Comparative analysis of the uterine and mammary gland effects of progesterone and medroxyprogesterone acetate

Objectives

In combined hormone replacement therapy (HRT) progestins are used to inhibit estradiol-activated uterine epithelial cell proliferation. In comparison to estradiol-only therapy, combined HRT leads to enhanced proliferation of mammary epithelial cells. In a quantitative mouse model, we assessed the balance between uterine and undesired mammary gland effects for two progestins that are widely used in HRT, progesterone and medroxyprogesterone acetate.

Study design

Mice were ovariectomized and after 14 days they were treated subcutaneously with either vehicle, estradiol (100 ng) or estradiol plus increasing doses of progesterone or medroxyprogesterone acetate for three weeks.

Main outcome measures

Measures for progestogenic mammary gland activity were stimulation of side-branching and stimulation of epithelial cell proliferation. Progestogenic activity in the uterus was assessed by measuring inhibition of estradiol-activated uterine epithelial cell proliferation. ED₅₀ and ID₅₀ values for the distinct readouts were obtained and dissociation factors for uterine versus mammary gland activity were calculated.

Results

MPA demonstrated uterine activity and mitogenic activity in the mammary gland at the same doses. In contrast, progesterone showed uterine activity at doses lower than those leading to significant stimulation of epithelial cell proliferation in the mammary gland.

Conclusions

Progestins do not behave the same. Use of the natural hormone progesterone, but not MPA, in combined hormone therapy might offer a safety window between uterine effects and undesired proliferative activity in the mammary gland.     

Transduction pathways of GH in ovine mammary acini involving regulated and functional growth hormone receptors

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50–60,000?Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50–60,000?Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.     

Transduction pathways of GH in ovine mammary acini involving regulated and functional growth hormone receptors

We have investigated the localization and regulation of growth hormone (GH) receptor-related proteins in the ovine mammary gland. Using a new rabbit polyclonal antibody (7122A) directed against the recombinant extracellular domain of GH receptor (GHR-ECD) for western blot assays, we found two bands with apparent molecular weights of 70,000 and 50-60,000 Da in ovine mammary gland solubilized proteins. The 70,000-protein was consistent with a membrane GH receptor form deprived of post-translational modifications such as phosphorylation, glycosylation or ubiquitin binding. The 50-60,000 Da was consistent with soluble GH binding protein, generated by the cleavage of membrane GH receptor. The intensity of related GHR proteins increased slightly throughout mammary gland development and was correlated with the amount of GHR immunoreactivity observed in the mammary gland sections. Moreover, a temporal and spatial regulation of GHR immunoreactivity was found in alveolar epithelial cells. Clearly, marked GHR immunoreactivity was associated with the apical membranes of alveolar epithelial cells at lactation. The up-regulation of related GHR proteins during the differentiation of mammary tissue supports the hypothesis that GH may act specifically via its own receptors. In ovine mammary cells, GH was able to promote a time-dependent activation of MAP kinases such as prolactin (Prl) and placental lactogen (PL). GH was also able to promote slight and transient Stat5 DNA-binding activity. Differences in the time dependence of Stat5 DNA-binding activation by the three different ligands, GH, Prl and PL, were found. All these results emphasize the direct action of GH on ovine mammary cells and highlight the specificity of action of this ligand.     

Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1 gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1 gene is truncated upstream of the transmembrane domain, and the resulting Notch1 intracellular domain (Notch1(intra)), deleted of most extracellular sequences, is overexpressed. Although Notch1(intra) transforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivo was not studied. Therefore, we generated MMTV/Notch1(intra) Tg mice that overexpress murine Notch1(intra) in the mammary glands. We observed that MMTV/Notch1(intra) Tg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of beta-casein. Notch1(intra) repressed expression of the beta-casein gene promoter, as assessed in vitro with a beta-casein/luciferase reporter construct. The MMTV/Notch1(intra) Tg females developed mammary gland tumors, confirming the oncogenic potential of Notch1(intra) in vivo. Furthermore, MMTV/Notch3(intra) Tg mice exhibited a very similar phenotype. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.     

Dynamic regulation of the Stra13/Sharp/Dec bHLH repressors in mammary epithelium.

Mammary gland development is a dynamic process involving cyclical proliferation, cellular differentiation, and cell death. In this study, we have determined that expression of the Stra13/Sharp/Dec basic helix-loop-helix (bHLH) family is dynamically regulated in mammary epithelium. In cultured HC11 cells, epidermal growth factor (EGF) treatment rapidly induces Stra13 protein accumulation, which is blocked by the synthetic glucocorticoid, dexamethasone. Neither the induction of Stra13 by EGF nor its repression by dexamethasone correlates with changes in Stra13 mRNA levels. During mouse mammary gland development in vivo, Stra13 is highly expressed in epithelial ducts during puberty, and strongly induced in both ducts and alveoli during early involution, while the related Sharp-1 gene is highly expressed only during late stages of involution. Together, these data indicate that Stra13/Dec/Sharp-family bHLH repressors are dynamically regulated during mammary gland development and may function to regulate apoptosis in this tissue.     

Immunohistochemical expression of monoclonal antibodies (epi-1 and myo-1) derived from human breast cancer against rat mammary tumors.

Immunohistochemical expression of monoclonal antibodies epi-1 and myo-1 derived from human breast cancer cell line (HBC-4W) was examined for DMBA-induced rat mammary tumors. Antibody epi-1 reacted with luminal epithelial cells while antibody myo-1 reacted with myoepithelial cells of the mammary glands in rats, respectively. The reactions with both antibodies were markedly visible, in particular, in the normal mammary gland, tumor-like lesions and benign epithelial mammary tumors in rats, which showed clear two-cell-type structures. Among malignant mammary tumors, adenocarcinoma was strongly positive with antibodies epi-1 and myo-1. However, squamous cell carcinoma and adenoacanthoma mainly reacted with antibody epi-1. On the other hand, the intercellular matrices of pleomorphic cell sarcoma and stromal areas of the normal mammary gland or epithelial tumors were positive with antibody myo-1.     

Circadian clock and cell cycle gene expression in mouse mammary epithelial cells and in the developing mouse mammary gland.

Mouse mammary epithelial cells (HC-11) and mammary tissues were analyzed for developmental changes in circadian clock, cellular proliferation, and differentiation marker genes. Expression of the clock genes Per1 and Bmal1 were elevated in differentiated HC-11 cells, whereas Per2 mRNA levels were higher in undifferentiated cells. This differentiation-dependent profile of clock gene expression was consistent with that observed in mouse mammary glands, as Per1 and Bmal1 mRNA levels were elevated in late pregnant and lactating mammary tissues, whereas Per2 expression was higher in proliferating virgin and early pregnant glands. In both HC-11 cells and mammary glands, elevated Per2 expression was positively correlated with c-Myc and Cyclin D1 mRNA levels, whereas Per1 and Bmal1 expression changed in conjunction with beta-casein mRNA levels. Interestingly, developmental stage had differential effects on rhythms of clock gene expression in the mammary gland. These data suggest that circadian clock genes may play a role in mouse mammary gland development and differentiation.     

Conserved antigen expression in epithelial tumors recognized by monoclonal antibody 4A9 generated against canine mammary carcinoma cells

Hybridoma-derived murine monoclonal antibodies (MoAbs) were generated by fusing P3X63-Ag8.653 myeloma cells with splenic cells from BALB/c mouse which had been immunized with viable canine mammary adenocarcinoma cells, CMT-2. Fifteen MoAbs were shown to react with immunizing cells in indirect immunofluorescence (IFA) and enzyme-linked immunosorbent (ELISA) assays. The reactivity of one IgM MoAb, designated 4A9, was evaluated. The antigen recognized by 4A9 on CMT-2 cells appeared to be localized both in cell membrane and cytoplasm against fixed and unfixed preparations by IFA. The 4A9 MoAb was found to bind with four of five canine mammary carcinoma cell lines while no binding was detected with normal fibroblastic cell lines. In vivo tissue distribution of 4A9 antigen was evaluated by indirect immunoperoxidase (IP) assay against formalin-fixed, paraffin-embedded sections of normal and neoplastic tissues. 4A9 MoAb reacted strongly to moderately with 75% of mammary carcinomas, moderately to weakly with 57% of benign mammary tumors, and strongly with squamous cell and perianal gland carcinomas (100%), interstitial cell tumors (100%), transitional cell carcinomas (43%), lung adenocarcinomas (40%), colon carcinomas (33%), and pancreatic adenocarcinomas (20%). Moderate to weak staining was detected with granulosa cell tumors (25%) and apocrine gland adenocarcinomas (50%). Strong reactivity with perianal gland carcinomas contrasted to no reactivity with perianal gland adenomas. No immunostaining was detected with a large variety and number of normal adult and fetal tissues tested; negligible and very restricted staining was observed in a few adult and fetal tissues. Normal mammary gland was negative. Since the antigen is expressed on the cell surface and in the cytoplasm of most mammary carcinoma cells and a variety of other epithelial tumor cells, the 4A9 antibody may have potential application in diagnosis and management of canine mammary cancer and a variety of other epithelial tumors.     

Transforming growth factor-alpha promotes mammary tumorigenesis through selective survival and growth of secretory epithelial cells.

Transforming growth factor (TGF)-alpha stimulates the growth and development of mammary epithelial cells and is implicated in the pathogenesis of human breast cancer. In this report we evaluate the consequences of overexpressing TGF-alpha in the mammary gland of transgenic mice and examine associated cellular mechanisms. When operating on a FVB/N genetic background (line MT100), TGF-alpha induced the stochastic development of mammary adenomas and adenocarcinomas f secretory epithelial origin in 64% of multiparous females. In contrast, tumors were exceedingly rare in virgin MT100 females, MT100 males, and multiparous FVB/N females. In MT100 females multiple foci of hyperplastic secretory lesions preceded the development of frank tumors; these initial lesions appeared during the involution period after the first lactation. Serial transplantation of these hyperplasias indicated an absence of proliferative immortality. Nevertheless, they gave rise to tumors at a low frequency and after a prolonged latency in virgin hosts; in multiparous hosts, tumors developed earlier and at a high incidence. The TGF-alpha transgene was highly expressed in hyperplasias and tumors but not in virgin and nonlesion-bearing tissue, suggesting that TGF-alpha overexpression provides a selective growth advantage. TGF-alpha also induced at lactation a 6.4-fold increase in DNA synthesis in MT100 epithelial cells, many of which were binucleated. MT100 mammary tissue experienced an obvious delay in involution, resulting in the postlactational survival of a significant population of unregressed secretory epithelial cells. In contrast, another line of transgenic mice on a CD-1 genetic background (MT42), in which TGF-alpha overexpression induced liver but not mammary tumors, failed to demonstrate postlactational epithelial cell survival. These data show that TGF-alpha promotes mammary tumorigenesis in multiparous MT100 mice by stimulating secretory epithelial cell proliferation during lactation and prolonging survival during involution. These points support the notion that TGF-alpha can act as a mitogen and also as a differentiation factor in mammary epithelium.     

Progesterone analogues similarly modulate endometrial matrix metalloproteinase-1 and matrix metalloproteinase-3 and their inhibitor in a model for long-term contraceptive effects.

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in normal menstruation, while MMP-1 and MMP-3 production by human endometrial stromal cells (HESCs) is repressed in vitro by progesterone. We postulated that the repression by synthetic progestins of MMP production from HESCs may not be fully maintained in the long term, and that this may account for the disturbed uterine bleeding patterns in women using long-acting progestins. In this study, a long-term HESC culture model was established to compare the effects of natural progesterone and a number of synthetic analogues (ORG2058, medroxyprogesterone acetate, norethindrone acetate, levonorgestrel and drospirenone) on the production by these cells of MMP-1 and MMP-3 and TIMP-1. Zymographic and enzyme-linked immunosorbent analysis of culture medium after 2 weeks showed that both natural progesterone and all of the synthetic progestins tested maintained a significant inhibition of MMP-1 and MMP-3 production. Production of mRNA for MMP-1 and MMP-3 was also suppressed by all progestins, while TIMP production was increased. Thus, menstrual bleeding disturbances which occur during the use of synthetic progestins is not likely to result directly from changes in the effect of long-term progestin exposure on MMP-1 or MMP-3 or TIMP-1 production by HESCs.     

Essential function of Wnt-4 in mammary gland development downstream of progesterone signaling

Female reproductive hormones control mammary gland morphogenesis. In the absence of the progesterone receptor (PR) from the mammary epithelium, ductal side-branching fails to occur. We can overcome this defect by ectopic expression of the protooncogene Wnt-1. Transplantation of mammary epithelia from Wnt-4(-)/(-) mice shows that Wnt-4 has an essential role in side-branching early in pregnancy. PR and Wnt-4 mRNAs colocalize to the luminal compartment of the ductal epithelium. Progesterone induces Wnt-4 in mammary epithelial cells and is required for increased Wnt-4 expression during pregnancy. Thus, Wnt signaling is essential in mediating progesterone function during mammary gland morphogenesis.     

On the possible role of mammary-derived growth hormone in human breast cancer

The incidence of breast cancer has risen worldwide, especially in countries where it used to be low, very probably as a result of economic prosperity and changes in life-style. In women, the available data have resulted in the concept of progression from normal breast development to cancer through precursor lesions sensitive to hormones and growth factors that can be produced locally in the mammary gland, acting as paracrine or autocrine stimulating agents. The local endocrine environment in the breast can be different from the situation in the circulation. In the dog, growth hormone (GH) can be produced locally in the mammary glands and its production can be stimulated by progestins. This GH probably plays a paracrine role in the progesterone-induced proliferation and differentiation of mammary epithelium. There is increasing evidence that the local mammary progestin/GH-axis is operational not only in dogs but also in human breast cancer. No data are yet available on the production of mammary-derived GH in women.     

Malignant myoepithelioma with a squamous epithelial component in the mammary gland of a cynomolgus monkey

A solid mass arising from the mammary gland was found in a 7-year-old female cynomolgus monkey. Histologically, the mass consisted of 2 components: spindle-shaped or ovoid sarcomatous cells and squamous epithelial cells. Metastatic nodules noted in the lung, liver and the gallbladder had the same histological features as the mammary mass. Immunohistochemistry revealed that the sarcomatous cells were positive for alpha-smooth muscle actin (alpha-SMA), vimentin, calponin, S-100 protein, epithelial membranous antigen (EMA), cytokeratin (large spectrum) and cytokeratin 14 (CK 14) in the cytoplasm, and p53, erbB-2 and progesterone receptor in the nuclei, but negative for desmin and estrogen receptor. The squamous epithelial cells were positive for EMA, cytokeratin (large spectrum) and CK 14, but negative for the rest. Both sarcomatous and squamous epithelial components were negative for glial fibrillary acidic protein (GFAP). Based on histological and immunohistochemical features, the present case was diagnosed as a malignant myoepithelioma with a squamous epithelial component in the mammary gland with distant metastases.     

Effects of progestins of human proliferative endometrium: an in vitro model of potential clinical relevance

Endometrium biopsy is a useful indicator of endometrium proliferation and is clinically relevant to diagnose cell proliferation and to evaluate response to progestin treatment and to monitor hormone replacement therapy. The aim of our study was to investigate the in vitro effects of progesterone and synthetic progestins on endometrium explants with a particular focus on estradiol receptor (ER) and progesterone receptor (PR) expression which reflects through cell secretion the hormone treatment efficiency. Most widely used progestagens belonging to three distinctive groups were investigated, i.e, medroxyprogesterone acetate (MPA), norethindrone acetate (NOR) and nomegestrol acetate (TX) which are respectively pregnane, 19-nortestosterone and norpregnane derivatives. We used organ culture from human proliferative endometrium, in which tissue integrity, particularly gland/stroma relationships are preserved. Progestins induce epithelial cell secretion and most effects were observed at the highest concentration tested (10(-7) M) and by TX and MPA on homogeneous and on heterogeneous (including also secretory glands) proliferative endometrium respectively. In these conditions, ER as well as PR expression were decreased on both glandular and stromal cells. In contrast, progesterone at 10(-7) M significantly decreased only PR, in glands and in stroma of homogeneous proliferative endometrium, and just in stroma of heterogeneous endometrium. NOR exhibited less effects. At lower concentrations (10(-8) M, 10(-9) M), significantly less effects were observed by synthetic progestins on proliferative endometrium. The experiments show that the different types of progestins do not exhibit in vitro similar effects. Since progestins variably act on proliferative endometrium, the exposure of endometrium explants to progestins may be a useful tool to predict clinical response to hormone therapy (individual "hormonogram") and to monitor endometrium proliferation.     

Development of mouse models of malignant phyllodes tumors by transplantation of syngeneic mammary gland cells expressing mutant H‐Ras

Phyllodes tumors (PTs) are rare fibroepithelial tumors of the breast with epithelial and stromal components, and surgical resection is the standard and only available treatment for malignant PTs. To provide a better understanding of these tumors, we developed mouse models that recapitulate the pathological and clinical properties of human malignant PTs. Mouse undifferentiated mammary gland cells were infected with a retrovirus encoding the human oncoprotein H‐Ras^G12V, and the infected cells were transplanted orthotopically into the mammary fat pads of syngeneic mice. The transplanted cells showed a high tumorigenic activity, with the resulting tumors manifesting pathological characteristics including stromal overgrowth similar to those of human malignant PTs. The tumors also showed high rates of both local recurrence and lung metastasis. Our models may prove useful for studies of the pathophysiology of malignant PTs as well as facilitate the development of new treatments.     

Vanadate disrupts mammary gland development in whole organ culture.

Protein tyrosine kinases and phosphatases are signaling molecules involved in all aspects of development, including proliferation, differentiation, and apoptosis. How disruption of protein tyrosine phosphatase affects mammary gland development is not entirely clear. We examined the effects of sodium vanadate, which is known to primarily inhibit tyrosine phosphatases, in mouse mammary gland development in whole organ culture. Mammary epithelial differentiation was effectively inhibited by vanadate in a dose-dependent manner as indicated by lack of epithelial alveoli compared to the contralateral non-treated gland controls. Mammary glands in the differentiation medium after four days in the presence of vanadate did not differentiate into alveoli. Instead, they exhibited prominent terminal end buds and lost the distinctive epithelial structures. The inhibitory effect of vanadate on mammary epithelial cell differentiation was irreversible after one day of treatment. Immunohistochemical staining for PCNA (Proliferating Cell Nuclear Antigen) showed that vanadate-treated glands exhibited elevated proliferation signals in the differentiation medium. Expression of beta-casein protein in the vanadate-treated glands decreased dramatically and progressively. Short-term exposure (up to 72 hours) of mammary glands to vanadate resulted in an increase in mammary epithelial cell density and loss of organization of the mammary structures. TUNEL assay of mammary glands with prolonged exposure to vanadate revealed widespread apoptosis. Furthermore, some cells were still proliferating or expressing beta-casein after prolonged exposure to vanadate. Taken together, these data indicate that vanadate treatment blocks mammary epithelial cell differentiation and promotes abnormal proliferation and apoptosis, likely through the inhibition of protein tyrosine phosphatase-mediated signaling.     

Cellular localization of aquaporins along the secretory pathway of the lactating bovine mammary gland: An immunohistochemical study

In this study we examined the cellular localization of aquaporins (AQPs) along the secretory pathway of actively lactating bovine mammary glands using immunohistochemistry. Mammary tissues examined included secretory ducts and acini, gland cisterns, teats, stromal and adipose tissues. Aquaporin 1 (AQP1) was localized in capillary endothelia throughout the mammary gland in addition to myoepithelial cells underlying teat duct epithelia. AQP2 and AQP6 were not detected and AQP9 was found only in leukocytes. AQP3 and AQP4 were observed in selected epithelial cells in the teat, cistern and secretory tubuloalveoli. AQP5 immunopositivity was prominent in the cistern. AQP3 and AQP7 were found in smooth muscle bundles in the teat, secretory epithelial cells and duct epithelial cells. These immunohistochemical findings support a functional role for aquaporins in the transport of water and small solutes across endothelial and epithelial barriers in the mammary gland and in the production and secretion of milk.     

Immunohistochemical detection of progesterone and cellular proliferation in canine mammary tumours

Immunohistochemical expression of progesterone and the cellular proliferation marker Ki-67 was assessed in formalin-fixed, paraffin wax-embedded samples from 43 canine mammary tumours. Benign tumours showed high surface membrane progesterone expression (mean 196.42+/-25.91 positively labelled cells per 500 tumour cells) and low cellular proliferation (52.14+/-16.73 positively labelled cells per 500 tumour cells), whilst malignant tumours had low progesterone expression (68.19+/-17.53 positively labelled cells per 500 tumour cells) and higher cellular proliferation (141.72+/-23.65 positively labelled cells per 500 tumour cells), the difference being statistically significant (P<0.005) in both cases. These findings suggest that the majority of progesterone receptors in canine mammary tumour tissue are not associated with bound progesterone. The progression towards malignancy in spontaneously arising canine mammary tumours therefore appears to be associated with a decrease in steroid hormone dependency. Progesterone expression was also noted in the cytoplasm of tumour cells where it may be associated with a cellular repair mechanism. This hypothesis is supported by the finding of significantly higher progesterone content in the cytoplasm of benign tumour cells.