Ji-Ping Wang

Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.

Estradiol Hypersensitivity and Mitogen-Activated Protein Kinase Expression in Long-Term Estrogen Deprived Human Breast Cancer Cells in Vivo

Women with breast cancer who have responded to initial hormonal therapy frequently experience additional remissions upon further endocrine manipulation. We postulate that hypersensitivity to estradiol (E2) may serve as a mechanistic explanation for these secondary responses. We previously provided evidence of hypersensitivity using an in vitro breast cancer model system and demonstrated the role of mitogen-activated protein kinase (MAP kinase) in the process of adaptation to long-term estradiol deprivation. In the present study, we wished to demonstrate that hypersensitivity to E2 could occur under more complex in vivo conditions and that MAP kinase activation is enhanced under these circumstances. We used an MCF-7 breast cancer model system involving long-term estradiol deprived (LTED) cells to produce xenografts in nude mice and an E2 clamp method to precisely control sex steroid levels. The E2 clamp was designed to maintain plasma E2 at a series of doubling doses from 1.25 pg/ml to 20.0 pg/ml in oophor...

Long-term Treatment with Tamoxifen Facilitates Translocation of Estrogen Receptor α out of the Nucleus and Enhances its Interaction with EGFR in MCF-7 Breast Cancer Cells

The therapeutic benefit of tamoxifen in patients with hormone-dependent breast cancer is limited by acquired resistance to this drug. To investigate the biological alterations responsible for tamoxifen resistance, an in vitro model was established. After 6-month continuous exposure to tamoxifen (10−7 mol/L), growth of MCF-7 breast cancer cells was no longer inhibited by this antiestrogen. Although there was no significant increase in the basal levels of activated mitogen-activated protein kinase (MAPK), tamoxifen-resistant (TAM-R) cells exhibited enhanced sensitivity to epidermal growth factor (EGF) and estradiol stimulated activation of MAPK. Tamoxifen elicited rapid phosphorylation of MAPK, in contrast to its antagonistic activity in control cells. Blockade of the EGF receptor (EGFR)/MAPK pathway caused more dramatic inhibition of growth of TAM-R cells than the control cells. An increased amount of estrogen receptor α (ERα) was coimmunoprecipitated with EGFR from TAM-R cells although the total levels of these receptors were not increased. Notably, ERα seemed to redistribute to extranuclear sites in TAM-R cells. Increased ERα immunoreactivity in the cytoplasm and plasma membrane of TAM-R cells was shown by fluorescent microscopy and by Western analysis of isolated cellular fractions. In TAM-R cells, an increased amount of c-Src was coprecipitated with EGFR or ERα. Blockade of c-Src activity resulted in redistribution of ERα back to the nucleus and in reduction of its interaction with EGFR. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that enhanced nongenomic function of ERα via cooperation with the EGFR pathway is one of the mechanisms responsible for acquired tamoxifen resistance. [Cancer Res 2007;67(3):1352–60]

Estrogen receptor-dependent and independent mechanisms of breast cancer carcinogenesis

Long term exposure to estrogens is associated with an increased risk of breast cancer. The precise mechanisms responsible for estrogen mediated carcinogenesis are not well understood. The most widely accepted theory holds that estradiol (E(2)), acting through estrogen receptor alpha (ERα), stimulates cell proliferation and initiates mutations arising from replicative errors occurring during pre-mitotic DNA synthesis. The promotional effects of E(2) then support the growth of cells harboring mutations. Over a period of time, sufficient numbers of mutations accumulate to induce neoplastic transformation. Laboratory and epidemiological data also suggest that non-receptor mediated mechanisms resulting from the genotoxic effects of estrogen metabolites are involved in breast cancer development. This manuscript critically reviews existing data implicating both ER-dependent and -independent mechanisms. The weight of evidence supports the possibility that both mechanisms are involved in the carcinogenic process. In addition, estrogen metabolites likely modulate stem cell functionality and cancer progression. The roles of ER dependent and independent actions in the carcinogenic process are pertinent to the consideration of breast cancer preventative agents as anti-estrogens block only receptor mediated pathways whereas the aromatase inhibitors block both.

Role of MAP kinase in the enhanced cell proliferation of long term estrogen deprived human breast cancer cells

Women with estrogen receptor (ER) positive breast cancers frequently respond initially to inhibition of estrogen action but later relapse with re-growth of tumor. Previously, we have utilized MCF-7 human breast cancer cells deprived of estradiol long term (LTED cells) as the model system to study the regrowth phenomenon and have demonstrated that these cells exhibited increased cell proliferation rate and increased ER functionality during the adaptive processes. In this report, we examined the hypothesis that the mitogen-activated protein kinase (MAP kinase) signal was involved. We found that activated MAP kinase was elevated in LTED cells and that the MAP kinase specific inhibitor PD98059 was able to inhibit the elevated MAP kinase and [3H]thymidine uptake in LTED cells, suggesting mediation of DNA synthesis and proliferation by the MAP kinase pathway. Other MAP kinase upstream inhibitors, including genestein, RG13022, and mevastatin were also able to inhibit the [3H]thymidine uptake in LTED cells. Interestingly, the antiestrogen, ICI 182,780 was able to block the activated MAP kinase in LTED cells. Treatment with PD98059 did not block elevated basal ERE-CAT activity while at the same time inhibiting [3H]thymidine uptake in LTED cells. Furthermore, treatment with PD98059 partially blocked the E2-stimulated ERE-CAT activity and [3H]thymidine uptake in both LTED and in wild type cells, indicating that both MAP kinase-dependent and MAP kinase-independent pathways are involved in the transactivation function of ER. Taken together, our data suggest that the MAP kinase pathway is, in part, involved in the adaptive process which results in enhanced DNA synthesis and cell proliferation in the absence of exogenous estrogen in LTED 3ptcells.

Adaptive hypersensitivity to estradiol: potential mechanism for secondary hormonal responses in breast cancer patients ☆

Women with hormone dependent breast cancer initially respond to hormone deprivation therapy with tamoxifen or oophorectomy for 12-18 months but later relapse. Upon secondary therapy with aromatase inhibitors, patients often experience further tumor regression. The mechanisms responsible for secondary responses are unknown. We postulated that hormone deprivation induces hypersensitivity to estradiol. Evidence of this phenomenon was provided in a model system involving MCF-7 cells grown in vitro and in xenografts. To determine if the ER transcriptional process is involved in hypersensitivity, we examined the effect of estradiol on ER reporter activity, PgR, PS2, and c-myc as markers and found no alterations in hypersensitive cells. Next, we examined whether MAP kinase may be upregulated in the hypersensitive cells as a reflection of increased growth factor secretion or action. Basal MAP kinase activity was increased both in vitro and in vivo in hypersensitive cells. Proof of principle studies indicated that an increase in MAP kinase activity induced by TGFalpha administration caused a two- to three-fold shift to the left in estradiol dose response curves in wild type cells. Blockade of MAP kinase with PD98059 returned the shifted curve back to baseline. These data suggested that MAP kinase overexpression could induce hypersensitivity. To determine why MAP kinase was increased, we excluded constitutive receptor activity and growth factor secretion by the demonstration that the pure anti-estrogen, ICI 182780, could inhibit MAP kinase activation. We also excluded hypersensitivity to estradiol induced growth factor secretion, and thus MAP kinase activation, since estradiol stimulated MAP kinase at 24, 48, and 72 h at the same concentrations in hypersensitive as in wild type cells. Surprisingly, a series of experiments suggested that MAP kinase increased in hypersensitive cells as a result of estrogen activation via a non-genomic pathway. We examined the classical signal pathway in which SHC is phosphorylated and binds to SOS and GRB-2 to activate Ras, Raf, and MAP kinase. With 5-20 min of exposure, estradiol caused binding of SHC to the estrogen receptor, phosphorylation of SHC, binding of GRB-2 to SOS, and activation of MAP kinase. All of these affects could be blocked by ICI 182780. Taken together, these observations suggest that the cell membrane ER pathway may be responsible for upregulation of MAP kinase and hypersensitivity in cells adapted to estradiol deprivation.

Adaptive hypersensitivity following long-term estrogen deprivation involvement of multiple signal pathways

Long-term estrogen deprivation causes hypersensitivity of MCF-7 cells to the mitogenic effect of estradiol (E2) which is associated with activation of mitogen-activated protein kinase (MAPK). However, several lines of evidence indicate that MAPK activation is not the exclusive mechanism for E2 hypersensitivity and multiple signal pathways might be involved. The current study explores the possible role of the PI3 kinase (PI3K) pathway in development of E2 hypersensitivity. Basal PI3K activity in long-term estrogen deprived MCF-7 cells (LTED) was elevated as evidenced by increased phosphorylation of three downstream effectors, Akt, p70 S6 kinase, and eukaryotic initiation factor-4E binding protein (4E-BP1), which was blocked by the specific inhibitor of PI3K, LY294002. Dual blockade of both MAPK and PI3K completely reversed E2 hypersensitivity of LTED cells. Enhancement in aromatase activity is another phenomenon accompanied with E2 hypersensitivity. In aromatase over-expressing MCF-7 cells, aromatase activity was reduced by inhibitors of MAPK and PI3K suggesting the involvement of protein phosphorylation in the regulation of aromatase activity. Our data suggest that in addition to the MAP kinase pathway, activation of the PI3 kinase pathway is involved in E2 hypersensitivity, which develops during adaptation of MCF-7 cells to the low estrogen environment.

Macrophages, estrogen and the microenvironment of breast cancer

Estrogen is a major mitogenic stimulus to established breast cancer. Estrogen sources include ovarian, extraglandular sites and breast tissue. Which source primarily maintains benign and breast cancer tissue estrogen concentrations remains unclear. While macrophages may comprise up to 50% of the mass of breast carcinomas, previous studies neglected to study them as possible sources of estrogen. We present evidence that breast macrophages constitute an in situ source of estradiol and that the amount produced is sufficient to mediate cellular proliferation. We utilized immunohistochemistry and RT-PCR to study cell-specific aromatase expression in (i) 29 breast biopsies, (ii) human monocytes/macrophages and (iii) a myeloid cell line (THP-1) capable of differentiating into macrophages. Use of a breast cancer cell line (MCF-7) provided biologic confirmation of the role of aromatization in cell proliferation. We demonstrated considerable amounts of immunoreactive-aromatase (irARO) in breast tissue macrophages and a positive correlation between the proportion of irARO present in macrophages and lesion severity. Using in vitro techniques, we demonstrated that monocytes and THP-1 cells require differentiation into macrophages to produce aromatase in amounts approaching placental levels. The amount of estrogen produced by THP-1 cells stimulated MCF-7 cells to proliferate, an effect blocked by aromatase inhibitors. Estrogen production by macrophages in breast tissue appears sufficient to stimulate the proliferation of adjacent epithelial cells and to autoregulate cytokine production. These findings represent a new dimension of cellular regulation in breast tissue with major biologic implications, amenable to pharmacological manipulation.

Mechanisms of acquired resistance to endocrine therapy in hormone-dependent breast cancer cells

Acquired resistance is a major problem limiting the clinical benefit of endocrine therapy. To investigate the mechanisms involved, two in vitro models were developed from MCF-7 cells. Long-term culture of MCF-7 cells in estrogen deprived medium (LTED) mimics aromatase inhibition in patients. Continued exposure of MCF-7 to tamoxifen represents a model of acquired resistance to antiestrogens (TAM-R). Long-term estrogen deprivation results in sustained activation of the ERK MAP kinase and the PI3 kinase/mTOR pathways. Using a novel Ras inhibitor, farnesylthiosalicylic acid (FTS), to achieve dual inhibition of the pathways, we found that the mTOR pathway plays the primary role in mediation of proliferation of LTED cells. In contrast to the LTED model, there is no sustained activation of ERK MAPK but enhanced responsiveness to rapid stimulation induced by E(2) and TAM in TAM-R cells. An increased amount of ERalpha formed complexes with EGFR and c-Src in TAM-R cells, which apparently resulted from extra-nuclear redistribution of ERalpha. Blockade of c-Src activity drove ERalpha back to the nucleus and reduced ERalpha-EGFR interaction. Prolonged blockade of c-Src activity restored sensitivity of TAM-R cells to tamoxifen. Our results suggest that different mechanisms are involved in acquired endocrine resistance and the necessity for individualized treatment of recurrent diseases.

Estrogen production via the aromatase enzyme in breast carcinoma: Which cell type is responsible?

Studies of breast tumor homogenates from women with breast cancer have demonstrated the synthesis of estrogens in situ through the enzyme aromatase. The present series of investigations sought to determine which cell type within the tumor is responsible for local estrogen biosynthesis, and whether or not the amount produced is biologically important. Accordingly, we utilized an indirect immunohistochemical scoring method (H-score) to determine the relative amount of enzyme present in tumor epithelial and stromal cells. This revealed a value of 13 for tumor stromal cells and 4.8 for the epithelial component. Contributing to this difference is the fact that a greater percentage of cells in the tumor were stromal (45%) than epithelial (37%). To obtain direct evidence that tumor stromal cells could synthesize estrogens, we isolated and grew these cells in tissue culture. Stromal cells originating from within the tumor could be stimulated by known enhancers of transcription to produce nearly as much aromatase as is found in placental microsomes. Stromal cells isolated from benign tissue distal to the tumor exhibited properties similar to those of the tumor stroma. Epithelial cells, in contrast, did not respond to these enhancers and had low levels of aromatase basally. To obtain proof of the principle that local estrogen synthesis can be biologically meaningful, we measured tumor tissue estradiol levels and growth rates in aromatase-transfected MCF-7 cells implanted into nude mice. Local synthesis resulted in tumor levels ranging from 300 to 800 pg/g and growth rates substantially higher than in non-aromatase-containing tumors. These data suggest that tumor stromal cells contribute the major portion of estrogen synthesized in tumors, and that this local synthesis can increase tumor estradiol levels and growth rates.