Mechanisms of normal and malignant breast epithelial growth regulation

In this presentation we review information highlighting the multiple roles of both steroidal and polypeptide regulators of mammary epithelial cell growth with some additional emphasis on the work of our laboratory. The effects of both classes of hormones are complex and involve multiple interactions with epithelial components (malignant or normal) and the stromal compartment. Estrogens induce growth regulatory polypeptide growth factors which are responsible for many of the induced phenotypic effects in hormone-dependent breast cancer. Progression of hormone-dependent breast cancer to hormone independence probably involves multiple genetic mechanisms of oncogene activation, loss of the estrogen receptor, or loss of hormone responsivity of other gene products. Initial carcinogenesis and progression of mammary epithelium to cancer probably also requires both proliferative stimuli (estrogen, polypeptide growth factors) and genetic damage, leading to qualitatively different hormonal responses (hormone responsive cancer). New therapeutic strategies based on these biological considerations are emerging, including a variety of approaches which interfere at multiple points with ability of ligand to induce receptor signaling.     

Hormones and breast cancer in vitro

Breast cancer is characterized by hormonal regulation. The current article reviews the role of estrogen and polypeptide growth factors in control of proliferation and basement membrane invasion of breast cancer cells in vitro. The role of antiestrogens to regulate proliferation, invasion, and growth factor secretion is further highlighted. Finally, the use of in vitro cultures of breast cancer cells to model steps in the malignant progression of the disease is emphasized. The availability of hormone dependent and independent breast cancer cell lines should allow screening for better antiestrogens, antimetastatic drugs, and antagonists of local action of growth factors.     

Phenol red in the culture medium strongly affects the susceptibility of human MCF-7 cells to roscovitine

Estrogens play an important role in the growth and terminal differentiation of the mammary gland. Prolonged exposure to estrogens seems to predispose women to breast cancer. It recently became evident that not only the intrinsic hormonal status but also external factors such as the occurrence of pharmaceuticals and chemicals with hormone activity in the environment may put women at greater risk of developing breast cancer. We focused on the interference of endocrine disruptors in breast cancer therapy. We observed that phenol red added to the culture medium strongly promoted the cell proliferation and cell cycle progression of human cells expressing the estrogen receptor, and affected their susceptibility to chemotherapy.     

Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids

Endogenous ovarian estrogens and progestins appear to play a critical role in the development and progression of breast cancer. Local productions of growth factors probably also contribute to malignant proliferation, while production and activation of collagenolytic enzymes may be equally critical for local invasive processes. The current review focuses on characterization of growth factor-receptor systems operant in normal and malignant breast epithelium. In addition, the determinants of local invasion are reviewed: attachment, modality, and proteose secretion. Finally, data are discussed concerning the regulation of both proliferation and invasion by hormones and antihormonal agents in hormone-dependent breast cancer. The results suggest new potential pharmacologic targets to explore to suppress onset and progression of breast cancer.     

Stimulatory and inhibitory growth factors and breast cancer

While steroid hormones act as endocrine effectors of growth and development of normal breast and of carcinogenesis and progression of malignant breast, recent evidence suggests that local hormonal effectors also exist. These are the growth regulatory growth factors. This article summarizes current status of our understanding of structure and function of growth factors secreted by the normal and malignant mammary epithelium. While growth inhibitory factors and their receptors generally suppress development of the transformed phenotype and promote differentiation, growth stimulatory factors and their receptors may be necessary for both normal proliferation and early stages of malignant progression of breast cancer. Overexpression of two receptors, c-erbB-2 and EGF receptor, have also been associated with poor prognosis in the clinical disease.     

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Ar^fl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.     

Heregulin is sufficient for the promotion of tumorigenicity and metastasis of breast cancer cells in vivo

Resistance of breast carcinomas to hormonal therapy is a clinical obstacle for the treatment of breast cancer. The molecular mechanisms and the factors involved in the progression of tumors from an estrogen (E2)-dependent to an E2-independent phenotype are not entirely understood. Heregulin (HRG) is a pleiotropic growth factor that binds to the erbB family of receptors, which are correlated with breast cancer progression and an aggressive phenotype in the breast carcinomas overexpressing the receptors. Previous studies in transgenic mice have shown that HRG is sufficient to induce mammary gland transformation and proliferation in the presence of hormonal stimulation. However, these studies did not address the important issue of the E2 independence that is part of the progression of breast cancer. In this study, we investigated the role of HRG in E2 independence. We were able to determine that HRG up-regulation was sufficient for the development of mammary tumors in the absence of E2 stimulation, a situation that mimics the progression of the human disease. We demonstrated that in ovariectomized nude mice, HRG induced E2 independence and antiestrogen resistance and promoted metastasis and preneoplastic transformation of the adjacent mouse mammary tissue. We show that one of the mechanisms by which HRG achieves the aggressive phenotype may be mediated via an increase in activated mitogen-activated protein kinase, an increase in a matrix-degrading enzyme, MMP-9, and the overexpression of vascular endothelial growth factors. The up-regulation of these genes occurred in the absence of any additional stimulation, in an autocrine manner. Our data provide new insights into the mechanisms of breast cancer progression in vivo, and reinforce the important role that HRG plays in this process.     

Cancers du sein hormono-dépendants: quels biomarqueurs tissulaires utiles dans la prise en charge ?

Breast cancer is the second most frequently diagnosed cancer in women, in France. It accounts for almost 37% of new cancer diagnosed in women. Despite a decrease of the mortality during the last 15 years, breast cancer remains the first cause of cancer death in women and a public health problem. Historically, there are two very different types of cancers based on their content of hormone receptors (HR): estrogen receptor (ER) and progesterone receptor (PR). Nearly 70% of breast tumors are HR-positive, called “hormone-dependent” because their growth depends on steroid hormones. Their treatment includes the first targeted therapy: the hormone therapy. Initially, the clinical benefits of hormone therapy were demonstrated on patients whose HR content was evaluated by quantitative ligand-binding (LBA) or immunoassays (EIA). Today, the hormone dependence is determined by testing the presence of the receptors by immunohistochemistry, a semi quantitative evaluation of content, on fixed tissue section. But this evaluation of HR, seems not to be sufficient to predict the sensitivity or resistance to hormonal treatment. These technical evolutions of RH mesurement and the loss of quantitative assessement remain an open question. Also the discovery of many other players in the estrogen signaling complicates the understanding of this tumor subtype. Thus, determination of the hormonal dependence remains a unresolved problem. The discovery of new potential relevant biomarkers and new technologies allows us to reconsider the RH question and to move towards the most personalized medicine.     

Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells

There is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.     

Hormone/growth factor interactions mediating epithelial/stromal communication in mammary gland development and carcinogenesis

Epithelial/mesenchymal interactions begin during embryonic development of the mammary gland and continue throughout mammary gland development into adult life. Stromal and epithelial growth factors that may mediate interactions between these compartments of the mammary gland are reviewed. Since mammogenic hormones are the primary regulators of mammary gland development, special consideration is given to hormonal regulation of growth factors in order to explore the integration of hormones and growth factors in the regulation of mammary gland growth and neoplasia. Examination of hormonal regulation of the fibroblast growth factor (FGF)-7/FGFR2-IIIb receptor system in the mammary gland reveals that mammogenic hormones differentially regulate the synthesis of stromal growth factors and their epithelial receptors. These effects serve to optimize the action of estrogen and progesterone on mammary gland development and illustrate that the ratio of these two hormones is critical in regulating this growth factor axis. The role of stromal/epithelial mitogenic microenvironments in modulating the genotype and phenotype of preneoplastic and neoplastic lesions by chemical carcinogens is discussed. Finally, changes in growth factor expression during mammary tumor progression are described to illustrate the relative roles that stromally-derived and epithelial-derived growth factors may play during progression to hormone independent tumor growth.     

Analysis of mammary tumors for cytochemical evidence of endogenous mammary peroxidase.

The purpose of this study was to determine whether or not endogenous mammary peroxidase can serve as a cytochemical marker to distinguish ovarian hormone-dependent from ovarian hormone independent mammary tumors. Spontaneous mammary tumors arising in virgin C3H and GR mice (hormone independent tumors) and hormone-dependent mammary tumors arising during pregnancy in GR mice were examined. None of these tumors contained mammary peroxidase. Mammary tumors induced in Sprague-Dawley rats with methylnitrousourea (MNU) and dimethylbenzanthracene (DMBA) were also examined. These tumors included hormone-dependent and hormone independent ones. Several of the DMBA-induced hormone-dependent tumors contained a few peroxidase-positive cells, but the hormone independent tumors were negative. All of the MNU-induced tumors examined were negative for mammary peroxidase. Twenty human breast tumors (malignant and non-malignant) removed from women at surgery, were also negative for mammary peroxidase. Our results indicate that endogenous mammary peroxidase cannot be used to distinguish hormone-dependent from hormone independent mammary tumors.     

Metabotropic Glutamate Receptor-1 Contributes to Progression in Triple Negative Breast Cancer

TNBC is an aggressive breast cancer subtype that does not express hormone receptors (estrogen and progesterone receptors, ER and PR) or amplified human epidermal growth factor receptor type 2 (HER2), and there currently exist no targeted therapies effective against it. Consequently, finding new molecular targets in triple negative breast cancer (TNBC) is critical to improving patient outcomes. Previously, we have detected the expression of metabotropic glutamate receptor-1 (gene: GRM1; protein: mGluR1) in TNBC and observed that targeting glutamatergic signaling inhibits TNBC growth both in vitro and in vivo. In this study, we explored how mGluR1 contributes to TNBC progression, using the isogenic MCF10 progression series, which models breast carcinogenesis from nontransformed epithelium to malignant basal-like breast cancer. We observed that mGluR1 is expressed in human breast cancer and that in MCF10A cells, which model nontransformed mammary epithelium, but not in MCF10AT1 cells, which model atypical ductal hyperplasia, mGluR1 overexpression results in increased proliferation, anchorage-independent growth, and invasiveness. In contrast, mGluR1 knockdown results in a decrease in these activities in malignant MCF10CA1d cells. Similarly, pharmacologic inhibition of glutamatergic signaling in MCF10CA1d cells results in a decrease in proliferation and anchorage-independent growth. Finally, transduction of MCF10AT1 cells, which express c-Ha-ras, using a lentiviral construct expressing GRM1 results in transformation to carcinoma in 90% of resultant xenografts. We conclude that mGluR1 cooperates with other factors in hyperplastic mammary epithelium to contribute to TNBC progression and therefore propose that glutamatergic signaling represents a promising new molecular target for TNBC therapy.     

Endogenous human prolactin and not exogenous human prolactin induces estrogen receptor alpha and prolactin receptor expression and increases estrogen responsiveness in breast cancer cells

Prolactin (PRL) and estrogen act synergistically to increase mammary gland growth, development, and differentiation. Based on their roles in the normal gland, these hormones have been studied to determine their interactions in the development and progression of breast cancer. However, most studies have evaluated only endocrine PRL and did not take into account the recent discovery that PRL is synthesized by human mammary cells, permitting autocrine/paracrine activity. To examine the effects of this endogenous PRL, we engineered MCF7 cells to inducibly overexpress human prolactin (hPRL). Using this Tet-On MCF7hPRL cell line, we studied effects on cell growth, PRLR, ER alpha, and PgR levels, and estrogen target genes. Induced endogenous hPRL, but not exogenous hPRL, increased ER alpha levels as well as estrogen responsiveness in these cells, suggesting that effects on breast cancer development and progression by estrogen may be amplified by cross-regulation of ER alpha levels by endogenous hPRL. The long PRLR isoform was also upregulated by endogenous, but not exogenous PRL. This model will allow investigation of endogenous hPRL in mammary epithelial cells and will enable further dissection of PRL effects on other hormone signaling pathways to determine the role of PRL in breast cancer.     

Estrogens and women's health: interrelation of coronary heart disease, breast cancer and osteoporosis

The determinants of blood levels of estrogen, estrogen metabolites, and relation to receptors and post-transitional effects are the likely primary cause of breast cancer. Very high risk women for breast cancer can now be identified by measuring bone mineral density and hormone levels. These high risk women have rates of breast cancer similar to risk of myocardial infarction. They are candidates for SERM therapies to reduce risk of breast cancer. The completion of the Women's Health Initiative and other such trials will likely provide a definite association of risk and benefit of both estrogen alone and estrogen-progesterone therapy, coronary heart disease, osteoporotic fracture, and breast cancer. The potential intervention of hormone replacement therapy, obesity, or weight gain and increased atherogenic lipoproteinemia may be of concern and confound the results of clinical trials. Estrogens, clearly, are important in the risk of bone loss and osteoporotic fracture. Obesity is the primary determinant of postmenopausal estrogen levels and reduced risk of fracture. Weight reduction may increase rates of bone loss and fracture. Clinical trials that evaluate weight loss should monitor effects on bone. The beneficial addition of increased physical activity, higher dose of calcium or vitamin D, or use of bone reabsorption drugs in coordination with weight loss should be evaluated. Any therapy that raises blood estrogen or metabolite activity and decreases bone loss may increase risk of breast cancer. Future clinical trials must evaluate multiple endpoints such as CHD, osteoporosis, and breast cancer within the study. The use of surrogate markers such as bone mineral density, coronary calcium, carotid intimal medial thickness and plaque, endothelial function, breast density, hormone levels and metabolites could enhance the evaluation of risk factors, genetic-environmental intervention, and new therapies.     

Cyclin-dependent kinase (cdk) inhibitors/cdk4/cdk2 complexes in early stages of mouse mammary preneoplasia.

The level of circulating ovarian hormones (estrogen and progesterone) alone or in combination with pituitary hormones have a potent mitogenic impact in the normal mammary gland, and they also play a pivotal role in the development and progression of mammary carcinoma. The differential effects of hormones on the molecular components of cyclin-dependent kinase (cdk) complexes in mammary epithelium of the hormone-dependent ductal outgrowth line, EL11, and the hormone-independent alveolar outgrowth line, TM2L, were the focus of this study. The two outgrowth lines, which represent early stages in mammary hyperplasia, were compared with normal mammary gland at different hormonal conditions: control, hormone stimulated by pituitary isograft, and hormone depleted by ovariectomy. Hormonal stimulation by a pituitary isograft resulted in DNA synthesis and lobuloalveolar development of normal mammary ducts, DNA synthesis but no lobuloalveolar development in the EL11 ductal outgrowth, and no changes either in DNA synthesis or in lobuloalveolar morphology in the TM2L outgrowth. The levels of cdk4- and cyclin D1-associated kinase activities were correlated with cell proliferation in only the alveolar phenotypes (i.e., in only hormonally stimulated normal virgin gland and in alveolar mammary outgrowth), whereas cyclin D2-dependent kinase activity was correlated with cell proliferation in only the alveolar preneoplasia. p16(INK4a) and p21(Cip1) protein levels were decreased at the earliest stages of preneoplasia, i.e., at immortalization, and were independent from changes in cyclin D1, which occurred later in preneoplasia. Although all cdk inhibitors changed in concordance with hormonal status reflected by proliferation levels, p27(Kip1) was the only cdk inhibitor that was up-regulated at the earliest stages of preneoplasia and may have a unique role in blocking alveolar differentiation in response to the loss of one or more of the cell cycle-negative regulators. We hypothesize that up-regulation of p27(Kip1) prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

Steroid receptors in mammary glands and tumors of hysterectomised rats

Epidemiological survey suggests that longer exposure of the breast to sex steroids may be one of the factors involved in increased risk for cancer. Using an experimental model of bilaterally hysterectomised rats, the sex steroid receptors in the mammary glands during growth as well as incidence and hormonal characteristics of chemically induced mammary tumors have been studied. Though steroid receptors were detectable in the mammary glands of the model earlier than in intact rats, the incidence or hormone dependency of the mammary tumors in the two groups were not considerably altered.