Identification of a unique epigenetic sub-microenvironment in prostate cancer

The glutathione S-transferase P1 (GSTP1) gene promoter is methylated in tumour cells in more than 90% of prostate carcinomas. Recently, GSTP1 promoter methylation was identified in tumour-associated stromal cells in addition to the tumour epithelium. To define the extent and location of stromal methylation, epigenetic mapping using pyrosequencing quantification of GSTP1 promoter methylation and an anatomical three-dimensional reconstruction of an entire human prostate specimen with cancer were performed. Normal epithelium and stroma, tumour epithelium, and tumour-associated stromal cells were laser capture-microdissected from multiple locations throughout the gland. As expected, the GSTP1 promoter in both normal epithelium and normal stromal cells distant from the tumour was not methylated and the tumour epithelium showed consistently high levels of promoter methylation throughout. However, tumour-associated stromal cells were found to be methylated only in a localized and distinct anatomical sub-field of the tumour, revealing the presence of an epigenetically unique microenvironment within the cancer. Morphologically, the sub-field consisted of typical, non-reactive stroma, representing a genomic alteration in cells that appeared otherwise histologically normal. Similar epigenetic anatomical mapping of a control prostate gland without cancer showed low background methylation levels in all cell types throughout the specimen. These data suggest that stromal cell methylation can occur in a distinct sub-region of prostate cancer and may have implications for understanding tumour biology and clinical intervention.     

Dietary 4-HPR suppresses the development of bone metastasis in vivo in a mouse model of prostate cancer progression

The effects of the synthetic retinoid N-(4-hydroxyphenyl) retinamide (4-HPR) on prostate cancer metastasis in vivo were evaluated in the mouse prostate reconstitution (MPR) model. MPRs were produced by infection of either heterozygous (+/−) or nullizygous (−/−) p53-mutant fetal prostatic epithelial cells with the recombinant retrovirus Zipras/myc 9. Previous studies have documented that loss of p53 function potentiates metastasis in this model system. MPRs were grafted into homozygous (+/+) p53 male mice, fed a 4-HPR containing diet or a control diet and maintained until the status of tumor progression dictated sacrifice. Under these experimental conditions, treatment with 4-HPR did not have a significant effect on primary tumor wet weight for either p53 +/− or p53 −/− MPRs. For, p53 +/− MPRs the animals fed the 4-HPR diet had a slight improvement in survival and a significant reduction in the number of mesenteric metastases (P=0.0477, t-test). Notably, in p53 +/− MPRs the incidence of metastasis to lumbar spine and sternum was 92% in the control animals compared to 54% in the 4-HPR treated animals (P=0.035, χ²-test). In p53 −/− MPRs there was a trend toward a reduction in the number of soft tissue metastases to lung and liver in the 4-HPR group relative to the control diet group and a statistically significant reduction in the incidence of metastasis to bone was demonstrated in that 50% of control animals versus 30% of 4-HPR treated p53 −/− animals harbored bone metastases (P=0<0.05, χ²-test). Cell lines were established from portions of the primary tumor and from selected metastatic deposits in each experimental group. Clonal analysis, by retroviral integration pattern, indicated increased clonal diversity in both the primary tumors and metastasis-derived cell lines from 4-HPR treated animals relative to the control animals. In vitro treatment with 4-HPR did not reveal discriminating differences between cell lines derived from primary tumors and bone metastases or control and treatment groups in regard to growth arrest or apoptotic responses. Overall these studies indicate limited anti-tumor and anti-metastatic activity in this highly aggressive in vivo mouse model of prostate cancer, yet 4-HPR treatment significantly suppressed the development of bone metastases in p53 +/− and p53 −/− MPRs revealing a novel and potentially clinically useful activity of this retinoid. This revised version was published online in July 2006 with corrections to the Cover Date.     

Establishment and validation of an in vitro co-culture model to study the interactions between bone and prostate cancer cells

Bone is the preferred site for prostate cancer (PCa) metastases. Once the tumor has established itself within the bone there is virtually no cure. To better understand the interactions between the PCa cells and bone environment in the metastatic process new model systems are needed. We have established a two-compartment in vitro co-culturing model that can be used to follow the trans-activation of bone and/or tumor cells. The model was validated using two PCa tumor cell lines (PC-3; lytic and LNCaP; mixed/osteoblastic) and one osteolytic inducing factor, 1,25-dihydroxyvitamin D₃ (D3). Results were in accordance with the expected bone phenotypes; PC-3 cells and D3 gave osteolytic gene expression profiles in calvariae, with up-regulation of genes needed for osteoclast differentiation, activation and function; Rankl, CathK, Trap and MMP-9, and down-regulation of genes associated with osteoblast differentiation and bone mineralization; Alp, Ocl and Dkk-1. LNCaP cells activated genes in the calvarial bones associated with osteoblast differentiation and mineralization, with marginal effects on osteolytic genes. The results were strengthened by similar changes in protein expression for a selection of the analyzed genes. Furthermore, the osteolytic gene expression profiles in calvarial bones co-cultured with PC-3 cells or with D3 were correlated with the actual ongoing resorptive process, as assessed by the release of collagen fragments from the calvariae. Our results show that the model can be used to follow tumor-induced bone remodeling, and by measuring changes in gene expression in the tumor cells we can also study how they respond to the bone microenvironment.     

Host matrix metalloproteinase-9 contributes to tumor vascularization without affecting tumor growth in a model of prostate cancer bone metastasis

Matrix metalloproteinases (MMPs) have been associated with initiation, progression and vascularization of a number of tumors. However, clinical trials using MMP inhibitors failed to meet expectations. Previously, we demonstrated the potential importance of MMP-9 activity in experimental prostate cancer bone tumor tissue. However, the particular roles of host- and tumor-derived MMP-9 remains to be defined. Herein, we examined the role of host MMP-9 in subcutaneous and intraosseous growth of the human androgen independent prostate cancer cell line PC3 in MMP-9 deficient mice. In the subcutaneous model, the tumor incidence in the control (RAG-1 ^ko/ko ) and experimental (RAG-1 ^ko/ko /MMP-9 ^ko/ko ) group was 100%, with similar tumor growth kinetics and microvascular densities. In the intraosseous tumor model, the tumor incidence was higher in RAG-1 ^ko/ko /MMP-9 ^ko/ko mice than in RAG-1 ^ko/ko mice (67% and 39%, respectively), though no statistical differences were found. The intraosseous tumor areas were similar in both groups, and the number of tumor-associated osteoclasts did not differ significantly. However, the microvascular density of intraosseous tumors was higher in RAG-1 ^ko/ko than in RAG-1 ^ko/ko /MMP-9 ^ko/ko mice, though no changes in tumor growth could be detected. In an in vitro assay, we found that bone marrow (BM) cells increased the invasiveness of PC3 cells, and that this enhancement was independent of MMP-9 expression by marrow cells. Our results with the RAG-1 model suggest that host-derived MMP-9 is neither necessary nor sufficient for subcutaneous or intraosseous PC3 tumor growth, osteoclastic response, or in vitro invasiveness of tumor cells.     

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.     

Matrix metalloproteinase activity and osteoclasts in experimental prostate cancer bone metastasis tissue

Previously, we and others showed that broad spectrum pharmaceutical inhibition of matrix metalloproteinase (MMP) activity reduces intraosseous tumor burden and bone degradation in animal models of bone metastasis. Herein, we used specific assays to measure net enzymatic activities of individual MMPs during colonization of bone by prostate cancer cells. PC3 cells were injected into the marrow of human fetal femurs previously implanted in SCID mice. Net MMP-9 activity in bone tissues peaked 2 weeks after injection, coinciding with a wave of osteoclast recruitment. In contrast, MMP-2 and MT1-MMP activity did not change. In vitro, co-culture of PC3 cells with bone tissue led to activation of pro-MMP-9 and increases in secreted net MMP-9 activity. Activation of pro-MMP-9 was prevented by metalloprotease inhibitors but not by inhibitors of other classes of proteases. Ribozyme suppression of MMP-9 expression in PC3 cells did not affect pro-MMP-9 activation or net MMP-9 activity and did not affect the phenotype of bone tumors. siRNA targeting of MMP-9 expression in preosteoclasts in vitro demonstrated that tumor-induced preosteoclast motility was dependent on MMP-9 expression. These data suggest that osteoclast-derived MMP-9 may represent a potential therapeutic target in bone metastasis and provide a rationale for the development of MMP-9-specific inhibitors.     

PET/CT对小动物前列腺癌骨转移的观察研究

目的　探讨小动物PET/CT扫描在小鼠前列腺癌骨转移检测中的可行性。 方法　裸鼠23只,随机分为对照组2只,前列腺原位注射组、左心室注射组、胫骨腔内注射组每组各7只。分别采用前列腺原位注射、左心室内注射、胫骨腔内注射的方法建立前列腺癌骨转移动物模型。建模成功后饲养40天,原位注射及左心室注射组采用PET/CT扫描检测,胫骨腔注射组采用小动物高分辨率CT检测,对可疑的骨转移灶行解剖学观察及HE染色明确。 结果　前列腺原位注射组肿瘤细胞聚集腹腔软组织内生长,均未发现明显骨质破坏（0/7）;左心室注射组均发生皮下等软组织转移（7/7）, PET/CT检测出一例胫骨上端骨质破坏,组织学检测证实为骨转移灶(1/7);胫骨腔注射组所有动物均形成明显骨质破坏（7/7）且高分辨率CT检测见骨破坏分级良好。 结论　小动物PET/CT扫描能够良好的显示转移灶在小鼠体内的定位,且能良好的显示骨破坏情况,因此该技术在检测小动物骨转移上有着良好的可行性。     

Hyaluronidase expression induces prostate tumor metastasis in an orthotopic mouse model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.     

Differentially expressed genes in two LNCaP prostate cancer cell lines reflecting changes during prostate cancer progression

Prostate cancer tends to become transformed to androgen-independent disease over time when treated by androgen-deprivation therapy. We used two variants of the human prostate cancer cell line LNCaP to study gene expression differences during prostate cancer progression to androgen-independent disease. Production of prostate-specific antigen was regarded as a marker of androgen-dependence and loss of prostate-specific antigen was regarded as a marker of androgen-independence. mRNA from both cell lines was used for cDNA microarray screening. Differential expression of several genes was confirmed by Northern blotting. Monoamine oxidase A, an Expressed Sequence Tag (EST) similar to rat P044, and EST AA412049 were highly overexpressed in androgen-dependent LNCaP cells. Tissue-type plasminogen activator, interferon-inducible protein p78 (MxB), an EST similar to galectin-1, follistatin, fatty acid-binding protein 5, EST AA609749, annexin I, the interferon-inducible gene 1-8U, and phospholipase D1 were highly overexpressed in androgen-independent LNCaP cells. All studied genes had low or no expression in PC-3 cells. The EST similar to rat P044, the EST similar to galectin-1, follistatin, annexin I, and the interferon-inducible gene 1-8U were also expressed in benign prostatic hyperplasia tissue. The Y-linked ribosomal protein S4, Mat-8, and EST AA307912 were highly expressed in benign prostatic hyperplasia tissue. Additionally, both confirmation of differential expression in Northern blots and in situ hybridization were carried out for monoamine oxidase A, the EST similar to rat P044, the EST similar to galectin-1, fatty acid-binding protein 5, and the interferon-inducible gene 1-8U. We identified several potential prostate cancer markers, indicating that the method used is a useful tool for the screening of cancer markers, but other methods, such as in situ hybridization, are needed to further investigate the observations.     

A novel orthotopic model of breast cancer metastasis to bone

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.     

miR-96与转移抑制因子1是前列腺癌骨转移潜在的生物学标志物

目的探讨miR-96和转移抑制因子1(MTSS1)在前列腺癌(PCa)组织中的表达及其临床意义。方法收集PCa患者标本89例,癌旁组织作为正常对照,分别用原位杂交及免疫组化检测PCa组织及癌旁组织中miR-96和MTSS1的表达水平,并对二者进行相关性分析。结果 89例PCa组织中miR-96阳性表达率为87.64%,显著高于癌旁组织(P0.01);而MTSS1蛋白阳性表达率为16.85%,显著低于癌旁组织(P0.01);miR-96的表达随着Gleason评分和临床分期演进而增加(P0.05),有骨转移的miR-96的表达高于无骨转移组(P0.05);MTSS1的表达随着Gleason评分和临床分期演进而下调,且有骨转移的MTSS1的表达显著低于无骨转移组(P0.01);miR-96表达与MTSS1的表达呈显著负相关(P0.01)。结论 miR-96与MTSS1可能是PCa骨转移的重要生物学标志。     

Activin A circulating levels in patients with bone metastasis from breast or prostate cancer

Recent studies have highlighted that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily, may be involved in the regulation of osteoblastic activity and in osteoclast differentiation. Therefore, we have investigated the clinical significance of its circulating levels in patients with bone metastasis. Activin A serum concentrations were determined, by a commercially available enzyme-linked immunosorbent assay kit, in 72 patients with breast cancer (BC) or prostatic cancer (PC) with (BM+) or without (BM−) bone metastases, in 15 female patients with age-related osteoporosis (OP), in 20 patients with benign prostatic hypertrophy (BPH) and in 48 registered healthy blood donors (HS) of both sex (25 female and 23 male). Activin A serum concentrations were significantly increased in BC or PC patients as compared to OP (P < 0.0001) or BPH (P = 0.045), respectively, or to sex matched HS (P < 0.0001). Additionally, these levels resulted more elevated in PC patients as compared to BC patients (P = 0.032). Interestingly, Activin A was significantly higher in BM+ patients than in BM− patients (BC, P = 0.047; PC, P = 0.016). In BC patients, a significant correlation was observed only between Activin A and number of bone metastases (P = 0.0065) while, in PC patients, Activin A levels were strongly correlated with the Gleason score (P = 0.011) or PSA levels (P = 0.0001) and, to a lessen extent, with the number of bone metastases (P = 0.056). Receiver operating characteristic curve (ROC) analysis showed a fair diagnostic accuracy of Activin A to discriminate between BM+ and BM− patients (BC: AUC = 0.71 ± 0.09, P = 0.03; PC: AUC = 0.73 ± 0.081, P = 0.005). These findings indicate that Activin A may be implicated in the pathogenesis of bone metastasis. Therefore, this cytokine may be considered a novel potential target for a more selective therapeutic approach in the treatment of skeletal metastasis and may be also useful as additional biochemical marker of metastatic bone disease.     

Identification of estrogen-responsive genes involved in breast cancer metastases to the bone

Bone metastasis is the most common metastasis in breast cancer patients. Clinical observations propose strong association between estrogen receptor (ER)-positive tumors and the development of bone metastases. We hypothesized of biologically diverse sets of hormone-dependent tumors predisposed to bone metastases and of possible role of ER-signaling pathways in the development and progression of bone metastases. We developed a novel in vitro estrogen (E2)-responsive model system, in which breast cancer cells and bone cells express high levels of either ERα or ERβ. Using co-culture approach and gene array technology we identified E2-responsive genes involved in the interaction between cancer cells and bone cells. We detected 13 genes that were altered solely by ERα and 11 genes that were regulated solely by ERβ in cancer cells. Only 5 genes were modified by both ERα and ERβ. Interestingly, the majority of genes in bone cells were altered through ERβ. Two genes, namely MacMarcks and Muc-1, whose changes in expressions in cancer cells in response to E2 were highly significant, were selected for immunohistochemical analysis using tissue microarrays of 59 infiltrating ductal carcinomas. Our results indicated that both MacMarcks and Muc-1 were expressed at high frequency in ER-positive tumors. The correlation between ERα- and ERβ-status of hormone-dependent tumors with combined expression of these two markers might suggest a more aggressive tumor phenotype associated with bone metastases. Further analysis of tissues with clinicopathological characteristics and known bone metastatic disease will indicate potential prognostic values of these and other markers in the development of bone metastases in a subgroup of “bad” hormone-dependent breast cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

BMP7, a putative regulator of epithelial homeostasis in the human prostate, is a potent inhibitor of prostate cancer bone metastasis in vivo

Bone morphogenic protein 7 (BMP7) counteracts physiological epithelial-to-mesenchymal transition, a process that is indicative of epithelial plasticity. Because epithelial-to-mesenchymal transition is involved in cancer, we investigated whether BMP7 plays a role in prostate cancer growth and metastasis. BMP7 expression in laser-microdissected primary human prostate cancer tissue was strongly down-regulated compared with normal prostate luminal epithelium. Furthermore, BMP7 expression in prostate cancer cell lines was inversely related to tumorigenic and metastatic potential in vivo and significantly correlated to E-cadherin/vimentin ratios. Exogenous addition of BMP7 to human prostate cancer cells dose-dependently inhibited transforming growth factor beta-induced activation of nuclear Smad3/4 complexes via ALK5 and induced E-cadherin expression. Moreover, BMP7-induced activation of nuclear Smad1/4/5 signaling transduced via BMP type I receptors was synergistically stimulated in the presence of transforming growth factor beta, a growth factor that is enriched in the bone microenvironment. Daily BMP7 administration to nude mice inhibited the growth of cancer cells in bone. In contrast, no significant growth inhibitory effect of BMP7 was observed in intraprostatic xenografts. Collectively, our observations suggest that BMP7 controls and preserves the epithelial phenotype in the human prostate and underscore a decisive role of the tumor microenvironment in mediating the therapeutic response of BMP7. Thus, BMP7 can still counteract the epithelial-to-mesenchymal transition process in the metastatic tumor, positioning BMP7 as a novel therapeutic molecule for treatment of metastatic bone disease.     

Identification of key genes and pathways in castrate-resistant prostate cancer by integrated bioinformatics analysis

ObjectiveTo identify hub genes and pathways involved in castrate-resistant prostate cancer (CRPC).MethodsThe gene expression profiles of GSE70768 were downloaded from Gene Expression Omnibus (GEO) datasets. A total of 13 CRPC samples and 110 tumor samples were identified. The differentially expressed genes (DEGs) were identified, and the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analysis was performed. Protein-protein interaction (PPI) network module analysis was constructed and performed in Cytoscape software. Weighted correlation network analysis (WGCNA) was conducted to determine hub genes involved in the development and progression of CRPC. The gene expression profiles of GSE80609 were used for validation.ResultsA total of 1738 DEGs were identified, consisting of 962 significantly down-regulated DEGs and 776 significantly upregulated DEGs for the subsequent analysis. GO term enrichment analysis suggested that DEGs were mainly enriched in the extracellular matrix organization, extracellular exosome, extracellular matrix, and extracellular space. KEGG pathway analysis found DEGs significantly enriched in the focal adhesion pathway. PPI network demonstrated that the top 10 hub genes were ALB, ACACB, KLK3, CDH1, IL10, ALDH1A3, KLK2, ALDH3B2, HBA1, COL1A1. Also, WGCNA identified the top 5 hub genes in the turquoise module, including MBD4, BLZF1, PIP5K2B, ZNF486, LRRC37B2. Plus, the Venn diagram demonstrated that HBA1 was the key gene in both GSE70768 and GSE80609 datasets.ConclusionsThese newly identified genes and pathways could help urologists understand the differences in the mechanism between CRPC and PCa. Besides, it might be promising targets for the treatment of CRPC.     

Estradiol increases ER-negative breast cancer metastasis in an experimental model

Breast cancer (BC) is the most common cancer affecting women in the United States and metastatic breast cancer is the leading cause of death. The role estradiol plays in ER-positive BC is well-documented, but the way it contributes to ER-negative BC remains unclear. In the present study, we utilized an experimental model of BC metastasis into lung by injecting ER-negative murine 4T1 cells into mice via the lateral tail vein. A 56 % metastasis occurrence rate following the injection of 5 × 10³ cells was observed, thus this cell number was selected to study the potential stimulatory effect of estradiol on ER-negative BC metastasis. Female ovariectomized mice were randomized into estradiol and control groups with 16 mice per group, and estradiol pellets were implanted subcutaneously in the estradiol group. Results demonstrated that estradiol accelerated BC metastasis as indicated by bioluminescent imaging. In addition, estradiol enhanced metastatic tumor colony formation and increased the size of tumor nodules in the lungs, which were due, in part, to the increase in proliferative cells in the metastatic tumors. In vitro, estradiol increased the motility and invasion of 4T1 cells, and the stimulatory effect on cell motility was not blocked by ICI 182, 780, confirming that ER was not involved in the process. Results from the present study suggest that estradiol plays a role in ER-negative BC metastasis in whole animals.