Sex hormone-induced mammary carcinogenesis in female Noble rats: the role of androgens

B. Xie, S.W. Tsao and Y.C. Wong1

Department of Anatomy, Faculty of Medicine, University of Hong Kong, Li Shu Fan Building, 5 Sassoon Road, Hong Kong


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is the most common cancer and the second most frequent cause of cancer death in women. Despite extensive research, the precise mechanisms of breast carcinogenesis remain unclear. We have shown that in female rats, treatment with a combination of oestrogen and testosterone can induce a high incidence of mammary cancer. The dosage of testosterone affects only the latency period of mammary cancer, not the final incidence. Based on these observations, we hypothesize that oestrogen and androgens may act in concert on the mammary gland to induce mammary carcinogenesis, with oestrogen serving as the predominant initiator whereas the androgen acts as a major promoter. In the present study, we report the changes in morphology of the mammary gland with special emphasis on the perialveolar or interlobular stroma after treatment with various sex hormone protocols. Our data showed that after treatment with testosterone, either alone or in combination with 17ß-oestradiol, there was overexpression of the androgen receptor in alveolar or ductal epithelial cells. Concurrent with strong expression of the androgen receptor in epithelium, there was also an increase in the amount of perialveolar and interlobular connective tissue, a decrease in surrounding adipose tissue and an increase in proliferation rate of fibroblast-like cells in the stroma. All these changes were blocked by simultaneous implantation of flutamide, indicating that androgens play a crucial role in the process despite the absence of androgen receptors in stromal cells. We further measured the mammary gland density (MGD), in order to determine the ratio of fatty to non-fatty tissue. The data showed that MGD values were significantly higher in animals treated with testosterone alone or in combination with 17ß-oestradiol than in those treated with 17ß-oestradiol alone or in controls. Furthermore, treatment with different doses of testosterone resulted in an increase in MGD in a dose-dependent manner. These findings highlight the effect of androgens on the stroma, probably through a paracrine action of epithelial cells. The stroma may, in turn, promote mammary carcinogenesis in a reciprocal fashion.

Abbreviations: AR, androgen receptor; E2, 17ß-oestradiol; ER, oestrogen receptor; IGF-I, insulin-like growth factor I; IGV, integrated grey value; MGD, mammary gland density; MGV, mammary gland volume; MGW mammary gland weight; RGB, red, green and blue; T, testosterone; TGF, transforming growth factor.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is the most common cancer and the second most frequent cause of cancer death in women (1). The American Cancer Society has estimated a 30% incidence of breast cancer (178 700 new breast cancer cases) and ~16% of all cancer related mortality (43 500 cancer related deaths) in 1998.

A number of factors, including nulliparity, late parity, early menarche, late menopause and family history of breast cancer, are linked to increased risk of breast cancer. In contrast, early parity, late menarche, early menopause, duration of lactation and hormone deprivation are associated with a lower risk. These epidemiological observations highlight the role of oestrogens and oestrogen receptors (ERs) in the development of breast cancer (27). On the other hand, androgens have also been implicated as a possible carcinogenic factor in breast cancer (8). This proposal is mainly based on the fact that the incidence of breast cancer is relatively high in post-menopausal women, when androgenic hormone levels are high (9). Furthermore, administration of androgens for cystic disease of the breast has been shown to increase the risk of breast cancer (10). It has been reported that pre-menopausal women with breast cancer are associated with higher plasma androgen and progesterone levels (11). Moreover, Japanese women, with a lower breast cancer risk than their American and British counterparts, have lower plasma androgen levels (12,13). More recently, a number of researchers have demonstrated evidence that, among all plasma steroids, the relationship between testosterone (T) levels and breast cancer is strongest (14). These epidemiological findings, though providing clues linking androgens to breast carcinogenesis, have shed little light on the underlying mechanism(s).

Androgens mediate their function by binding to the intracellular androgen receptor (AR). In studies of AR distribution in human tissues, Ruizeveld de Winter et al. (15) and Janssen et al. (16) have reported that most alveolar and duct cells in the mammary gland express the AR, whereas myoepithelial and stromal cells are negative for the AR. In western blot analyses, Kimura and Mizokami (17) have also detected abundant expression of the AR in T47D and MCF-7 breast cancer cells. However, the roles of androgens and the AR in development and carcinogenesis of the mammary gland have, unfortunately, been neglected.

We have observed that in female rats, treatment with a combination of oestrogen and T can induce a high incidence of breast cancer with a shortened latency period (18). Similar results have been observed by other researchers in male Noble rats (19). We have further demonstrated that the dose of T affects only the latency period of mammary cancer, not the final incidence (18,20). In their study on male Noble rats, Liao et al. (21) also observed that androgens could shorten the latency period, enhance tumour size and increase the incidence of mammary cancers.

Some researchers have suggested that oestrogens and their metabolites could cause both non-genotoxic and genotoxic effects in hamster liver and kidney as well as rat liver carcinogenesis (22). Other researchers have further suggested that the carcinogenic activity of oestrogens might be mediated or modulated by specific oestrogen-metabolizing enzymes in target cells (23). More recently, Yu and co-workers (24) found that after activation, 17ß-oestradiol (E2) is able to bind to DNA and E2–DNA adducts can be detected by 32P-post-labelling. They further reported that when female ACI rats are given intramammary injections of E2 and activated E2, identical DNA adducts are formed in vivo and that the activated E2 is at least 25 000-fold more effective in forming DNA adducts in mammary glands than E2 (25), suggesting that E2 may act as an initiator in breast carcinogenesis.

Based on this background, we hypothesize that oestrogen and androgen may act in concert on the mammary gland to induce breast carcinogenesis, with oestrogen serving as the predominant initiator whereas androgen acts as a promoter. In order to verify this hypothesis, we have carried out a series of comparative studies to examine the morphological changes, expression of growth factors and their receptors, expression of oncogenes and a few other parameters. In this paper, we report the changes in morphology of the mammary gland and the stroma after treatment with various sex hormone protocols. Our data show that androgens have a major proliferative effect on mammary perialveolar connective tissue, together with a concurrent reduction in fatty tissue, and that these changes, which were totally blocked by flutamide, were closely associated with mammary carcinogenesis. These findings highlight the actions of androgens on the stroma, probably through a paracrine influence of the mammary epithelium. The stroma, in turn, may promote mammary carcinogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Young female Noble rats (3 months old) were housed three to a cage under standard conditions (22 ± 2°C, 40–70% relative humidity, 12 h light/12 h dark). Animals were fed with standard rat chow with tap water ad libitum. Their body weights were recorded weekly. All surgical operations were carried out under pentobarbitone anaesthesia. Under proper aseptic conditions, a small incision was made in the left inguinal region and hormone-filled Silastic tubing was inserted s.c. The rats were killed at the end of the experiment by cervical dislocation.

Hormone treatments
At 3 months of age sexually mature female Noble rats, weighing 180–210 g, were randomly divided into six groups (24 animals/group) and received the following treatments. (i) Group 1 (1E2 + 4T). The rats were surgically implanted s.c. in the left inguinal region with four 2.0 cm lengths of Silastic tubing (i.d. 1.6 mm, o.d. 3.2 mm, sealed with RTV-108 silicone rubber adhesive sealant; General Electric, Waterford, NY) tightly packed with testosterone propionate (each 2 cm tube containing 30 mg, total 120 mg) (Sigma, St Louis, MO) and one 1.0 cm Silastic tube containing E2 benzoate (22 mg) (Sigma) for 1, 3, 7 or 13 weeks. (ii) Group 2 (1E2). The rats were implanted s.c. with one 1.0 cm Silastic tube of E2 (22 mg) for 1, 3, 7 or 13 weeks. (iii) Group 3 (4T). The rats were implanted s.c. with four 2.0 cm Silastic tubes of testosterone for 1, 3, 7 or 13 weeks. (iv) Group 4 (control). The rats received empty Silastic tubes sealed with the same sealant for 1, 3, 7 or 13 weeks. (v) Group 5 (flutamide treatment). The rats were treated with flutamide plus 1E2 + 4T, 1E2, 4T or empty capsules for 7 weeks. Flutamide was packed in four 2.0 cm long Silastic tubes. (vi) Group 6 (tamoxifen treatment). The rats were treated with tamoxifen plus 1E2 + 4T, 1E2, 4T or empty capsules for 7 weeks. Tamoxifen (Sigma) was packed in one 1.0 cm Silastic tube. Six rats (n = 6) were used at each of the four time points or for each treatment in all groups. All tubes were replaced at 12 week intervals.

In order to investigate the correlation between the number of T implants and mammary gland density (MGD) we also carried out an experiment treating the rats with 2, 4 or 6 T implants (abbreviated to 2T, 4T and 6T, respectively).

The tumours used were induced by treating the rats with 1E2 + 4T for >3 months (18,20).

Autopsy and histological examination
The rats were killed at 1, 3, 7 or 13 weeks after the initiation of treatment. The left thoracic mammary glands were removed and fixed immediately in 10% phosphate-buffered formalin (pH 7.2), trimmed and embedded in paraffin. Paraffin sections (4 µm) were prepared and stained with haematoxylin and eosin for histopathological examination, using the criteria and classification of breast pathology as outlined by Rosen (26). Mammary tumours were obtained from animals treated with 1E2 + 4T for >3 months and similarly processed.

Immunocytochemistry
The avidin–biotin complex (ABC) method was used for all immunocytochemical examination. Briefly, the slides were deparaffinized, rehydrated in water and incubated with 3% H2O2 for 20 min to block endogenous peroxidase activity. After pre-incubation with normal serum for 20 min at room temperature, the primary antibody was applied and incubated overnight at 4°C. The sections were then incubated with the appropriate biotinylated secondary antibody at 1:200 dilution followed by peroxidase-conjugated avidin–biotin complex according to the manufacturer's instructions (Elite ABC kit; Vector, Peterborough, UK) and diaminobenzidine (Dako, Carpinteria, CA). The sections were then counterstained with Meyer's haematoxylin. Between each change of incubation the sections were rinsed three times in phosphate-buffered saline for 5 min each.

To examine expression of the AR, ER and Ki-67, antigens were retrieved by heating the slides in citrate buffer (pH 6.0) with a MW oven (800 W, 2.45 gHz; National Co., Tokyo, Japan) for 15 min at full power.

The primary antibodies used were: monoclonal ER antibody (C-314 and C-311, dilution 1:50) raised against the N-terminal domain of ER-{alpha} of human origin (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal AR antibody (N-20, dilution 1:200) raised against the N-terminal of the AR of human origin (Santa Cruz Biotechnology); monoclonal Ki-67 antibody (NCL-Ki67-MM1, dilution 1:500) reacting to nuclear antigen (Ki-67) present in all phases of the cell cycle (Novocastra, Newcastle upon Tyne, UK).

For negative controls, normal rabbit or normal mouse sera were used instead of the corresponding primary antibodies. In addition, pre-absorption of antisera to the ER and AR with a 50-fold concentration of specific peptides was also used to ensure the specificity of each antibody.

To evaluate the Ki-67 labelling index, in each group 500 epithelial cells and 500 stromal cells were counted in randomly chosen fields at x400 magnification. The Ki-67 labelling index was expressed as the number of clearly labelled Ki-67-reactive nuclei in 500 cells counted.

Image analysis
The intensity of immunoreactivity for AR and ER in `pre-malignant' mammary epithelial cells and cancer cells was quantified by measuring the integrated grey values (IGV) of red, green and blue colours (RGB), which were expressed in composite units to represent the intensity of immunoreactivity (27). Briefly, microscopic images were studied with a Zeiss Axiolab microscope (Carl Zeiss, Jena, Germany) equipped with a JVC TK-C1380 video camera (Victor Co. of Japan, Tokyo, Japan) connected to a colour video monitor. The Leica Qwin image processing and analysis system (Leica Imaging Systems, Cambridge, UK) was used for morphometric analysis. To determine the intensity of immunoreactivity, we evaluated 10 randomly chosen fields of each immunoperoxidase-stained section at x200 magnification. In each group, 10 similar randomly chosen fields at x200 magnification were selected on each of the slides from all animals. A density threshold (RGB) was set to quantify the positive immunoperoxidase reaction product. The threshold was selected to exclude the background haematoxylin counterstain. The same threshold and system settings were used for all slides quantified. The number of pixels falling within the threshold, indicating the quantity of immunoperoxidase reaction product, was recorded for each field. The intensity of immunoreactivity for each slide was expressed as the mean IGV of RGB ± SD.

Mammary gland density measurement
Right thoracic and right inguinal mammary glands were carefully dissected out together with their enclosing fat pad and weighed (mammary gland weight, MGW). The mammary gland volume (MGV) was determined by the underwater weighing method as described by Wang et al. (28). The mammary gland density (MGD) was determined by the equation: MGD = MGW/MGV (g/cm3)

Statistical analysis
Data on the Ki-67 index, the IGV of AR and ER and MGD were given as means ± SD. ANOVA was used to compare the means of Ki-67 labelling index, the means of IGV of AR and ER and the means of MGD in the four groups investigated. We used the statistical software program SPSS 7.0 to generate the analysis (29). P < 0.05 was considered to be statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Histopathology
The histological pattern of the mammary gland in the age-matched control animals showed all the characteristic features of an adult inactive breast, namely the glands had a sparse cluster of epithelial tubules surrounded by small amounts of connective tissue which was in turn embedded in a large fat pad. The epithelial ducts had a narrow, small lumen, lined by cuboidal cells with darkly stained nuclei. The acinar epithelial cells were small and apparently inactive (Figure 1DGo).



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Fig. 1. Effects of sex hormones on the histology of mammary gland. (A) 1E2 + 4T (7 weeks). Note that the dilated alveolar ducts are lined by large `clear' cells, with evidence of secretion in the lumen. The perialveolar tissue consists mainly of fibrous connective tissue, with very few adipocytes. (B) 1E2 (7 weeks). The changes are mainly in dilated alveolar ducts. The perialveolar connective tissue is not as prominent as seen in (A). (C) 4T (7 weeks). The mammary gland again has dilated alveolar ducts and prominent perialveolar connective tissue, with very few fat cells. (D) Control (7 weeks). Mammary tissue from a control animal to show the typical sparse, poorly developed alveolar ducts and fatty perialveolar stroma. (E) 1E2 + 4T (7 weeks). This micrograph shows a typical dysplastic area in the alveolar duct. Note that dysplastic cells (arrows) are characterized by pleomorphic nuclei. (F) 1E2 + 4T (10 weeks). Note the typical multifoci dysplastic changes (arrowheads) in the dilated alveolar duct. Note also the abundant connective tissue in this gland.

 
The mammary glands of rats implanted with the combination 1E2 + 4T had a more extensively branched and dilated alveolar or ductal system against a background of connective tissue stroma. After treatment with the hormones, there was an apparent increase in size of tubuloalveolar structures, consisting mainly of hypertropic `clear' epithelial cells (Figure 1AGo). Interestingly, there was a significant increase in the amount of perialveolar as well as interlobular stroma connective tissue, accompanied by a decrease in the quantity of surrounding fat tissue (Figure 1AGo).

Similar morphological changes of mammary glands were observed in the 4T-treated group, except that the alveolar ducts were less dilated. However, the increase in perialveolar and interlobular stromal tissue was more significant (Figure 1CGo). On the other hand, in the E2 (1E2 group) treatment group, the changes were mainly restricted to epithelium. There was no significant increase in both the amount of connective tissue and the surrounding fat (Figure 1BGo).

The alveolar ducts showed a variable degree of epithelial dysplasia after treatment with 1E2 + 4T for 7 and 10 weeks (Figure 1E and FGo, respectively). Focal areas of dysplastic cells were seen interspersed among relatively normal glandular cells. Dysplastic cells were characterized by a loss of polarity and pleomorphic nuclear morphology. Irregular proliferation of epithelium within ducts was often accompanied by an apparent increase in secretory activity (Figure 1A and EGo). We also observed a marked dilation of ducts resulting from the accumulation of secretory material and foamy cell debris (Figure 1A and EGo). Typically, patches of dysplastic and carcinomal cells were seen initially as focal thickening of epithelium extending into the lumen (Figure 1E and FGo). There was no epithelial dysplasia present in tamoxifen- or flutamide-blocked groups in the same treatment periods (data not shown).

We further measured the MGD in order to correlate the changes in amount of stroma connective tissue to fatty tissue in mammary gland. The results showed that the MGD in the 1E2 + 4T and 4T groups were significantly higher than in the control group (Figure 2AGo). However, there was no significant difference between the 1E2 and control groups. When the animals were treated simultaneously with flutamide and 1E2 + 4T or 4T, a total blockage of the increase in MGD was observed. The end result after treatment with flutamide was that there was no difference among the four groups in MGD values. On the other hand, simultaneous treatment with tamoxifen and the corresponding treatment protocols gave rise to a slight increase in MGD in each group (Figure 2AGo). In order to examine the effects of different doses of T on the changes in MGD, rats were treated with different doses of T (i.e. 0T, 2T, 4T and 6T). The MGD value increased in a dose-dependent manner (Figure 2BGo).



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Fig. 2. Effects of sex hormones on MGD. The data are the means of MGD. Error bars represent the SD. (A) Effects of different sex hormones and their receptor blockers on mammary gland density. Bars with different lower case letters are significantly different from one another at P < 0.05 among different sex hormone treatments. Bars with different capital letters are significantly different from one another at P < 0.05 within the same hormone treatment combined with different blocking methods. (B) Effects of different doses of T on mammary gland density. Bars with different letters are significantly different from one another at P < 0.05.

 
Androgen and oestrogen receptors
In mammary glands of rats treated with 1E2 + 4T (Figure 3AGo) and 4T (Figure 3CGo), strong AR immunoreactivity was observed in the ductal or alveolar epithelial cells, whereas in the control group (Figure 3DGo) the ductal or alveolar epithelial cells showed only low to moderate AR immunoreactivity. Interestingly, in the 1E2 group (Figure 3BGo), the ductal epithelial cells showed only very weak reactivity. Negative immunoreactivity was observed in myoepithelial and stromal cells in all four groups. We have observed an uneven distribution of AR immunoreactivity in mammary tumours induced by a combination of T and E2. It appeared that in many cells, AR immunoreactivity was stronger in tumour cells in the peripheral region than in the central region of the tumour (Figure 3EGo).



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Fig. 3. Effects of sex hormones on expression of the AR in the mammary gland. (A) 1E2 + 4T (7 weeks). Strong AR immunoreactivity is observed in the ductal or alveolar epithelial cells. Almost all stromal cells are AR negative. (B) 1E2 (7 weeks). The ductal epithelial cells show only very weak reactivity with this antibody. (C) 4T (7 weeks). Strong AR immunoreactivity is observed in the ductal or alveolar epithelial cells. Again, most stromal cells are AR negative. (D) Control (7 weeks). The ductal or alveolar epithelial cells show only low to moderate AR immunoreactivity. Negative immunoreactivity was observed in myoepithelial and stromal cells in all four groups. (E) In mammary tumours AR immunoreactivity is much stronger in the peripheral region (arrows) than in the central region of tumour cells.

 
On the other hand, strong ER immunoreactivity was observed in mammary glands of rats treated with 1E2 and empty implants, in the ductal or alveolar epithelial cells (Figure 4B and DGo). In rats treated with 1E2 + 4T (Figure 4AGo) and 4T (Figure 4CGo), only moderate immunoreactivity was observed in ductal or alveolar epithelial cells. It seemed that some stromal cells also showed moderate immunoreactivity to ER, especially in mammary glands of rats treated with 1E2 + 4T (Figure 4AGo). In mammary tumour, moderate ER immunoreactivity was observed in tumour cells but not in adjacent myoepithelial and stromal cells (Figure 4EGo).



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Fig. 4. Effects of sex hormones on expression of the ER in mammary gland. (A) 1E2 + 4T (7 weeks). Moderate ER immunoreactivity can be seen in ductal or alveolar epithelial cells. (B) 1E2 (7 weeks). Strong ER immunoreactivity is observed in mammary glands in the ductal or alveolar epithelial cells. (C) 4T (7 weeks). Moderate ER immunoreactivity can also be seen in ductal or alveolar epithelial cells. (D) Control (7 weeks). Strong ER immunoreactivity is observed in mammary glands in the ductal or alveolar epithelial cells. It seems that some stromal cells also showed moderate immunoreactivity to ER, especially in mammary glands of rats treated with 1E2 + 4T, as shown in (A). (E) In mammary tumour, moderate ER immunoreactivity is observed in tumour cells but not in adjacent myoepithelial cells and stromal cells.

 
Because almost all mammary epithelial cells expressed AR and ER, we evaluated the expression of AR and ER by measuring the intensity of immunoreactivity rather than counting the labelling index of AR and ER. The results of image analysis for the intensity of AR and ER immunoreactivity are summarized in Table IGo. T treatment, either alone or in combination with E2, resulted in a significantly higher IGV of AR than E2 treatment alone or no treatment. The IGV of AR was significantly lower in the 1E2 group than in the control group, indicating that treatment with E2 may suppress the expression of AR. The highest IGV for AR was observed in fully developed mammary cancers. In contrast, the IGV of ER in fully developed mammary cancers was lowest among the five investigated groups. Furthermore, E2 treatment alone resulted in the highest IGV of ER. The IGV of ER was significantly lower in the 1E2 + 4T and 4T groups than in the 1E2 and control groups, indicating that T treatment may suppress the expression of ER. The differences in IGV of ER among the 1E2 + 4T and 4T group and fully developed mammary cancers were not significant.


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Table I. The IGV of AR and ER in mammary glands and mammary cancersa
 
Ki-67 index
After treatment with the corresponding hormonal protocols for 1 week, the Ki-67 labelling indices of mammary epithelial cells were not different among the three experimental groups, but each was significantly higher than that of the control group (Figure 5AGo). However, at week 3 after treatment there was a sharp drop in Ki-67 labelling index of the 4T group, from 467 ± 21 to 207 ± 26. From then on, the Ki-67 index was maintained at a steady high level. A similar downward trend was also observed for the E2-treated group, but the high Ki-67 index of the 1E2 + 4T group was maintained throughout. The increase in Ki-67 labelling index in epithelium was blocked by simultaneous treatment with tamoxifen (Figure 5BGo) but not by flutamide (Figure 5CGo), suggesting that the increase in Ki-67 labelling in epithelium was mediated through the ER, not via the AR.



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Fig. 5. Effects of sex hormones on proliferation of mammary gland epithelial cells. The data show the means of Ki-67 labelling indices, with the error bars representing SD. (A) These sets of histograms show the Ki-67 labelling indices of epithelial cells treated with different hormonal protocols for different durations. Within the same treatment period, histograms denoted with different letters are significantly different at P < 0.05, while those with the same letters are not significant. (B) These sets of histograms show the effect of blocking with tamoxifen for 7 weeks. The results show that tamoxifen is effective in blocking the Ki-67 indices of epithelial cells in all groups. Bars with different letters indicate that indices are significantly different at P < 0.05. (C) These sets of histograms show the effect of blocking by flutamide for 7 weeks. Again, bars with different letters indicate that indices are significantly different at P < 0.05.

 
In contrast, treatment with 1E2 + 4T and 4T alone for 3 weeks produced a significantly higher Ki-67 labelling index of the stroma than that produced by 1E2 alone (Figure 6AGo). Furthermore, there was a time-dependent pattern of increase in Ki-67 labelling index seen in the 1E2 + 4T and 4T groups (Figure 6AGo). The increase in Ki-67 labelling index in the stroma was inhibited by simultaneous treatment with flutamide (Figure 6BGo), indicating that the increase was mediated by the AR of the epithelium.



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Fig. 6. Effects of sex hormones on proliferation of mammary gland stromal cells. The data show the means of Ki-67 labelling indices, with the error bars representing SD. (A) These sets of histograms show the Ki-67 indices in stromal cells at different periods of treatment by different protocols of hormones. Histograms with different letters indicate that proliferation indices of stromal cells between groups and at different time points are significant at P < 0.05. (B) These sets of histograms represent the blocking effect on stromal cells of flutamide treatment. The results show that flutamide can effectively reduce the Ki-67 indices of stromal cells (P < 0.05).

 
Figure 7Go shows samples of results for Ki-67 index in the 1E2 + 4T (Figure 7AGo) and 1E2 + 4T + flutamide (Figure 7BGo) groups, as well as in mammary cancers (Figure 7CGo). In the 1E2 + 4T group, the Ki-67-positive immunoreactivity appeared not only in epithelial cells but also in stromal cells (Figure 7AGo), whereas in the 1E2 + 4T + flutamide group, the immunoreactivity was mainly restricted to epithelial cells (Figure 7BGo), indicating that stromal cells were not stimulated to proliferate. In mammary cancer, both cancer cells and surrounding stromal cells expressed Ki-67 very strongly (Figure 7CGo).



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Fig. 7. The immunoreactivity of Ki-67 in mammary glands treated with sex hormones and in breast cancer induced by a combination of T and E2. (A) In the 1E2 + 4T group, the Ki-67-positive immunoreactivity appears not only in epithelial cells but also in stromal cells. (B) In the 1E2 + 4T + flutamide group, the immunoreactivity is mainly restricted to epithelial cells. (C) In mammary cancer, both cancer cells (arrows) and surrounding stromal cells (arrow heads) express Ki-67 very strongly.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have previously shown that treatment with a combination of oestrogen and T can induce a higher incidence of breast cancer with a shortened latency period (18,20). Based on these observations, we have proposed that oestrogen and androgen act in concert on breast carcinogenesis, with oestrogen possibly being the predominant initiator whereas the androgen acts as a major promoter for mammary gland carcinogenesis. Here, we have further shown that the promoting role of androgen in mammary gland carcinogenesis may be mediated through the stromal cells by indirectly stimulating proliferation of fibroblasts of the perialveolar and interlobular stroma. However, the mechanism involved in this process has not been clearly established at this stage.

Our results demonstrate that the AR is expressed in alveolar or ductal epithelial cells but not in myoepithelial and stromal cells. This is consistent with the results reported by other researchers (1517,3032). More importantly, this study has revealed that animals treated with 1E2 + 4T and 4T have strong AR reactivity, whereas those treated with 1E2 alone have only weak AR reactivity. On the other hand, the ER immunoreactivity of mammary gland epithelial cells is stronger in the 1E2 and control groups than in the 1E2 + 4T and 4T groups (Table IGo). This finding seems to suggest that expression of AR can be inhibited by high levels of oestrogen while expression of ER can be inhibited by high levels of androgen.

Our observation of strong ER immunoreactivity in the control group was initially somewhat puzzling, as it does not reconcile with clinical observations in humans. We have thus tried two anti-ER antibodies, which both showed a strong immunoreactivity in normal mammary gland epithelial cells. However, when we applied the same antibodies to normal human breast samples, negative immunoreactivity was consistently observed (unpublished observation). It would appear that the strong ER reactivity may be unique to the Noble rat and this may be one of the reasons why Noble rats are more sensitive to oestrogen stimulation than other strains of rat (33,34).

We have further observed that the presence of T, either alone or in combination with E2, gives rise to a significant increase in the number of fibroblasts in the periductal and interlobular stroma, together with a significant decrease in surrounding fat cells. According to Wisser et al. (35), fat-free body mass has a density of 1.100 g/cm3 and the body density is inversely correlated with the percentage of body fat. This observation indicates that the density of tissues is dependent upon the ratio of fat to non-fat cells. Based on this notion, we have measured the MGD to confirm our microscopic findings. In the 1E2 + 4T and 4T groups, the MGD values approached 1.100 g/cm3, but in the 1E2 group, as well as the control, group the MGD values were ~0.890 g/cm3, suggesting that the ratio of non-fat to fat cells in the mammary gland had increased after treatments containing T. Furthermore, treatment with different doses of T results in an increase in MGD in a dose-dependent manner, indicating that T may be responsible for the increased ratio of non-fat to adipose tissue. This is consistent with our morphological observation that under the influence of T, the fatty stroma of the mammary gland is replaced by fibrous non-fatty stroma.

We have attempted to determine the specific cell type which may be stimulated by E2 and T. Our data show that the Ki-67 labelling index of the epithelium is affected by E2, while the labelling index of the stromal cells is affected by T. More importantly, in the stroma almost all Ki-67-positive cells belong to fibroblasts. Very little or no Ki-67-positive immunoreactivity was observed in fat cells. This would mean that T affects the proliferation of fibroblastic cells, but not fat cells.

As indicated earlier, the AR is located only in alveolar or ductal epithelial cells. Despite the absence of AR, the proliferation of stromal fibroblasts can be stimulated by T, presumably through the receptor on alveolar epithelial cells. This effect of T can be blocked by flutamide. The mechanism(s) by which the stromal fibroblasts are stimulated to proliferate remains unknown. We speculate that the increase in proliferative index in stromal cells may be due indirectly to an action of androgen through its AR in alveolar epithelial cells. One of the possibilities may be through cytokines or growth factors secreted by alveolar or ductal epithelial cells in response to T stimulation.

We believe that this may be due to paracrine influences of factors secreted by epithelial cells, such as transforming growth factor (TGF) ß. TGF-ß1 is an important growth factor involved in epithelial–stromal interaction. The important point concerning TGF-ß1 in epithelial–stromal interaction in carcinogenesis is its various effects on tumour host tissue, particularly with respect to angiogenesis, immunosuppression, fibroblast activation and restructuring of the tumour extracellular matrix. Although TGF-ß1 is a powerful inhibitor of normal epithelial cell proliferation, its effect on epithelial tumour cells is slight in adenomas and totally lost in carcinomas (36). In our studies, we have observed that breast cancer cells expressed high levels of TGF-ß1. Furthermore, after prolonged treatment with 1E2 + 4T, there was apparent overexpression of TGF-ß1 in epithelial cells (37). Taken together, we believe that the following scenario may occur in the mammary gland under T stimulation. T may act through the AR on mammary epithelial cells. Upon stimulation by T, the epithelial cells secrete TGF-ß1, which can act on stromal cells to stimulate growth of the stroma. This may explain growth of the stroma on androgen stimulation. Moreover, if initiation of neoplastic growth renders the initiated cells, at least partially, resistant to growth inhibition by TGF-ß1 in epithelial carcinogenesis, the presence of TGF-ß1 may exert a selective pressure favouring outgrowth of the initiated clone and facilitating the promotion process (38). These observations suggest that the paracrine loops between the stromal and epithelial or tumour cells may have a reciprocal proliferative effect.

Interestingly, we have also observed that very strong AR immunoreactivity is found at the periphery of the tumour cells adjacent to the proliferative stroma in many mammary tumours (Figure 3EGo). This observation supports our conviction that tumour cells may stimulate active growth of surrounding stromal tissues.

There is accumulating evidence that stroma fibroblasts play a crucial role in both normal development and in carcinogenesis of the mammary gland. The stroma of the mammary gland accounts for >80% of the resting breast volume (39). This stroma consists of fat cells, fibroblasts, blood vessels and a macromolecular network composed of glycoproteins and proteoglycans, known collectively as the extracellular matrix. Fibroblasts can respond to hormonal stimulation by expression and release of a panel of growth factors, such as TGF-{alpha} and TGF-ß, epidermal growth factor, fibroblast growth factors I and II, insulin-like growth factors I (IGF-I) and II and vascular endothelial growth factor (40). Each of these stromal factors may contribute to the regulation of epithelial growth in a paracrine fashion, although some of them could contribute to the regulation of breast cancer cells in an autocrine fashion (41). In their review on stromal–epithelial interactions in the normal and neoplastic prostate, Hayward et al. (42) proposed the concept of `tumour stroma' as an essential component of the carcinogenic process. Wang and Wong (43) have reported a switch from paracrine to autocrine regulatory activity of IGF-I in rat prostate carcinogenesis, through its receptor. Dynamic interaction of emerging or established carcinoma cells with the stroma has led to the concept that the stroma is not an inert scaffold supporting the epithelial neoplasm, but instead has an active, essential role in carcinogenesis and tumourigenesis (4446). Studies of malignant breast tumours have also led to the concept of the activated or abnormal tumour fibroblast (44,47,48). Our observations on changes in the stroma upon hormonal treatment are consistent with the theme outlined. We thus believe that this epithelial–stromal interaction is of crucial importance in mammary carcinogenesis.

Johnston et al. (49) have shown that conditioned medium from a 3T3L1 mouse preadipocyte cell line can inhibit growth and thymidine incorporation of cultured human MCF-7 breast cancer cells. In contrast, conditioned medium from the parent 3T3 fibroblast cell line led to stimulation of breast cancer cell growth and thymidine incorporation. These in vitro studies have suggested that adipocytes and fibroblasts in the breast stroma can modulate tumour growth. In an attempt to grow a breast cancer in the nude mouse, many laboratories (including ours) have had difficulties in obtaining tumour growth even with hormone implants (50). However, when co-injected with fibroblasts, breast cancer cells could grow easily in nude mice. These in vitro studies have provided further evidence to support the hypothesis that fibroblasts play a very important role in the development of mammary tumours.

Intact female rat mammary glands are characterized by sparse clusters of epithelial tubules embedded in small amounts of connective tissue surrounded by a large fat pad. After treatment with sex hormones, the incidence of breast cancer increases significantly, especially in the 1E2 + 4T group. More importantly, the dose of T affects only the latency period of breast cancer, not the final incidence. No breast cancer was detected in the age-matched untreated control group over the same experimental period (18,20). Taking the results of the present study into consideration, our studies have provided evidence not only for a promoting role of androgens and the AR in breast carcinogenesis, but also to support the concept that the stroma has an active, essential role in carcinogenesis and tumourigenesis, although certain key elements remain unknown.


    Notes
 
1 To whom correspondence should be addressed Email: ycwong{at}hkucc.hku.hk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 9, 1999; revised April 27, 1999; accepted April 28, 1999.