there is a role for decorin in the cross-linking process but this has not yet been confirmed in vivo (165). Studies indicate that decorin also has more complex interactions, depending on the cell type, that involve different pathways and compensatory mechanisms (166). Decorin modulates the signaling of several receptors involved in cell growth and survival (167). It inhibits the effects of transforming growth factor (TGFβ) and thus prevents fibrosis in mice muscle wounds (168). It interacts directly with EGFR, resulting in activation followed by down-regulation of the receptor (163) with an antiproliferative effect.

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Decorin can also activate caspase 3 (169). Decorin deficiency can result in tumorigenesis (163), and decorin powerfully inhibits tumor cell growth and migration by interacting in tumor stroma deposition and cell signaling pathways. Decorin shows a synergistic effect with carboplatin in inhibiting ovarian cancer cell growth (170).

Proteoglycans and breast cancer A number of studies have indicated substantial differences in the level of expression of particular classes of proteoglycans and GAGs between normal mammary gland tissue and mammary tumor tissue. Syndecan-1 was activated in tumor tissue compared to normal breast tissue, whereas decorin expression decreased in cancer tissue (171). Baba reported over-expression of both syndecan-1 and syndecan-4 in ER-negative breast cancer, with a strong association between HSPG and Ki67 levels (148). Syndecan-1 levels were 10-fold higher in tumor tissue than in normal tissue in postmenopausal women, and had been redistributed from the epithelium to stromal tumor tissue (172). Troup et al. reported reduced expression of SLRPs, associated with poor outcomes, in node-negative invasive breast cancer (173). Injection of decorin protein into mammary carcinoma rodent models resulted in a marked reduction in both primary tumor tissue and metastatic spread compared to control animals (174). Decorin resides in the tumor microenvironment, modulates the signaling of EGFR, and exerts its antitumor activity by inhibition of key receptors through down-regulation (11). In an animal model, decorin interfered with cross talk between the EGFR and the androgen receptor in prostate carcinoma cells, with a reduction in tumor growth (175).

Inflammation and the mammary gland: involution In the mammary gland, when lactation is finished and the involution process starts, the microenvironment shares similarities with the inflammatory process (176). The pro-inflammatory milieu in the breast, although it is physiologically normal, promotes tumor progression. In a study by McDaniel and Schedin on rat mammary glands, ECM from nulliparous and parous rats was exposed to normal epithelial cells (MCF-12A) and a tumor cell line (MDA-MB-231). The nulliparous matrix promoted normal ductal elongation of the normal cells and suppressed invasion of tumor cells in the tumor cell line. The involution matrix in parous rats, however, failed to support the duct development and promoted invasion of the tumor cells (177). When nulliparous and involution matrices were premixed with tumor cells and injected into mammary fat pads, metastasis was higher in the involution matrix group than in the nulliparous matrix group. The composition and

30

function of the ECM seem to facilitate the epithelial proliferation and differentiation that occur during pregnancy, lactation and involution (178, 179). During involution, the mammary gland undergoes massive remodeling with cell death of the secretory epithelium, elevated MMP activity, and high levels of fibrillar collagen and bio-active proteolytic fragments of laminin and fibronectin in the stroma (47). The poor prognosis of pregnancy-associated breast cancer among young women may be a result of the activated stroma, where physiological changes in mammary ECM composition contribute to mammary carcinogenesis (47). Cabodi and Taverna have reported activation of inflammatory cascades resulting in tumor progression as one of three new concepts in breast cancer tumorigenesis. The other two new concepts are tumorigenesis by epigenetic events and tumor growth controlled by cancer stem cell-specific inflammatory stimuli (180).

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Aims of the studies

General aim The overall aim of the study was to examine the effects of endogenous and exogenous sex steroid hormones on the human female breast.

Specific aims I. To examine the effects of endogenous and exogenous hormones on ERα

and PR content in non-cancerous female breast tissue.

II. To compare the gene expression and protein level of decorin, syndecan-1 and syndecan-4 in healthy breast tissue in the follicular and luteal phases of the menstrual cycle.

III. To compare the gene expression and protein level of the proteoglycans

decorin, syndecan-1 and syndecan-4 and the androgen receptor after short-term use of OC with that in the normal menstrual cycle.

IV. To analyze associations between mammographic breast density and

markers of proliferation, apoptosis, the androgen receptor and the studied proteoglycans (syndecan-1, syndecan-4 and decorin), in pre-menopausal women.

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Materials and methods

Study design A randomized study was carried out to investigate the effects of endogenous and exogenous steroid hormones on human non-cancerous breast tissue. Pre- and postmenopausal women on the waiting list for mammoplastic surgery for breast reduction at the Department of Plastic Surgery, University hospital, Uppsala, Sweden, were recruited to take part in the study. The first part of the study was undertaken between 1996 and 1998 in a collaborative work with the Department of Plastic Surgery, the Department of Radiology, and the Department of Women´s and Children´s health at Uppsala University Hospital, and the Fox Chase Cancer Institute (Professor J. Russo, Philadelphia, Penn., USA). The study “restarted” with professor Gunvor Ekman-Ordeberg in 2007, with the aim to study the effects of proteoglycans in the female breast. The breast tissue from the same women included in the first part of the study was examined. The study was approved by the Ethics Committee of the Faculty of Medicine, Uppsala University, Uppsala, Sweden.

Participating women Premenopausal women were eligible if they had regular menstrual periods (28 ±7 days), had not used contraception or any other hormonal treatment during the previous 3 months, were not pregnant, had no history of cardiovascular disease, diabetes, cancer, liver disease or deep-vein thrombosis, and had a normal gynecological examination.

Postmenopausal women were eligible if they had had their last menstrual period more than 6 months ago, had not been using HT for the last 3 months, had no hormone-dependent cancer, diabetes liver disease or deep-vein thrombosis, and had a normal gynecological examination.

After oral and written information and consent, women who met these inclusion criteria were given a face-to-face interview to check their reproductive, menstrual and medical histories, anthropometric measures, alcohol consumption, smoking, use of hormones and family history of breast cancer, and non-eligible women were excluded.

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Randomization and interventions The premenopausal women were divided into two groups according to parity (nulliparous and parous), with ten women from each group scheduled for surgery in the follicular phase, ten in the luteal phase and ten after OC use for two months (30+30).

The postmenopausal women were divided into two groups according to parity (nulliparous and parous). In the beginning of the study it became obvious that none of the postmenopausal nulliparous women on the waiting list showed an interest to join the study or have an operation done, while only parous postmenopausal women were included. Ten women received ET and ten women received EPT for the two months preceding the operation (10+10).

A breast sample (needle biopsy) and a blood sample were taken from all participating women at the same time as the preoperative mammogram, see flow chart.

T he B iom a m stu dy . T he st ud yi nc lud ed 79 w om e n.

R an d om iz ati on

Ma mm o gr ap hy + ne ed le bi op sy

B lo od s am pl es

2 Mo nt hs

O pe ra tio n + ti ssu e sam p lin gB lo od sa m ple s

N ull ipa ro us

n= 23

F oll icu lar p has e n= 8

L ute al p ha se n = 7

F oll icu lar p has e n= 8

P ar ou s

n =3 6

F oll icu lar p has e n= 14

L ute al p ha se n= 10

F oll icu lar p has e n= 12

Lu te al ph ase

F oll icu la rp ha se

O C

Po stm e no pa us al w om en P os tm en op au sal n= 10 P os tm en op au sal n =1 0

Pr em e no pa us alw o me n

O C

O C

Es tra di ol

E st rad io l+M P A

F CL F

F OC

F LL FF OC

The Biomam study. The study included 79 women.

Randomization

Mammography + needle biopsy

Blood samples

2 Months

Operation + tissue samplingBlood samples

Nulliparous

n=23

Follicular phase n=8

Luteal phase n=7

Follicular phase n=8

Parous

n=36

Follicular phase n=14

Luteal phase n=10

Follicular phase n=12

Luteal phase

Follicular phase

OC

Postmenopausal women Postmenopausal n=10 Postmenopausal n=10

Premenopausal women

OC

OC

Estradiol

Estradiol+MPA

FCLFFOC

FLLFFOC

Figure 4. Flow chart for the main study. Blood samples, tissue samples taken at the day of mammography and the day of operation.

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Exogenous hormones given in the study The premenopausal women randomized to OC were given one monophasic OC containing 30 g ethinylestradiol and 150 g levonorgestrel daily for two months. Postmenopausal women were randomized to either ET (estradiol valerate 2 mg daily for two months) or EPT (estradiol valerate 2 mg daily for two months plus MPA 10 mg daily for 14 days of each cycle given sequentially).

Sampling of breast tissue

Core Needle biopsy A pilot study was performed before the BIOMAM randomized study to investigate safety and discomfort issues for the women associated with the core biopsy. In this study, two to five core needle biopsies were taken. The women reported very little discomfort, and bleeding and hematoma were unusual (pers. com. E.Thurfjell). Core needle biopsies allow histological evaluation and examination of mRNA gene expression. The biopsy was taken preferably from the right breast, with an automatic double spring gun “Manan” pro-Mag 2.2 with a 22 mm stroke lens and a 14 Gauge needle (2.1mm: X16 cm with a 17 mm notch U.S. Biopsy division of Promix Inc USA). Needle biopsy samples were frozen at -70° and formalin-fixed.

Breast reduction plastic surgery The reduction mammoplasties were performed according to routine procedures. Premenopausal women were operated on in either the follicular phase (day -13 to day -1) or the luteal phase (day +1 to day +13), where the expected day of ovulation was set at day 0, taking into account the individual length of the menstrual cycle. The intention was that surgery would be performed in the follicular phase (day 8-12) or the luteal phase (day 18-21); however, surgical planning was of course also dependent on the theatre schedule. Postmenopausal women were operated on after two months of hormonal treatment.

Immediately after surgery, parenchymal tissue was dissected from adipose tissue, frozen in liquid nitrogen and stored at – 70 o until analysis. Breast tissue was also fixed in para-formaldehyde, and mounted on paraffin-blocks and sectioned (5μm) and mounted on Fisher slides. The slides were stored in a dark room at a cool temperature.

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Mammography and mammographic assessments A preoperative mammogram was taken two months before the planned operation. Mammography examinations comprised mediolateral oblique and craniocaudal views of both breasts. A needle biopsy was performed in preferably the right breast at the same time. Mammograms were assessed using the scale adapted by Lundström et al. (Karolinska Institute) which requires a 20% change in density for each class, although we incorporated all densities > 40% together, thus dividing the women into three classes: 0 < 20%, 20 < 40% and > 40%. The mean value of three measurements of each mammogram was used in the analysis. Mean breast density (BD %), was the mean of left and right breast and we also used breast density class (< 20% vs. > 40%).

Tissue preparation and the enzyme immunoassay (EIA) Specimens weighing 0.15 - 0.3g were cut into smaller pieces on ice. The minced tissue was transferred to a prechilled (dry ice) capsule containing a Teflon ball. The capsule was frozen in liquid nitrogen and the specimen was pulverized in a dismembranator for 30 s. The procedure was repeated once with intermediate freezing in nitrogen. The pulverized tissue was then transferred to a prechilled tube and suspended in 10 vol Tris buffer. The suspension was centrifuged at 23.500 x g for 30 min at +4° C. After centrifugation, the ER and PR concentrations in the cytosol were determined by commercial monoclonal receptor EIA kits (Abbott-ER-EIA and PR-EIA, Abbott Diagnostica, Weisbaden, Delkenheim, Germany) and the protein concentrations were determined according to the method of Bradford (181) using the Bio-Rad Assay (Bio-Rad, Munich, Germany). The results were expressed as fmol receptor/mg protein. For cytosol with a protein concentration of 1mg/ml, the sensitivity of the assay was 1.5 fmol/ ml for ER and 1.7 fmol / ml for PR. Each assay included control samples for estimation of reproducibility, which were within the stipulated values.

Immunohistochemistry (IHC) The paraffin-embedded breast tissue samples were preheated for 30 minutes at 60 o C in an oven. The slides were then heated with Diva Decloaker (Biocare Medical, Concord, CA) in a 2100-Retriever (Histolab, Gothenburg, Sweden) for 20 minutes for antigen retrieval, after which the tissue sections were rinsed in Hot Rinse (Biocare Medical) to guarantee complete deparaffinization. After rinsing in Tris-buffered saline (TBS) (Biocare Medical), endogenous peroxidase activity was quenched in all sections by incubation in Peroxidazed

36

(Biocare Medical) for 5 minutes in darkness at room temperature. After incubation with Background Sniper (Biocare Medical) for 10 minutes, the sections were incubated for 60 minutes with the primary monoclonal antibodies for decorin (concentration 1mg/ml, Code No 270425, Clone 6-B-6, 1:3000; Seikagaku, Tokyo, Japan), syndecan-1 (Code No MCA681, Clone No B-B4 1:400; Serotec, Oxford, England), the androgen receptor (concentration 414 g/ml, Code No M3562, 1:200, DAKO CA,USA), and Ki67 (concentration 80 g/ml, Code NCL-Ki67-MM1, 1:100, Novocastra, UK), and the primary polyclonal antibodies for syndecan-4 (concentration 200 gIgG/1ml, Code No Sc-9499, 1:800; Santa Cruz, CA, USA) and cleaved caspase-3 (Code #9661 , 1:200, Cell Signaling Technology, Boston, USA); see Figure 5. Subsequently, the biotin-free detection system mouse-probe HRP polymer kit MACH 3™ (Biocare Medical, Walnut Creek, CA) was used for decorin, syndecan-1, the androgen receptor and Ki67, while the Goat-HRP polymer kit MACH 3™ was used for syndecan-4, and the MACH 3TM Rabbit-HRP polymer kit was used for caspase-3. The reaction was developed using the diaminobenzidine DAB kit (Biocare Medical, Walnut Creek, CA) and TBS was used to rinse between each step. The sections were then counterstained with hematoxylin. Finally, the slides were mounted using Pertex® (Histolab products AB, Gothenburg, Sweden).

In each assay, controls for specificity were carried out using primary antibodies preincubated with blocking peptides for syndecan-4 (Santa Cruz, Biotechnology) and cleaved caspase 3 (Cell Signaling) and with mouse IgG1 negative controls for syndecan-1, the androgen receptor, Ki 67 and decorin. In all cases, immunoreactivity in the breast biopsy was classified blind by two independent observers (EA, GH) using conventional light microscopy. The staining intensity (SI) was graded on a scale of (0) = no staining, (1) = weak staining, (2) = moderate staining and (3) = strong staining. At least 2 lobuli and ductuli and > 200 cells had to be found in each sample to be considered. The distribution pattern, measured as the percentage of positive stained cells (PP) was depicted as 0 = 0%, 1= <10%, 2 = 11- 50%, 3 = 51- 80% and 4 = > 81%.

In studies III and IV, a semi-quantitative method (the IRS score) previously described by Remmele and Stegner 1987 (182), was used to evaluate the SI and the PP of the IHC staining as follows: IRS = SI x PP. In our analysis, the mean values of the results from the two observers (GH, EA) were used.

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Assay ID used in real-time RT-PCR and antibodies used in Immunohistochemistry

Real Time PCR Antibodies (IHC)

Assay ID Reference Sequence database accession number

No, manufacturer Type Dilution

Syndecan-1 Hs 00174579_m1 NM_001006946.1 MCA 681, Serotec, Oxford, England

Mouse monoclonal 1:400

Syndecan-4 Hs 00161617_m1 NM_002999.2 Sc-9499, Santa Cruz, CA, USA

Goat polyclonal 1:800

Androgen receptor Hs 00907244_ml NM_001011645.1 M3562, DAKO,

CA, USA Mouse

monoclonal 1:200

Decorin Hs 00370385_m1 NM_133503.2 270425, Seikagaku, Tokyo, Japan

Mouse monoclonal 1:3000

Ki67 Hs01032443_m1 NM_001145966.1 NCL-Ki67MM1, Novocastra, UK

Mouse Monoclonal 1:100

Caspase 3 Hs00263337_m1 NM_032991.2 #9661, Cell Signaling Rabbit polyclonal 1:200

Figure 5.

Tissue homogenization and extraction of RNA The biopsies were homogenized frozen using a dismembranation apparatus (Retsch KG, Haan, Germany) to achieve a fine powder from which the total RNA was extracted using a Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Reverse Transcription (RT) The concentration of total RNA was determined by Nanodrop™ 1000 and the quality of the total RNA was verified by running an agarose gel. The RT reaction used 1 g total RNA, 1 l (200 ng) of pd (N)6 Random Hexamer 5'-Phosphate primers (Amersham Biosciences, Pistacaway, NJ, USA), 1 l of 10 mM dNTP (Amersham Biosciences), and sterile water to 12 l. The mixture was incubated for 5 min at 65°C, cooled and centrifuged. The reaction mixture, consisting of 4 l of 5 × First-Strand Buffer, 2 l of 0.1 M DTT (Invitrogen, Carlsbad, California), and 1 l (40 U/ l) of Protector Rnase Inhibitor (Roche, Mannheim, Germany), was added and the resulting mixture was incubated for 2 min at 25°C. 1 l (200 U/ l) of Superscript™ Rnase H-

Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) was mixed into each tube, which was then incubated for 10 minutes at 25 °C. The RT step

38

was carried out at 42 °C for 50 min, followed by heating at 70°C for 15 min to inactivate the enzyme. The cDNA was stored at -70°C until used.

Real-Time PCR The levels of mRNA encoding decorin, Ki67, caspase, androgen receptor, syndecan-1, and syndecan-4 were quantified using the Applied Biosystems 7300 Real-Time PCR system (Applied Biosystems, Foster city, CA, USA). All determinations were performed in triplicate in the Taqman Universal PCR master Mix (Applied Biosystems, Foster city, CA, USA) on 96-well optical PCR plates. Appropriate primers and probes were obtained from commercially available Taqman® gene expression assays (Applied Biosystems). Assay IDs and Reference Sequence database accession numbers are presented in Figure 5. Ribosomal 18 S RNA (Assay ID 4319413E, Applied Biosystems), hormone-stable and expressing ribosomal RNA, was used as a house-keeping gene. Each reaction used 5 µl diluted cDNA corresponding to 10 ng total RNA in 12.5 µl Universal Master Mix, 1.25 µl assay mixture and 6.25 µl sterile water. The real-time PCR reaction was carried out according to the manufacturer´s protocol. The threshold cycles (CT), at which an increase in reporter fluorescence above the baseline signal was first detected, were determined. 18S was used as endogenous control and for normalization (ΔCT) of the mRNA levels for the gene of interest. Serial dilutions of breast tissue cDNA were made from total RNA (Ambion, Austin TX, US) to validate the quality of the experiment.

Calculations for real-time RT-PCR In Study II and III, calculations of relative gene expressions were carried out using the CT method, where the women in the follicular menstrual phase were used as a control group. Because the amount of product doubles in each cycle, the relative gene expression was calculated using the formula 2- T, given in the manufacturer´s instructions. In study IV calculations were based on CT.

Serum analysis Blood samples were taken on the same the day as the mammography. Blood samples were also collected the day before the main operation when the woman arrived for the preoperative appointment. Commercial kits were used for analyzing the serum levels of E2 and progesterone (Amerlite®, Johnson &Johnson clinical diagnostics Ltd, Amersham, UK), SHBG (Delphia, Wallac Oy, Turku, Finland), and total testosterone (Immulite, Siemens, Siemens

39

medical Solutions Diagnostics, Los Angeles, CA, USA). The analyses were performed according to the manufacturer´s manual. The detection limits and within- and between-assay coefficients of variation were: 50 pmol/L, 8% and 9.1% for E2, 0.11 nmol/L, 7.5% and 8% for progesterone, 3 nmol/L, 6% and 8% for SHBG, and 0.3 nmol/L, 7.4% and 7.7% for total testosterone.

Statistical analysis

Study I Parametric (Student t test for independent samples) and non-parametric (two–sample Wilcoxon rank sum test) tests were used to compare study groups with respect to baseline characteristics and primary study outcome. Non-parametric analysis of variance (ANOVA), based on individual scores, was performed to study the simultaneous effects of age, BMI, parity and treatment regimens on the content of ER and PR. A parametric ANOVA was applied to the raw data after applying square root transformation. Associations were tested using Spearman rank correlation. Statistical analysis was performed using the Stata Statistical Software version 7, 2001; Stata Corporation, College Station, TX, USA).

Study II The matched-pair Wilcoxon signed rank test was employed to compare the paired IHC results. Comparisons between the two independent groups in the real-time RT-PCR test were carried out using the Mann-Whitney U test.

Study III The matched-pair Wilcoxon signed rank test was used to compare the paired results for both IHC and real-time RT-PCR. The Mann Whitney U test was used for the unpaired analysis.

Study IV Values were expressed as medians (range) or as distributions. Non-parametric methods were used because of the ordinal scaled and/or skewed distributed variables. Comparisons among groups were made with the Mann-Whitney two-group test. Groups were compared with adjustment for age using Willet’s residual method (183). Associations between variables were examined with Spearman rank correlation coefficients or Spearman partial rank correlation coefficients with adjustment for age. A two-sided p value < 0.05 was

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considered a statistically significant result. All statistical analyses were performed with the statistical program packages of SAS or JMP (SAS Inst. Inc. NC, USA).

Ethics The studies were approved by the Ethics Committee of the Faculty of Medicine, Uppsala University, Uppsala, Sweden, and all women gave their informed consent to participate.

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Results

The prestudy power analysis, based on the expected change in the proliferation marker Ki67 of the breast tissue (positive staining for Ki 67 of around 1 %) and using calculations for 80% statistical power with a level of significance of 5%, indicated that 60 premenopausal and 40 postmenopausal women were required for the study. Due to lack of interest among the nulliparous postmenopausal women in participating in the study, it was not possible to obtain the required 100 participants for randomization. There were also few nulliparous premenopausal women on the waiting list, and little interest in an invitation to join the study. However, many women in the parous premenopausal group were interested in joining, and this randomization arm was increased. In total, 81 women were finally included.

Participating women in study I – IV and flow-chart A total of 60 premenopausal and 21 postmenopausal women were randomized. One premenopausal nulliparous woman was rejected for surgery because of weight gain after mammography but before the operation. One postmenopausal woman was excluded during exogenous hormone substitution because of symptoms of deep vein thrombosis, which could not be confirmed radiologically. This left 59 premenopausal and 20 postmenopausal eligible women.

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IHC (# 53) PCR (# 23) F = 38 F = 19L = 15 L = 4

Premenopausal women

N = 59

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Study 1

Study 2

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EIA (# 49)FL = 17 LF = 12 OC = 20

IHC (# 38) PCR (# 20) F vs. L F vs. LAll pairs 14 pairs

IHC (# 20) PCR (# 19) F vs. OC F vs. OC20 pairs 10 pairs

Menstrual cycle phases

Estrogen and progesterone receptors

Oral contraceptive (EE2/L-NG (30/150)

Mammographic breast density

Figure 6. Participating women in study I-IV. (E2Val = estradiolvalerate, MPA = medroxyprogesterone acetate, EE2= ethinylestradiol, L-NG = levonorgestrel)

Due to loss of samples in the transportation between the research centres, the remaining numbers of tissue samples were limited. Study I: Breast tissue from ten premenopausal women and four postmenopausal women was not included in the first study using enzyme immunoassay. This was because of adverse events, and practical circumstances beyond our control involving the handling and laboratory preparation of specimens or tissue. This left 49 premenopausal and 16 postmenopausal evaluable tissue samples for this study. Study II: Twenty-three women randomized to follicular-luteal and 15 randomized to luteal-follicular regimens were included in Study II. Breast tissue for the reverse-transcription polymerase chain reaction (RT-PCR) study was not found for nine parous and nine nulliparous premenopausal women. This left 38 evaluable tissue specimens for IHC and 20 for PCR, for this study. Study III: Twelve parous and eight nulliparous premenopausal women were randomized to two months of an OC in Study III. Breast tissue for the real-time RT-PCR was available, however, from 19 of these premenopausal women, and ten of these were paired.

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Study IV: Fifty-nine premenopausal women had a preoperative mammogram and biopsy, resulting in 55 evaluable mammograms and 53 cases where both a mammogram and IHC or PCR results were available. We were unable to locate some of the mammograms because the mammographic section at the Department of Radiology moved during the study period from its former location at the University hospital, Uppsala, to a private location (Avesina). The numbers of evaluable IHC slides (i.e readable in at least two fields) for the studied antibodies were: Ki 67 = 43, caspase 3 = 47, androgen receptor = 46, syndecan-1 = 42, syndecan-4 = 47 and decorin = 53. RT-PCR results for needle biopsies were evaluable for 23 of the women, 19 in the follicular and 4 in the luteal phase.

Findings

Study I Among premenopausal women, age was positively associated with content of ER (p = 0.0002). Premenopausal parous women had higher ER levels (p = 0.009) than nulliparous women, whereas no significant difference was found for PR. The ER levels were similar in the follicular and luteal phases (p = 0.50) while the PR levels were numerically lower in the luteal phase (P = 0.08). Women on OCs had lower levels of ER (p = 0.048) and PR (p = 0.007) than women in the follicular phase. The ERα levels did not differ significantly between postmenopausal women receiving ET and EPT, but PR levels were lower among those receiving EPT (p = 0.03). Levels of PR were lower in current smokers than in non-smokers (p = 0.02), a difference that remained statistically significant after adjustment for HT, pre- and postmenopausal status and menstrual cycle phase. No other association between ER or PR and risk factors for breast cancer was observed (age at menarche and menopause, age, BMI, height, alcohol consumption or family history of breast cancer).

Study II Gene expression of syndecan-4 was significantly lower in the luteal phase than in the follicular phase of the menstrual cycle in the total group (p = 0.03) and in parous women (p = 0.021). Gene expression levels for syndecan-1 (p = 0.034) and the SLRP decorin (p = 0.018) were also significantly lower in the luteal phase than the follicular phase in parous women. The distribution of decorin in the ECM was prominent in both the follicular and luteal phases. Immunoreactivity for syndecan-1 and -4 was clearly identified in the epithelial

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and basement membranes. There were no significant differences in the distribution of syndecan-1 between the menstrual cycle phases.

Study III Gene expression of decorin decreased five-fold after OC use compared to during the follicular menstrual phase (p = 0.002) Figure 7A. There was a numerical non-statistically significant down-regulation of gene expression after OC use for syndecan-1, syndecan-4 and the androgen receptor Figure 7B. There was a significant decrease in expression of androgen receptor protein after OC use compared to during the follicular phase (p = 0.002) Figure 8A. The immunoreactivity of decorin was strong in the ECM in the follicular phase and decreased after OC use (p = 0.018). Syndecan-4 protein levels decreased after OC use compared to the follicular phase (p = 0.003). Protein levels of syndecan-1 in the epithelium showed a tendency to increase with OC use (p = 0.057) Figure 8A. There were no differences in gene expression levels for syndecan-1, syndecan-4, the androgen receptor or decorin between the luteal phase and after short-term use of OC.

All control sections were negative. The inter-observer correlations were syndecan-1: rs = 0.73, p < 0.0001; syndecan-4: rs = 0.072, p = 0.0001; androgen receptor: rs = 0.57, p = 0.0006; and decorin: rs = 0.52, p = 0.0012.

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7A

7B

Follicular phase OC use0.45

0.50

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0.65

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Syndecan-1

10/d

elta

ct

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elta

ct

Decorin

Follicular phase OC use0.6

0.7

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1.0

p=0.002

10/d

elta

ct

Figure 7A. The gene expression of syndecan-1 and -4, the androgen receptor and Decorin after OC use, using real-time RT-PCR. 7B. Difference in gene expression between the follicular phase and after oral contraceptives (OC) visualized in box-plots.

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8A

Syndecan-1

Follicular phase OC use0

2

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scor

e

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scor

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8B

Figure 8A. Quantification of pair-matched immunohistochemistry results for syndecans-1, syndecans-4, the androgen receptor and Decorin in the follicular phase and after OC use. 8B. Immunhistochemical staining of the proteoglycans and the androgen receptor. The magnification used was 400X.

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Study IV We found no significant differences in mammographic BD (%) between the follicular and luteal phases, with a difference of only 1% between the phases.

Spearman rank correlation test: Mammographic BD (%) decreased significantly with age (rs = -0.62; p < 0.0001) and with BMI (-0.42; 0.002). Gene expression of the AR was positively associated with both increasing BD class (< 20% vs > 40%) and with BD (%) in the breast from which the needle biopsy was taken (rs = 0.43; p = 0.048, for both). After adjustment for age, this association was strengthened for both BD class (0.56; 0.044) and BD (%) (rs = 0.57; p = 0.044). In addition, after adjustment for age, caspase 3 was positively associated with BD (%), with borderline significance (0.55; 0.053), whereas the protein levels (IHC) for caspase 3 were negatively associated with BD class and BD% from the needle biopsy breast (-0.61; 0.026).

Group comparisons: The androgen receptor was numerically higher in more dense breasts, from both PCR and IHC results, with borderline significance (p = 0.056) for PCR.

For caspase 3, PCR indicated higher gene expression in more dense breasts, which was retained after adjustment for age with borderline significance (p = 0.056). However, IHC age-adjusted values indicated lower protein levels of caspase 3 in more dense breasts, with borderline significance for both BD class overall and for BD class in the needle biopsy breast (p = 0.05 and 0.049, respectively). Analysis of the PCR follicular phase data showed significantly higher caspase 3 levels in more dense breasts (p = 0.04) for both unadjusted and age-adjusted data, based on BD% (right and left breasts) as well as on BD% in the needle biopsy breast (p = 0.04 for all).

In this limited material, Ki67, syndecan–1, and syndecan –4 showed numerically higher gene expression (PCR) in more dense breasts (not significant), while protein expression (IHC) was numerically higher for Ki67 and syndecan-1 but lower for syndecan-4 (not significant). Decorin levels were unaffected by changes in BD for both PCR and IHC results.

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Discussion

General discussion In Study I, we evaluated the effects of endogenous and exogenous estrogen and progesterone/progestin on the breast to assess the impact of menstrual cycle phase, parity, OC use and HT on the levels of ER and PR. In the normal menstrual cycle, ER levels are down-regulated during the luteal phase (115, 184-187). We found numerically lower levels of ERα in the luteal phase, but the difference was not statistically significant. We also found reduced content of ER and PR after short term OC use compared with women in the follicular phase of their menstrual cycle who were not taking OCs, as has been previously described (104, 188). However, some investigators have reported increased levels of PR during OC use compared with the natural cycle (105).

In postmenopausal women, the PR content was significantly higher after exposure to ET than after EPT, but there were no significant differences in ER content. Hofseth and colleagues (189) also found that women on ET had higher PR levels than women on EPT, although Hargreaves and associates observed similarly up-regulated PR expression with both ET and EPT (190). In a study on surgically postmenopausal cynomolgus macaque monkeys, ET increased the PR content and EPT decreased the content of ER (186). The measured concentrations of ER and PR in our study were in a range similar to those in other studies on postmenopausal women (105). However, we had no information about the receptor levels in these postmenopausal women before the start of HT; therefore, the direct effects of HRT intervention could not be evaluated. The women were only exposed to exogenous hormones for 2 months, but this has been shown to be a sufficient duration for studying the effects on receptor content and proliferation in other studies (191).

The main carcinogenetic mechanism of ovarian hormones is thought to be related to receptor-mediated mitogenic activity (192, 193). There are two lines of evidence indicating that ER content is related to the risk of breast cancer. First, levels of ER in normal breast tissue in Australian women, among whom breast cancer incidence is high, are higher than those in women from Japan, where breast cancer incidence is low (113). Second, Khan and co-workers demonstrated that ER levels in `normal` breast tissue adjacent to a malignant tumor were higher than those in tissue in the proximity of benign lesions (115). However, the reasoning by Khan and co-workers is etiologically

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complicated since high receptor content in tissue adjacent to a neoplasm could be a consequence of the influence of intra-tumor growth factor production rather than the increased receptor content causing malignant transformation (110).

Countering the hypothesis that ER expression is a risk factor for breast cancer, it has been argued that ER -containing epithelial cells do not proliferate in response to estrogen and only 7% of normal breast cells harbor ER (22, 85, 194, 195). Also, breast epithelial proliferation peaks during the luteal phase of the menstrual cycle when ER is least expressed. Thus, the dogma has held that the effect of estrogen on breast epithelial division must be indirect (12, 112). Cells that contain ER are thought to respond to estrogen by secreting paracrine-signaling molecules, which could influence the proliferation of ER -negative epithelial cells.

The association between PR expression and breast cancer risk is even less well understood (196, 197). Studies have indicated that there are differences in the risk of breast cancer for different progestagens, which are able to independently induce proliferation in both malignant and benign human breast cells (198). Growth factors and progestogens (MPA) have additive synergistic effects on cell proliferation but use different pathways. MPA has stimulatory effects possibly through different kinases (i.e EGF, bFGF, IGF-1) (199).

Short term estrogen administration with or without progesterone decreased the incidence of mammary cancer in a rat study (200). Both high-dose estriol and E2 at normal pregnancy doses, with or without progesterone for 1-3 weeks, strongly decreased the incidence of BC. MPA at contraceptive doses did not increase the risk of carcinogenesis, but with doses 10 times greater there was a carcinogenic response. Russo and Russo have shown that treatment with human chorionic gonadotropin has the same protective effect on carcinogenesis in the rat breast (201). Thus, the dose, duration and nature of hormone treatment result in very different effects on breast tissue (200).

We found that PR levels were clearly lower in current smokers than in non-smokers. There is evidence of various effects of smoking on the risk of BC. The lowering of endogenous estrogen levels by current smoking could decrease the risk (202) and the genotoxic effects of tobacco metabolites on the breast epithelium could increase it (203). However, results from a collaborative re-analysis of the literature and a large population-based study from Sweden found no association at all between active smoking and the risk of BC, including its duration, intensity or timing (27, 204).

In Study II, we addressed the effects of ovarian hormones on premenopausal breast tissue in terms of the expression of the genes and proteins of three PGs during the menstrual cycle. Gene expression of decorin, syndecan-1 and syndecan-4 was significantly lower in the luteal phase than in the follicular phase of the menstrual cycle in parous women. Although the proteins were clearly identified by IHC, no significant differences between menstrual phases were observed.

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Earlier studies of the healthy human breast during the menstrual cycle have reported various histologic changes in the epithelium and stroma (84, 205). Ferguson et al. used indirect immunofluorescence to describe a pattern of increased staining of HSPG, laminin, fibronectin, and collagen in the ECM in weeks 1 and 4 of the menstrual cycle and reduced staining in weeks 2 and 3 (206). An ultrastructural study described an increase in the distance between the proteoglycan-collagen associations in the luteal phase, which was explained by higher water content (207). In addition to the epithelial changes during the menstrual cycle where small cuboidal epithelial cells during the first two weeks change to differentiated, columnar cells in the second half of the menstrual cycle (84), there are also changes in the stroma. The intralobular stroma is loose and more responsive to the ovarian hormones. The intralobular stroma contains numerous fibroblasts while the interlobular stroma contains very few (206). The fat cells surrounding the interlobular stroma contain HSPG (206). In Stoeckelhuber's study, the increase in distance was more prominent in the intralobular tissue than in the interlobular stroma. Most of the gene expression required for transformation of normal tissue to cancerous tissue in situ occurs in the stroma rather than in the epithelium (208). Stromal changes have been documented in both pre-invasive breast lesions and tumors (209).

The SLRP decorin is found abundantly in the ECM. Decorin is a powerful and effective agent against breast cancer because of its ability to inhibit both primary tumor growth and metastatic spreading by inactivating the oncogenic ERbB2 protein in breast carcinoma cells (210) and downregulating members of the ERb tyrosine kinase family, leading to growth inhibition (167). Previous studies have reported down-regulation of decorin in breast cancer tissue (171), a situation which has been associated with a poor prognosis (173).

The syndecans, which are HSPGs, can be either up-regulated or down-regulated during the progression of a malignancy. Both syndecan-1 and syndecan-4 are overexpressed in the estrogen receptor–negative, highly proliferative breast carcinoma subtype (148). Syndecan-1 can be redistributed from the epithelial cells to the stroma compartment in postmenopausal cancers (172, 211, 212). Increased expression of syndecan-1 has been associated with increased mammographic BD as well as re-distribution from the epithelium to the stroma among postmenopausal women (213).

The physiologic reduction in the expression of proteoglycan genes in the luteal phase of the menstrual cycle may be a consequence of the effect of ovarian hormones (206). Progesterone, elevated during the luteal phase, interacts with growth factors such as HGF, EGF and IGF-1 and gives rise to both synergistic and inhibitory effects, influenced by the composition of the ECM (214).

In Study III, we examined the effects of short-term OC use on the expression of the proteoglycans syndecan-1, syndecan-4 and decorin and the

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androgen receptor in human breast tissue from women of fertile age. Our hypothesis was that the additive effect of exogenous hormones on the fertile breast could down-regulate the expression and levels of the proteoglycans compared to those in the follicular and luteal phases. The study revealed that the gene expression of decorin was significantly lower after OC use than during the follicular phase of the normal menstrual cycle. Gene expression of syndecan-1, syndecan-4 and the androgen receptor tended to be lower after OC use, but the difference was not statistically significant. IHC showed lower levels of protein for syndecan-4, the androgen receptor and decorin after OC use, whereas syndecan-1 levels tended to increase after OC use. This is in accordance with results from Hofling et al. on oophorectomized cynomolgus monkeys. They found that the expression of syndecan-1 increased with two types of exogenous hormone treatment [conjugated equine estrogens (CEE) + MPA, and tibolone) and that levels of the androgen receptor were suppressed by CEE+MPA and increased by tibolone administration [Hofling, 2009]. The syndecans are cell surface matrix receptors that regulate the activation of various integrins on mammary carcinoma cells and fibroblasts (153, 215) and control the signaling of other cell surface receptors (216). Angiogenesis is increased in syndecan-1 deficient mice, and syndecan-1 over-expressing mice have abnormally dilated blood vessels (217, 218). Therefore, changes in expression of the syndecans could reduce cell adhesion and promote cancer cell invasion (219).

Changes in the expression of several proteoglycans have been associated with prognosis and clinical outcome in several types of cancer (219, 220). Various methods have been used to identify the tumor-growth-promoting and tumor-growth-inhibiting sequences in the heparane sulfate structure and to detect decorin, syndecan-1 and syndecan-4 in colon cancer, pancreatic cancer and fibrosarcoma tissue (221, 222). Shimada et al. have suggested that syndecan-1 could be involved in the conversion of androgen-dependent prostate cancer to androgen-independent cancer, thus providing a potential new target molecule for hormone-resistant prostate cancer therapy (223).

One important aspect of the effect of syndecans is the process of shedding, where part of the transmembrane-bound syndecan can be shed and become soluble. Nikolova et al. have pointed out that different effects occur when the membrane-bound syndecan-1 undergoes proteolytic conversion to the soluble version, switching from a proliferative to an invasive phenotype (224).

The SLRP decorin is a complex molecule (163) known to be involved with signaling, interacting with and controlling cell growth, morphogenesis and immunity. The SLRPs interact with tyrosine kinase and Toll-Like receptors, and take part in both cancer and innate immunity processes (163). The antiproliferative effects of decorin were first demonstrated by Yamaguchi et al. (225) and the molecule has also been shown to activate caspase 3, a key enzyme in programmed cell death (169). Decorin also suppresses prostate tumor growth through inhibiton of EGFR and androgen receptor pathways

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(226). In the normal cell, decorin signals through the IGF-1 receptor and favors cellular growth via anti-apoptotic and proliferative pathways (163).

Levo-norgestrel, the synthetic progestin used in study III, is a derivative of 19-nortestosterone. In a previous study on OC use with the same progestin, the proliferative activity (measured using Ki67) in breast tissue was increased in the first week of OC use compared to that in women in the follicular phase of the normal menstrual cycle (227). Further, Isaksson et al. have reported increased proliferation (Ki67) after OC use compared to the luteal phase of the menstrual cycle (191).

The observation that the ECM serves as a reservoir for cytokines and growth factors involved in glycosaminoglycan/proteoglycan composition by regulating its stiffness/proteolysis is even more interesting (79). Maller describes the ECM as having three different structures: the basal lamina, the intra- and interlobular stroma, and fibrous connective tissue. Many of the studies performed previously have used rodents, monkeys, or cell-lines. Although the epithelial structures are similar in different species, the amount of connective tissue and fat tissue surrounding the epithelium varies. More studies in humans are therefore required.

In Study IV, we examined mammographic BD and the correlations between gene expression and protein levels of markers of proliferation, apoptosis, the androgen receptor and three proteoglycans in non-cancerous breast tissue from pre-menopausal women. Our results are in accordance with previous reports of increased mammographic BD with age, decreased BD with increasing BMI (228), and similar BD in the follicular and luteal phases of the menstrual cycle (229, 230).

In our study, gene expression of caspase 3 tended to increase but protein levels were decreased in dense breasts, indicating decreased apoptosis. Similarly, in a study on postmenopausal women using HRT, where mammographic BD increased, the expression of the apoptosis marker Bcl-2 also indicated decreased apoptosis activity (67).

We found increased expression of the androgen receptor in the mammographically dense breast. This is likely to be the result of the increase in fibrous stroma in dense breasts, with the increase in fibroblasts possibly leading to an increase in aromatase enzyme (73), and subsequent potential for increased estrogen synthesis. Increased estrogen synthesis could stimulate the proliferation of stromal cells to produce IGF-1 or PGs, which could stimulate epithelial proliferation through paracrine actions (72). Conner et al. found the decline in free testosterone levels during HRT to be one of the most important factors for predicting increased mammographic BD (69). Androgens are able to act in a pro-estrogenic fashion after being aromatized to estrogens and, when sufficient estrogens are present, in an inhibitory fashion, preventing estrogenic stimulation of growth (122). Further, Löfgren et al. found levels of the AR to be, on average, 2-fold higher in cancerous breast tissue than in normal postmenopausal breast tissue (172).

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Our results also suggested a tendency for up-regulation of the gene for and higher protein levels of Ki67 in more dense breasts. Previous studies on BD have found an association between mammographic BD and histopathological changes in the breast (231, 232). Hawes et al. found the breast epithelial cells to be concentrated in high density areas, but the proliferation rate in these dense areas was lower than in less dense areas (233). Harvey et al. examined postmenopausal women with and without HT and found no correlation between ER and PR levels and breast density. However, in both users and non-users of HT, there was a clear positive correlation between BD and Ki67 (72). In contrast, Verheus et al. found little evidence for an association between Ki67 and mammographic BD (234).

Decorin, syndecan-1 and syndecan-4 levels showed no significant correlations with mammographic BD in our study and the risk of type II error. However, this finding must be interpreted with caution because of the limited sample size. Guo et al. examined the associations between mammographic BD, growth factors and stromal matrix proteins. They stained for cell nuclei, total collagen, TIMP-3 and IGF-1, and found that dense areas had a significantly greater nuclear area, and larger stained area of total collagen, IGF-1 and TIMP-3 (235). Since the mammary epithelium, the myoepithelial cells and the fibroblasts all communicate with paracrine signals, they hypothesized that growth factors within the stroma stimulate proliferation and the ECM and contribute to the composition of more dense tissue, thus affecting the epithelium. Since inflammation is a part of breast carcinogenesis, Reeves et al. examined IL-6, TNF , and CRP but found no substantial impact of these inflammatory parameters on mammographic BD (236).

Study limitations The study participants undergoing reduction mammary plastic surgery had a BMI above the normal limit of 25, which might have influenced the results and the generalizability of our results. The proportions of epithelium and adipose tissue can also differ in hyperplastic breasts from normal, since large breasts can contain more adipose tissue (234). The limited number of women in the study may have implications for the study results because of the risk of type II errors, especially for the non-significant findings. The inclusion criterion for ‘postmenopausal’ status was set at six months without menstrual bleeding, in contrast to the general definition of 12 months without menstrual bleeding. We therefore examined the level of follicle-stimulating hormone (FSH) in the group of postmenopausal women with less than a year since their last bleed but with postmenopausal values for FSH. The breast tissue biopsies were primarily intended to give a representative biopsy and were not specifically from a dense area.

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It was very difficult to obtain good quality results when staining caspase for IHC, despite the use of many different antibodies, dilutions, and time-limits and discussions with two pathologists. We could see staining in the nucleus and the cytoplasm and distinct nuclei staining was observed in the stroma.

Since the praffine-embedded slides were very old, even if stored in a cool temperature for many years, new slides from the paraffin-blocks was made for comparison. In the spring of 2010, fresh breast tissue from two women, having reduction plastic surgery performed was examined and compared with the old immunhistochemical stainings with similar staining results.

Endogenous androgen concentrations are low in women and conventional immunoassays commonly used are not optimal for analysis of low concentrations, and are less reliable than mass spectrometry (237). Because the decision on the date of the operation and serum sampling was in the hands of the operation coordinator, the luteal phase samples were sometimes obtained late in the luteal phase.

Future perspectives The importance of the ECM and the proteoglycans has become very clear during resent years. We have only examined three of the many proteoglycans involved in the intricate work of balancing the cell signalling and regulation of the breast tissue. The proteoglycans are involved in both acute and chronic inflammatory processes. The acute inflammatory process in the human breast is represented by the start of the lactogenesis and the involution of the breast tissue to end the lactation period. The proteoglycans may well be regulators of this delicate process. The question of how the regulation of inflammatory response could interact with the subsequent risk of mastitis and perhaps even breast cancer is even more exciting.

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General conclusions

I: In premenopausal women, estrogen receptor α (ERα) levels in the breast were positively associated with age.

PR levels were lower in current smokers than in non-smokers. ERα and progesterone receptor (PR) levels in the breast were lower after

short-term oral contraceptive use than in the follicular phase of the menstrual cycle.

The ERα content was similar between estrogen-only and estrogen-

progestogen therapy in postmenopausal women, whereas PR levels were lower among postmenopausal women on estrogen-progestin therapy.

II: Gene expression of syndecan–1, syndecan-4 and decorin were lower in

the luteal than in the follicular phase of the menstrual cycle, in parous premenopausal women.

There were no statistically significant differences in the protein levels of

the three proteoglycans between the follicular and luteal phases. III: Protein levels of syndecan–4, decorin, and the androgen receptor and gene

expression of decorin, were lower after short-term oral contraceptive use than during the follicular phase of the menstrual cycle.

IV: In premenopausal women, mammographic breast density decreased with

age and body mass index, but was similar for the menstrual phases. Increased breast density was associated with increased expression of the

androgen receptor gene. After adjustment for age, breast density was associated with lower protein level of caspase 3.

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Sammanfattning på svenska

Bröstcancer är den vanligaste cancern hos kvinnor i Sverige och västvärlden. Vi har idag kunskap kring många faktorer som ökar risken för ett insjuknande i bröstcancer. Till dessa kan nämnas tidig ålder för menstruationsstart, sent klimakterium (menopaus), att föda barn sent i livet, att inte föda barn alls. Där finns en lätt ökad risk efter användning av preventivmedel med p-piller med syntetiskt östrogen och progesteron. Hormonpreparat i klimakteriet ger också en riskökning där tillägget av progesteron ger den ökade bröstcancer risken. Vid bröströntgen (mammografi) har man sedan många år tillbaka noterat att en del kvinnobröst ser mer röntgenologiskt täta ut, det ger en vitare röntgenbild. Man vet att mammografiskt täta bröst utgör en påtaglig riskfaktor för att insjukna i bröstcancer. Vad denna röntgenologiska täthet utgör diskuteras fortfarande.

Hormon som påverkar celltillväxten i körtelcellerna och därmed ökar risken för felprogrammering, och mutationer är en uppenbar risk för cancertillväxt. Celltillväxt (proliferation) kan mätas med en markör som heter KI 67. Även graden av reglerad celldöd (apoptos) är av betydelse för den normala regleringen av antal celler i vävnaden. Apoptos kan bl.a mätas i mängden av ett enzym som heter caspase 3. Forskningen har inriktats mot studier på de receptorer som bl.a. styr effekten av östrogen och progesteron; östrogenreceptorn ER och progesteronreceptorn, samt den receptor som styr det manliga hormonet testosteron; androgen receptorn. Man har försökt att både i friska kvinnors bröstvävnad och i cancersjuka kvinnors bröst se på mängd och förekomst av dessa receptorer som ett mått på riskökning och i vilken grad dessa receptorer uttrycks i vävnaden. Det kommer bl.a. att styra behandlingens utformning vid bröstcancer. Det kvinnliga bröstet består av körtelvävnad som ligger i sk ” körtelbollar” lobuli som sedan går över i allt större körtelgångar för att till slut mynna i 15-20 små öppningar i bröstvårtan. Körtelvävnaden är inbäddad i både lucker och tät bindväv samt fettvävnad. I bindväven, också kallad extracellulärmatrix, består av ett skelett som består av collagen fibrer och kolhydratrika proteiner (proteoglykaner). Det är i denna grundsusbstans som vävnadens celler finns förankrade. Proteoglykanerna har stor inverkan på förändringar som sker i bröstet särskilt vid utveckling av tumörer.

Förståelsen för egentillverkade (endogen) och yttre tillförda (exogena) östrogen och progesteronhormoners inverkan på det friska kvinnliga bröstet har fört detta avhandlingsarbete till fyra delstudier med följande resultat:

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Vi fann att uttrycket av östrogenreceptorn ökade med ålder och var högre hos de kvinnor som fött barn. Progesteronreceptorn var lägre hos rökare. Progesteron receptorn, men inte östrogen receptor var lägre hos postmenopausala kvinnor med kombinerad östrogen-progestogen behandling, jämfört med enbart östrogenbehandling.

I den andra studien mätte vi mängden proteoglykaner och deras genetiska uttryck i bröstvävnad och fann lägre nivåer i den senare delen av menstruationscykeln.

I den tredje studien fann vi en sänkning av det genetiska uttrycket och förekomsten av proteoglykaner och androgen receptor efter kortvarigt p-piller intag jämfört med den första delen av menstruationscykeln.

I den fjärde studien fann vi en ökning av androgen receptorn hos kvinnor med tätare bröst och en viss påverkan på markören för celldöd (caspase 3). Vi kunde inte se någon förändring i uttryck av proteoglykaner eller tillväxtmarkören. Vissa analyser grundar sig på relativt få deltagare, varför fynden framförallt de icke-signifikanta resultaten måste tolkas med försiktighet.

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Acknowledgements

Jag vill framföra min tacksamhet till alla de som varit en del i detta avhandlingsarbete Ett stort tack till alla kvinnor som så beredvilligt ställde upp i BIOMAM studien, Ingemar Persson min första huvudhandledare som tillsammans med professor Russo i Philadelphia initierade och startade denna studie, Tord Naessén som tog över huvudhandledarskapet efter Ingemar Persson, och som med varlig hand fört mig framåt och nu på senare tid drivit på och försökt få mig att förstå att min tidsoptimism är stor, Cecilia Magnusson min bihandledare som med sin briljans i epidemiologi, engelska språket och personliga värme gjorde sitt yttersta för att föra mig framåt i början av denna studie och vid varje kontakt på senare år alltid varit genuint intresserad och omtänksam, Gunvor Ekman-Ordeberg min bihandledare från 2007, som gjöt nytt liv i detta avhandlingsarbete och utan vars entusiasm, vilja, generositet och kraft detta inte hade blivit möjligt; du är en fantastisk kvinna och jag är stolt över att få ha lärt mig både obstetrik och forskning av dig! Eva Andersson min medförfattare och ”lärare” i PCR, immunohistokemi, med ett leende löser Eva alla problem jag kunde hitta på vid lab.bänken samtidigt som hon tipsar om bra filmer på bio och bussförbindelser mellan Sthlm och Uppsala på samma gång, José Russo och Irma Russo som varmhjärtat och generöst tog emot mig vid sitt laboratorium vid Fox Chase Cancer Institute i Philadelphia, och skickade mig 106 lådor med paraffinerade glas för immunhistokemi att använda i mitt fortsatta arbete, Eva Lundström, min medförfattare och forskarkollega, för ditt know-how och ditt intresse. Det har betytt mycket! Ulla Geifalk för hjälp med serumanalyser i den första fasen av studien, och för hjälp i jakten på försvunna preparat, Sonia Diaz för allt stöd med tabellering, för din vänskap och ditt engagemang, Ulrika Lundkvist för att du introducerade mig till laborativt arbete, och för att du genomförde enzym-immunoassayen,

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Marja Boström för fint samarbete vid starten av BIOMAM studien vid fredagsmottagningarna på Plastiken där vi hade vår mottagning för de kvinnor som inkluderades i studien, tack för ditt lugn och för din vida kompetens, Bengt Gerdin och Lennart Olsén för visat intresse och trevligt samarbete ” borta på Plastiken” Erik Thurfjell och Absaleh Shahin för nålbiopsiprovtagning vid Mammografienheten, Birgitta Byström för ditt intresse och engagemang för mitt arbete och för fina historier på dalmål, Lambert Skoog, Edneia Tani för visat intresse för mina caspase-glas, Alla forskar vänner på FRH lab i Stockholm, Johanna, Angela, Aurelija, Lalit och Sujata Yvonne, Muhammed, och alla andra, tack för trevligt labbande, och vänlig atmosfär, Alla på KKs lab i Uppsala som vänligt bjudit in mig att laborera under tidigare år och mikroskopera på senare tid, Lars Berglund och Rino Bellocco för excellent statistical advice, Antona Wagstaff för en fantastisk språkgranskning, Bo Sultan verksamhetschef för Kvinnokliniken i Uppsala för att generöst gett mig tid för forskning och för visat intresse, Alla kolleger i Uppsala som arbetat istället för mig och peppat mig framåt, bland andra Barbro Woxler Lönnberg min rumskamrat som försett mig med frukt, Britta Nordström som läste manuskript och gav många kloka kommentarer, Pappa Torsten och mamma Mai, inte längre i livet men i mitt hjärta bevarade som de mest kärleksfulla och fina föräldrar man kan tänka sig, och mina tre systrar Kristina, Elisabeth och Lena med familjer, Olle, livskamrat och käre man, livet med dig är färgstarkt och spännande, Anna, Björn och Tora våra älskade barn, mina tre ögonstenar, de bästa som finns! och Totti, vår lagottohund för långa tysta promenader.

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