Estrogen independence and progression to a metastatic phenotype are hallmarks of therapeutic resistance and mortality in breast cancer patients. Metastasis has been associated with chemokine signaling through the SDF-1–CXCR4 axis. Thus, the development of estrogen independence and endocrine therapy resistance in breast cancer patients may be driven by SDF-1–CXCR4 signaling. Here we report that CXCR4 overexpression is indeed correlated with worse prognosis and decreased patient survival irrespective of the status of the estrogen receptor (ER). Constitutive activation of CXCR4 in poorly metastatic MCF-7 cells led to enhanced tumor growth and metastases that could be reversed by CXCR4 inhibition. CXCR4 overexpression in MCF-7 cells promoted estrogen independence in vivo, whereas exogenous SDF-1 treatment negated the inhibitory effects of treatment with the anti-estrogen ICI 182,780 on CXCR4-mediated tumor growth. The effects of CXCR4 overexpression were correlated with SDF-1–mediated activation of downstream signaling via ERK1/2 and p38 MAPK (mitogen activated protein kinase) and with an enhancement of ER-mediated gene expression. Together, these results show that enhanced CXCR4 signaling is sufficient to drive ER-positive breast cancers to a metastatic and endocrine therapy-resistant phenotype via increased MAPK signaling. Our findings highlight CXCR4 signaling as a rational therapeutic target for the treatment of ER-positive, estrogen-independent breast carcinomas needing improved clinical management.

Tamoxifen is the most commonly prescribed therapy for patients with estrogen receptor (ER)α-positive breast tumors. Tumor resistance to tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress human epidermal growth factor receptor 2 (HER2). Current preclinical models of HER2 overexpression fail to recapitulate the clinical spectrum of endocrine resistance associated with HER2/ER-positive tumors. Here, we show that ectopic expression of a clinically important oncogenic isoform of HER2, HER2Δ16, which is expressed in >30% of ER-positive breast tumors, promotes tamoxifen resistance and estrogen independence of MCF-7 xenografts. MCF-7/HER2Δ16 cells evade tamoxifen through upregulation of BCL-2, whereas mediated suppression of BCL-2 expression or treatment of MCF-7/HER2Δ16 cells with the BCL-2 family pharmacological inhibitor ABT-737 restores tamoxifen sensitivity. Tamoxifen-resistant MCF-7/HER2Δ16 cells upregulate BCL-2 protein levels in response to suppressed ERα signaling mediated by estrogen withdrawal, tamoxifen treatment or fulvestrant treatment. In addition, HER2Δ16 expression results in suppression of BCL-2-targeting microRNAs miR-15a and miR-16. Reintroduction of miR-15a/16 reduced tamoxifen-induced BCL-2 expression and sensitized MCF-7/HER2Δ16 to tamoxifen. Conversely, inhibition of miR-15a/16 in tamoxifen-sensitive cells activated BCL-2 expression and promoted tamoxifen resistance. Our results suggest that HER2Δ16 expression promotes endocrine-resistant HER2/ERα-positive breast tumors and in contrast to wild-type HER2, preclinical models of HER2Δ16 overexpression recapitulate multiple phenotypes of endocrine-resistant human breast tumors. The mechanism of HER2Δ16 therapeutic evasion, involving tamoxifen-induced upregulation of BCL-2 and suppression of miR-15a/16, provides a template for unique therapeutic interventions combining tamoxifen with modulation of microRNAs and/or ABT-737-mediated BCL-2 inhibition and apoptosis.

Glyceollins, a group of novel phytoalexins isolated from activated soy, have recently been demonstrated to be novel antiestrogens that bind to the estrogen receptor (ER) and inhibit estrogen-induced tumor progression. Our previous publications have focused specifically on inhibition of tumor formation and growth by the glyceollin mixture, which contains three glyceollin isomers (I, II, and III). Here, we show the glyceollin mixture is also effective as a potential antiestrogenic, therapeutic agent that prevents estrogenstimulated tumorigenesis and displays a differential pattern of gene expression from tamoxifen. By isolating the individual glyceollin isomers (I, II, and III), we have identified the active antiestrogenic component by using competition binding assays with human ER␣ and in an estrogen-responsive element-based luciferase reporter assay. We identified glyceollin I as the active component of the combined glyceollin mixture. Ligand-receptor modeling (docking) of glyceollin I, II, and III within the ER␣ ligand binding cavity demonstrates a unique type II antiestrogenic confirmation adopted by glyceollin I but not isomers II and III. We further compared the effects of glyceollin I to the antiestrogens, 4-hydroxytamoxifen and ICI 182,780 (fulvestrant), in MCF-7 breast cancer cells and BG-1 ovarian cancer cells on 17␤-estradiol-stimulated expression of progesterone receptor and stromal derived factor-1␣. Our results establish a novel inhibition of ER-mediated gene expression and cell proliferation/ survival. Glyceollin I may represent an important component of a phytoalexin-enriched food (activated) diet in terms of chemoprevention as well as a novel therapeutic agent for hormonedependent tumors.Breast cancer accounts for 25% of all female cancers, making it the most common cancer in women in the western world (Greenlee et al., 2000Lester, 2007 Ali and Coombs, 2002). For patients with hormone receptor-positive breast cancer, several promising endocrine agents are currently available with promising results. Therapies have been developed to reduce estrogen levels or to block signaling through estrogen receptors (ER) (Pink and Jordan, 1996;Howell, 2006). These agents include tamoxifen, a selective estrogen

Adult human mesenchymal stem cells (hMSCs) have been shown to home to sites of breast cancer and integrate into the tumor stroma. We demonstrate here the effect of hMSCs on primary breast tumor growth and the progression of these tumors to hormone independence. Co-injection of bone marrow-derived hMSCs enhances primary tumor growth of the estrogen receptor-positive, hormone-dependent breast carcinoma cell line MCF-7 in the presence or absence of estrogen in SCID/beige mice. We also show hormone-independent growth of MCF-7 cells when co-injected with hMSCs. These effects were found in conjunction with increased immunohistochemical staining of the progesterone receptor in the MCF-7/hMSC tumors as compared to MCF-7 control tumors. This increase in PgR expression indicates a link between MCF-7 cells and MSCs through ER-mediated signaling. Taken together, our data reveal the relationship between tumor microenvironment and tumor growth and the progression to hormone independence. This tumor stroma-cell interaction may provide a novel target for the treatment of estrogen receptor-positive, hormone-independent, and endocrine-resistant breast carcinoma.

To investigate the therapeutic effect of methylselenocysteine (MSC) combined with tamoxifen in MCF-7 breast cancer xenograft and the underlying mechanisms. MCF-7 breast cancer xenograft was established in ovariectomized female athymic nude mice and treated with tamoxifen and/or MSC. Tumor size was measured twice a week. Immunohistochemistry and TUNEL assays were used to measure ERalpha expression, ERalpha target genes (progesterone receptor (PR) and cyclin D1 expression), Ki-67 index, apoptosis and microvessel density. Combined treatment with tamoxifen and MSC synergistically inhibited tumor growth compared to MSC alone and tamoxifen alone. MSC alone or MSC + tamoxifen significantly reduced ERalpha, PR and cyclin D1, Ki67 index and microvessel density while increasing apoptosis in tumor tissues. These findings demonstrate synergistic growth inhibition of ERalpha positive breast cancer xenografts by combination of tamoxifen with organic selenium compounds. Organic selenium may provide added benefit when combined with tamoxifen in adjuvant therapy or prevention.

Endometriosis is associated with activation of local and systemic inflammatory mechanisms, including increased levels of chemokines and other proinflammatory cytokines. We have previously reported increased gene expression of chemokine receptor 4 (CXCR4), the receptor for CXCL12, in lesions of the rat model of endometriosis. The CXCR4-CXCL12 axis has been shown to have both immune (HIV infection, lymphocyte chemotaxis) and nonimmune functions, including roles in tissue repair, angiogenesis, invasion, and migration. There is evidence indicating that these mechanisms are also at play in endometriosis; therefore, we hypothesized that activation of the CXCR4-CXCL12 axis could be responsible, at least in part, for the survival and establishment of endometrial cells ectopically. Immunohistochemistry (IHC) showed that CXCR4 protein levels were significantly higher in endometriotic lesions compared to the endometrium of controls. Next, we determined basal gene and protein expression of CXCR4 and CXCL12 and regulation by estradiol (E2) and/or progesterone (P4) in endometrial cell lines using quantitative polymerase chain reaction (qPCR), and Western blots. Basal CXCR4 gene expression levels were higher in epithelial versus stromal cells; conversely, CXCL12 was expressed at higher levels in stromal vs epithelial cells. CXCR4 gene expression was significantly downregulated by ovarian steroid hormones in endometrial epithelial. These data suggest that steroid modulation of CXCR4 is defective in endometriosis, although the specific mechanism involved remains to be elucidated. These findings have implications for future therapeutic strategies specifically targeting the inflammatory component in endometriosis.

Nuclear hormone receptors, such as the estrogen receptors (ERs), are regulated by specific kinase signaling pathways. Here, we demonstrate that the p38 MAPK stimulates both ERalpha- and ERbeta-mediated transcription in MCF-7 breast carcinoma, Ishikawa endometrial adenocarcinoma, and human embryonic kidney 293 cells. Inhibition of this potentiation using the p38 inhibitor, RWJ67657, blocked estrogen-mediated transcription and proliferation. Activated ERs promote gene expression in part through the recruitment of the p160 class of coactivators. Because no direct p38 phosphorylation sites have been determined on either ERalpha or beta, we hypothesized that p38 could target the p160 class of coactivators. We show for the first time using pharmacological and molecular techniques that the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) is phosphorylated and potentiated by the p38 MAPK signaling cascade in vitro and in vivo. S736 was identified as a necessary site for p38 induction of GRIP1 transcriptional activation. The C terminus of GRIP1 was also demonstrated to contain a p38-responsive region. Taken together, these results indicate that p38 stimulates ER-mediated transcription by targeting the GRIP1 coactivator.

Altered death receptor signaling and resistance to subsequent apoptosis is an important clinical resistance mechanism. Here, we investigated the role of death receptor resistance in breast cancer progression. Resistance of the estrogen receptor alpha (ER)-positive, chemosensitive MCF7 breast cancer cell line to tumor necrosis factor (TNF) was associated with loss of ER expression and a multi-drug resistant phenotype. Changes in three major pathways were involved in this transition to a multidrug resistance phenotype: ER, Death Receptor and epithelial to mesenchymal transition (EMT). Resistant cells exhibited altered ER signaling, resulting in decreased ER target gene expression. The death receptor pathway was significantly altered, blocking extrinsic apoptosis and increasing NF-kappaB survival signaling. TNF resistance promoted EMT changes, resulting in a more aggressive phenotype. This first report identifying specific mechanisms underlying acquired resistance to TNF could lead to a better understanding of the progression of breast cancer in response to chemotherapy treatment.