Ability Of Breast Cancer Cells Research Articles

Epithelial–mesenchymal transition (EMT) is not sufficient for spontaneous murine breast cancer metastasis

Epithelial-mesenchymal transition (EMT) has been linked to metastatic propensity. The 4T1 tumor is a clinically relevant model of spontaneous breast cancer metastasis. Here we characterize 4T1-derived cell lines for EMT, in vitro invasiveness and in vivo metastatic ability. Contrary to expectations, 67NR cells, which form primary tumors but fail to metastasize, express vimentin and N-cadherin, but not E-cadherin. 4T1 cells express E-cadherin and ZO-1, but are migratory, invasive, and metastasize to multiple sites. 66cl4 cells form lung metastases and display a mixed phenotype, but are not as migratory or invasive as 67NR cells. These findings demonstrate that the metastatic ability of breast cancer cells does not strictly correlate with genotypic and phenotypic properties of EMT per se, and suggest that other processes may govern metastatic capability. Gene expression analysis of primary tumors did not identify differences in EMT markers, but did reveal candidate genes that may influence metastatic ability.

Comparative actions of progesterone, medroxyprogesterone acetate, drospirenone and nestorone on breast cancer cell migration and invasion

BackgroundLimited information is available on the effects of progestins on breast cancer progression and metastasis. Cell migration and invasion are central for these processes, and require dynamic cytoskeletal and cell membrane rearrangements for cell motility to be enacted.MethodsWe investigated the effects of progesterone (P), medroxyprogesterone acetate (MPA), drospirenone (DRSP) and nestorone (NES) alone or with 17β-estradiol (E2) on T47-D breast cancer cell migration and invasion and we linked some of these actions to the regulation of the actin-regulatory protein, moesin and to cytoskeletal remodeling.ResultsBreast cancer cell horizontal migration and invasion of three-dimensional matrices are enhanced by all the progestins, but differences are found in terms of potency, with MPA being the most effective and DRSP being the least. This is related to the differential ability of the progestins to activate the actin-binding protein moesin, leading to distinct effects on actin cytoskeleton remodeling and on the formation of cell membrane structures that mediate cell movement. E2 also induces actin remodeling through moesin activation. However, the addition of some progestins partially offsets the action of estradiol on cell migration and invasion of breast cancer cells.ConclusionThese results imply that P, MPA, DRSP and NES alone or in combination with E2 enhance the ability of breast cancer cells to move in the surrounding environment. However, these progestins show different potencies and to some extent use distinct intracellular intermediates to drive moesin activation and actin remodeling. These findings support the concept that each progestin acts differently on breast cancer cells, which may have relevant clinical implications.

MT1-MMP-Dependent Invasion Is Regulated by TI-VAMP/VAMP7

Proteolytic degradation of the extracellular matrix (ECM) is one intrinsic property of metastatic tumor cells to breach tissue barriers and to disseminate into different tissues. This process is initiated by the formation of invadopodia, which are actin-driven, finger-like membrane protrusions. Yet, little is known on how invadopodia are endowed with the functional machinery of proteolytic enzymes [1, 2]. The key protease MT1-MMP (membrane type 1-matrix metalloproteinase) confers proteolytic activity to invadopodia and thus invasion capacity of cancer cells [3-6]. Here, we report that MT1-MMP-dependent matrix degradation at invadopodia is regulated by the v-SNARE TI-VAMP/VAMP7, hence providing the molecular inventory mediating focal degradative activity of cancer cells. As observed by TIRF microscopy, MT1-MMP-mCherry and GFP-VAMP7 were simultaneously detected at proteolytic sites. Functional ablation of VAMP7 decreased the ability of breast cancer cells to degrade and invade in a MT1-MMP-dependent fashion. Moreover, the number of invadopodia was dramatically decreased in VAMP7- and MT1-MMP-depleted cells, indicative of a positive-feedback loop in which the protease as a cargo of VAMP7-targeted transport vesicles regulates maturation of invadopodia. Collectively, these data point to a specific role of VAMP7 in delivering MT1-MMP to sites of degradation, maintaining the functional machinery required for invasion.

Calpain 2 and PTP1B function in a novel pathway with Src to regulate invadopodia dynamics and breast cancer cell invasion

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.

PTHrP promotes homotypic aggregation of breast cancer cells in three-dimensional cultures

Parathyroid hormone-related protein (PTHrP) regulates growth and migration of adherent breast cancer cells. Here, we show that PTHrP also interferes with the ability of breast cancer cells to aggregate in suspension cultures. Cell colonies were significantly smaller when the expression of PTHrP or its target genes, integrin α6 or KISS-1, was suppressed by RNA interference. TGFβ1, a stimulator of PTHrP transcription, abolished the effect of PTHrP and KISS-1 specific siRNAs and increased ERK1/2 phosphorylation, whereas inhibition of ERK1/2 phosphorylation by U0126 reduced colony size. PTHrP and KISS-1 may regulate colony formation in 3D by influencing ERK1/2 phosphorylation.

Down-regulation of CXCR4 expression by siRNA inhibits invasive ability of breast cancer cells

OBJECTIVE To investigate the efficiency of gene silencing by CXCR4-siRNAs (small interfering RNA), and to examine the invasive ability and the expression of other metastatic associated genes in siRNA-treated breast cancer cells. METHODS Three siRNAs were designed and cloned into the pSilenc™ 3.1-H1 neo vector. The reconstructed plasmids were purified and trans-fected into the T47D breast cancer cell line, which highly expressed CXCR4. The amount of CXCR4 expression in the transfected cells was measured by flow cytometry and Real-time PCR. Cell invasive ability was evaluated us -ing 24-well Matrigel invasion chambers. In addition, the expression of other metastatic-associated genes, such as E-cad, IGFBP-5, FN and MMP-2, was assessed by Real-time PCR. RESULTS The suppression rates of CXCR4 mRNA expression reached 95.7%, 85.9% and 98.3%compared with control-siRNA cells in the 3 CXCR4-siRNA T47D cells respectively. FCM assays for CXCR4 protein expression showed a similar inhibitory effect. The invasion indexes of these CXCR4-siRNA cells were 0.037, 0.290 and 0.188respectively compared with control-siRNA cells. After treatment of the cells with CXCR4-siRNA, the expression of E-cad showed an upward tendency and that of IGFBP-5 had a downward trend, while alteration in expression of FN and MMP2 varied without a con -sistant effect. CONCLUSION CXCR4 plays an important role in modulating migra-tion of human breast cancer cells. Small interfering RNA can significantly silence the CXCR4 gene in the human T47D breast cancer cell line. The results of this study strengthen the need for further research on novel gene therapy against breast cancer metastasis.

Plasminogen binding and activation at the breast cancer cell surface: the integral role of urokinase activity

IntroductionThe regulation of extracellular proteolytic activity via the plasminogen activation system is complex, involving numerous activators, inhibitors, and receptors. Previous studies on monocytic and colon cell lines suggest that plasmin pre-treatment can increase plasminogen binding, allowing the active enzyme to generate binding sites for its precursor. Other studies have shown the importance of pre-formed receptors such as annexin II heterotetramer. However, few studies have used techniques that exclusively characterise cell-surface events and these mechanisms have not been investigated at the breast cancer cell surface.MethodsWe have studied plasminogen binding to MCF-7 in which urokinase plasminogen activator receptor (uPAR) levels were upregulated by PMA (12-O-tetradecanoylphorbol-13-acetate) stimulation, allowing flexible and transient modulation of cell-surface uPA. Similar experiments were also performed using MDA-MB-231 cells, which overexpress uPAR/uPA endogenously. Using techniques that preserve cell integrity, we characterise the role of uPA as both a plasminogen receptor and activator and quantify the relative contribution of pre-formed and cryptic plasminogen receptors to plasminogen binding.ResultsCell-surface plasminogen binding was significantly enhanced in the presence of elevated levels of uPA in an activity-dependent manner and was greatly attenuated in the presence of the plasmin inhibitor aprotinin. Pre-formed receptors were also found to contribute to increased plasminogen binding after PMA stimulation and to co-localise with uPA/uPAR and plasminogen. Nevertheless, a relatively modest increase in plasminogen-binding capacity coupled with an increase in uPA led to a dramatic increase in the proteolytic capacity of these cells.ConclusionWe show that the majority of lysine-dependent plasminogen binding to breast cancer cells is ultimately regulated by plasmin activity and is dependent on the presence of significant levels of active uPA. The existence of a proteolytic positive feedback loop in plasminogen activation has profound implications for the ability of breast cancer cells expressing high amounts of uPA to accumulate a large proteolytic capacity at the cell surface, thereby conferring invasive potential.

The Spemann organizer gene, Goosecoid , promotes tumor metastasis

The process of invasion and metastasis during tumor progression is often reminiscent of cell migration events occurring during embryonic development. We hypothesized that genes controlling cellular changes in the Spemann organizer at gastrulation might be reactivated in tumors. The Goosecoid homeobox transcription factor is a known executer of cell migration from the Spemann organizer. We found that indeed Goosecoid is overexpressed in a majority of human breast tumors. Ectopic expression of Goosecoid in human breast cells generated invasion-associated cellular changes, including an epithelial-mesenchymal transition. TGF-beta signaling, known to promote metastasis, induced Goosecoid expression in human breast cells. Moreover, Goosecoid significantly enhanced the ability of breast cancer cells to form pulmonary metastases in mice. These results demonstrate that Goosecoid promotes tumor cell malignancy and suggest that other conserved organizer genes may function similarly in human cancer.

Osteoprotegerin Overexpression by Breast Cancer Cells Enhances Orthotopic and Osseous Tumor Growth and Contrasts with That Delivered Therapeutically

Osteoprotegerin (OPG) acts as a decoy receptor for receptor activator of NF-kappaB ligand (RANKL), which is a pivotal molecule required for osteoclast formation. In vitro OPG inhibits osteoclast formation and in vivo (administered as Fc-OPG) it reduces hypercalcemia and the establishment of osteolytic lesions in mouse models of tumor cell growth in bone. Osteolysis can be induced by parathyroid hormone-related protein (PTHrP) produced by breast cancer cells that results in an increased osteoblastic RANKL/OPG ratio. We examined the effect of local tumor production of OPG on the ability of breast cancer cells to establish and grow in bone and mammary fat pad. MCF-7 cells or MCF-7 cells overexpressing PTHrP were transfected with full-length OPG and inoculated into the proximal tibiae of athymic nude mice. Mice injected with cells overexpressing PTHrP and OPG showed enhanced tumor growth, increased osteolysis (2-fold compared with MCF-7 cells overexpressing PTHrP), and altered histology that was reflective of a less differentiated (more aggressive) phenotype compared with MCF-7 cells. In contrast, administration of recombinant Fc-OPG reduced tumor growth and limited osteolysis even in mice inoculated with OPG overexpressing cells. Similarly, OPG overexpression by breast cancer cells enhanced tumor growth following orthotopic inoculation. These results indicate that OPG overexpression by breast cancer cells increases tumor growth in vivo and that there are strikingly different responses between therapeutically administered Fc-OPG and full-length OPG produced by tumor cells.

Inducible IκB Kinase/IκB Kinase ε Expression Is Induced by CK2 and Promotes Aberrant Nuclear Factor-κB Activation in Breast Cancer Cells

Aberrant activation of nuclear factor-kappaB (NF-kappaB) transcription factors has been implicated in the pathogenesis of breast cancer. We previously showed elevated activity of IkappaB kinase alpha (IKKalpha), IKKbeta, and protein kinase CK2 in primary human breast cancer specimens and cultured cells. A novel inducible IKK protein termed IKK-i/IKKepsilon has been characterized as a potential NF-kappaB activator. Here, we provide evidence that implicates IKK-i/IKKepsilon in the pathogenesis of breast cancer. We show IKK-i/IKKepsilon expression in primary human breast cancer specimens and carcinogen-induced mouse mammary tumors. Multiple breast cancer cell lines showed higher levels of IKK-i/IKKepsilon and kinase activity compared with untransformed MCF-10F breast epithelial cells. Interestingly, IKK-i/IKKepsilon expression correlated with CK2alpha expression in mammary glands and breast tumors derived from MMTV-CK2alpha transgenic mice. Ectopic CK2 expression in untransformed cells led to increased IKK-i/IKKepsilon mRNA and protein levels. Inhibition of CK2alpha via the pharmacologic inhibitor apigenin or upon transfection of a CK2 kinase-inactive subunit reduced IKK-i/IKKepsilon levels. Expression of a kinase-inactive IKK-i/IKKepsilon mutant in breast cancer cells reduced NF-kappaB activity as judged by transfection assays of reporters driven either by NF-kappaB elements or the promoters of two NF-kappaB target genes, cyclin D1 and relB. Importantly, the kinase-inactive IKK-i/IKKepsilon mutant reduced the endogenous levels of these genes as well as the ability of breast cancer cells to grow in soft agar or form invasive colonies in Matrigel. Thus, CK2 induces functional IKK-i/IKKepsilon, which is an important mediator of the activation of NF-kappaB that plays a critical role in the pathogenesis of breast cancer.

Anti-apoptotic Proteins Induce Non-random Genetic Alterations that Result in Selecting Breast Cancer Metastatic Cells

To shed light on the relationships between over-expression of anti-apoptotic proteins, genomic instability, and the metastatic ability of breast cancer cells, we analyzed genetic changes in tumors and metastases by orthotopically injecting MDA-MB 435 cells transfected with anti-apoptotic genes Bcl-xL or Bcl-2 into nude mice. Tumors and metastasis variants were extracted by primary culture from breast, bone, lung, and lymph node from mice with 435/Bcl-xL, 435/Bcl-2, and 435/Neo tumors. Using the Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), which permits the detection of allelic imbalances, we generated four different fingerprints utilizing four primers. We found that the genetic damage fraction (GDF) increased in 435/Bcl-2 (GDF=0.55) and 435/Bcl-xL cells (GDF=0.34), in regard to 435/Neo control cells (GDF=0.29), indicating that non-random genetic alterations occurred in cells secondary to Bcl-2 or Bcl-xL over-expression. Anti-apoptotic proteins render breast cancer cells susceptible to the in vivo acquisition of highly tumorigenic (Kruskal-Wallis, P=0.019) and metastatic (Kruskal-Wallis, P=0.004) activity. We therefore propose that genetic instability is a molecular mechanism favored by anti-apoptotic proteins involved in the selection of highly metastatic cells during tumorigenesis, a pathogenic event favoring the expansion of metastasis.

Metastatic breast cancer cells suppress osteoblast adhesion and differentiation

Bone is a primary target for colonization of metastatic breast cancer cells. Once present, the breast cancer cells activate osteoclasts, thereby stimulating bone loss. Bone degradation is accompanied by pain and increased susceptibility to fractures. However, targeted inhibition of osteoclasts does not completely prevent lesion progression, nor does it heal the lesions. This suggests that breast cancer cells may also affect osteoblasts, cells that build bone. The focus of this study was to determine the ability of breast cancer cells to alter osteoblast function. MC3T3-E1 osteoblasts were cultured with conditioned medium from MDA-MB-231 breast cancer cells and subsequently assayed for changes in differentiation. Osteoblast differentiation was monitored by expression of osteocalcin, bone sialoprotein and alkaline phosphatase, and by mineralization. Osteoblasts cultured with MDA-MB-231 conditioned medium did not express these mature bone proteins, nor did they mineralize a matrix. Inhibition of osteoblast differentiation was found to be due to transforming growth factor beta present in MDA-MB-231 conditioned medium. Interestingly, breast cancer conditioned medium also altered cell adhesion. When osteoblasts were assayed for adhesion properties using interference reflection microscopy and scanning acoustic microscopy, there was a reduction in focal adhesion plaques and sites of detachment were clearly visible. F-actin was disassembled and punctate in osteoblasts cultured with MDA-MB-231 conditioned medium rather than organized in long stress fibers. Taken together, these observations suggest that metastatic breast cancer cells alter osteoblast adhesion and prevent differentiation. These affects could account for the continued loss of bone after osteoclast inhibition in patients with bone-metastatic breast cancer.

Immunocytochemical demonstration of down regulation of HLA class-I molecule expression in human metastatic breast carcinoma.

Deficient expression of HLA class-I molecules observed in many cancers is suggested to influence both disease progression and potential efficacy of T cell-mediated immune therapy. Previous studies have attempted to correlate either primary breast cancer tumor grade with HLA class-I levels or the presence of HLA class-I-deficient cells in metastatic lesions with survival. In this study we evaluated the HLA class-I status of matched primary and secondary breast cancer lesions in order to ask the question: is metastasis of breast cancer associated with down-regulation of HLA class-I expression? Immunocytochemistry analysis shows a definitive correlation between diminished HLA class-I expression and dissemination of breast cancer to tumor-draining lymph nodes: both the total number of HLA class-I+ cells per sample and the levels of expression are dramatically decreased in secondary versus primary tumor lesions. These findings are consistent with the contention that the ability of breast cancer cells to escape the confines of the original tumor lesion requires down-regulation of HLA class-I expression and implies that enhancing HLA class-I expression in secondary breast cancer may have a beneficial effect on T-cell-mediated immunotherapy.

Loss of the tight junction protein claudin-7 correlates with histological grade in both ductal carcinoma in situ and invasive ductal carcinoma of the breast.

Claudins are transmembrane proteins that seal tight junctions, and are critical for maintaining cell-to-cell adhesion in epithelial cell sheets. However, their role in cancer progression remains largely unexplored. Here, we report that Claudin-7 (CLDN-7) expression is lower in invasive ductal carcinomas (IDC) of the breast than in normal breast epithelium, as determined by both RT-PCR (9/10) and Western analysis (6/8). Immunohistochemical (IHC) analysis of ductal carcinoma in situ (DCIS) and IDC showed that the loss of CLDN-7 expression correlated with histological grade in both DCIS (P<0.001, n=38) and IDC (P=0.014, n=31), occurring predominantly in high-grade (Nuclear and Elston grade 3) lesions. Tissue array analysis of 355 IDC cases further confirmed the inverse correlation between CLDN-7 expression and histological grade (P=0.03). This pattern of expression is consistent with the biological function of CLDN-7, as greater discohesion is typically observed in high-grade lesions. In line with this observation, by IHC analysis, CLDN-7 expression was lost in the vast majority (13/17) of cases of lobular carcinoma in situ, which is defined by cellular discohesion. In fact, inducing disassociation of MCF-7 and T47D cells in culture by treating with HGF/scatter factor resulted in a loss of CLDN-7 expression within 24 h. Silencing of CLDN-7 expression correlated with promoter hypermethylation as determined by methylation-specific PCR (MSP) and nucleotide sequencing in breast cancer cell lines (3/3), but not in IDCs (0/5). In summary, these studies provide insight into the potential role of CLDN-7 in the progression and ability of breast cancer cells to disseminate.

Overexpression of parathyroid hormone-related protein causes hypercalcemia but not bone metastases in a murine model of mammary tumorigenesis.

Several lines of evidence suggest that production of parathyroid hormone-related protein (PTHrP) by breast cancer cells contributes to the formation of bone metastases. However, it is not clear if PTHrP promotes access of cancer cells to the skeleton or if it simply promotes bone resorption around cells already within bone. To study the effects of PTHrP on the development of bone metastases, we treated mice overexpressing PTHrP in their mammary glands (K14-PTHrP transgenic mice) with 9,10-dimethyl-1,2-benz-anthracene (DMBA), a known mammary carcinogen. After DMBA treatment, K14-PTHrP mice showed a higher incidence of tumor formation and a shorter latency to tumor formation than wild-type littermates. Transgenic tumors expressed the K14-PTHrP transgene and secreted excess amounts of PTHrP. In response, tumor-bearing transgenic mice became hypercalcemic and had elevated circulating levels of PTHrP. Despite the development of visceral metastases, neither transgenic mice nor wild-type controls developed bone metastases. This was true even if tumor cells were introduced into the arterial circulation of immunodeficient mice. Our results are consistent with the emerging notion that the ability of breast cancer cells to produce PTHrP in response to cues from the bone microenvironment may be more important to the development of skeletal metastases than the production of PTHrP by cells within the primary breast cancer.

Vascular and Biology 02

Background: The chemokine receptor CXCR4 is linked to chemokine-mediated metastasis in breast cancer. The breast cancer microenvironment is rich in cytokines, known to have a regulatory effect on CXCR4. We examined the inflammatory cytokine regulation of CXCR4 surface expression and its effect on migration in MCF-7 and MDA MB231 cells. As heparin competitively antagonizes chemokines we evaluated its potential to block migration of these cell lines towards the chemokine SDF-1. Methods: Expression of CXCR4 was analysed by flow cytometry (R & D Mab 172) before and after stimulation with TNF-α or IFN-γ. The effects of TNF-α and heparin on migration were examined by chemotaxis assays. Results: TNF-α significantly reduced CXCR4 expression in both cell lines after 24 h (MCF-7: 83 per cent*; MDA MB231: 89 per cent*). In MCF-7 cells IFN-γ reduced expression (97 per cent*) by 24 h; a bi-phasic response was observed for MDA MB 231 cells, with a maximal reduction occurring after 16 h (83 per cent*), returning to unstimulated levels by 24 h. The reduction in CXCR4 expression by TNF-α was consistent with reduced migration towards SDF-1 (MCF-7 by 80 per cent*, MDA MB231 by 94 per cent*). Heparin also inhibited chemotaxis of MCF-7 (at 250 µg mL−1*) and MDA MB231 (at 100 µg mL−1 and 250 µg mL−1*). Conclusions: Regulation of the CXCR4 by proinflammatory cytokines alters the ability of breast cancer cells to migrate towards SDF-1. Heparin significantly reduces SDF-1 induced breast cancer cell migration, suggesting a potential therapeutic action in patients with breast cancer. Statistics: Student's one-tailed t-test. (*P < 0.05) (TNF-α: tumour necrosis factor alpha, IFN-γ: interferon gamma, SDF-1: stromal derived factor-1)

Tumors and Fibrinogen

The progression of a tumor from benign and localized to invasive and metastatic growth is the major cause of poor clinical outcome in cancer patients. Much like in a healing wound, the deposition of fibrin(ogen), along with other adhesive glycoproteins, into the extracellular matrix (ECM) serves as a scaffold to support binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during angiogenesis and tumor cell growth. Inappropriate synthesis and deposition of ECM constituents is linked to altered regulation of cell proliferation, leading to tumor cell growth and malignant transformation. Fibrin deposition occurs within the stroma of a majority of tumor types. In contrast, abundant FBG, not fibrin, is present within the stroma of breast cancers. It is thought to originate from exudation of plasma FBG and subsequent deposition into the tumor stroma and not endogenous synthesis and secretion of FBG by breast tumor cells. However, we show that MCF-7 human breast cancer cells synthesize and secrete FBG polypeptides, suggesting that the origin of FBG in the stroma of breast carcinoma may be due to endogenous synthesis and deposition. Moreover, FBG assembles into ECM as conformationally altered FBG, not as fibrin. Studies in our laboratory demonstrate that FBG alters the ability of breast cancer cells to migrate. Together, the results of studies from our laboratory, as well as the laboratories of others, indicate that the presence of fibrin(ogen) within the tumor stroma likely affects the progression of tumor cell growth and metastasis. This review focuses on FBG within tumors and its relationship with other tumor constituents, ultimately focusing on the role of FBG in breast cancer.

Osteopontin expression in a group of lymph node negative breast cancer patients.

The aim of this study was to examine the cellular distribution of osteopontin (OPN) protein [by immunohistochemical (IHC) analysis] and mRNA [by in situ hybridization (ISH)] in the primary tumors of lymph node negative (LNN) breast cancer patients and to determine whether the level of immunodetectable OPN may be associated with tumor aggressiveness. We examined OPN levels in tumors from 154 patients with LNN breast cancer who were followed for a median of 7 years (range 1.7-16.3 years). IHC staining for OPN was seen in tumor infiltrating macrophages and lymphocytes in 70% of these tumors, and in the carcinoma cells themselves in 26%. ISH was performed to determine cellular distribution of OPN mRNA expression in sections from selected tumors. OPN mRNA was detected in groups of tumor cells, individual tumor cells and tumor infiltrating macrophages and lymphocytes. Matched sections showed that some tumor cells with IHC staining for OPN protein were also positive for OPN mRNA by ISH, in contrast with previous studies which have shown OPN mRNA expression only in tumor infiltrating inflammatory cells. Our results thus indicate that OPN protein can be produced by breast cancer cells in vivo and suggest that it may also be taken up from the environment (i.e., secreted by inflammatory cells or other tumor cells). Tumor cell IHC staining intensity was then assessed using a semiquantitative scoring system. Univariate analysis showed tumor cell OPN positivity above an optimized cutpoint to be significantly associated with decreased disease-free survival (DFS) and overall survival (OS). The results of this pilot study thus suggest that the ability of breast cancer cells to either synthesize OPN or to bind and sequester OPN from the microenvironment may be associated with tumor aggressiveness and poor prognosis.

Bone sialoprotein expression in primary human breast cancer is associated with bone metastases development.

Breast cancer metastasizes to bone more frequently than to any other organ, and over 80% of advanced breast cancer patients develop bone metastases. Our recent demonstration that human breast cancer cells express bone sialoprotein (BSP), a bone matrix protein, provides a possible clue for the selective affinity of breast cancer cells for bone. We tested the hypothesis that detection of BSP in primary human breast cancer could be a potential indicator of the ability of breast cancer cells to metastasize to bone. BSP expression was evaluated in the primary breast cancers of 39 patients using immunoperoxidase and two specific anti-BSP antibodies. None of these patients presented clinically or scintigraphically detectable bone metastases at the time of surgery. In the course of their disease, 22 patients developed clinically diagnosed bone metastases. Expression of BSP in breast cancer cells from patients who developed bone metastases was significantly higher (p = 0.008, according to the Mann-Whitney test) than in patients with no bone involvement. No association was found between BSP expression in the primary breast lesions and axillary lymph node metastases. BSP expression was significantly increased in infiltrating ductal carcinoma compared with infiltrating lobular carcinoma (p = 0.0023). No correlation was found between immunoreactivity to BSP antibodies and estrogen receptor (ER) status, progesterone receptor (PR) status, or age. Our data suggest that BSP could help to identity which women will develop bone metastases and provide new bases for the understanding of the molecular mechanism(s) responsible for breast cancer cells osteotropism.

Positive regulation of migration and invasion by vasodilator-stimulated phosphoprotein via Rac1 pathway in human breast cancer cells

This study aimed to investigate the role of the cytoskeleton-associated protein vasodilator-stimulated phosphoprotein (VASP) on the migration and invasion of human breast cancer cells and its relationship to Rac1 which is a member of the Rho family and has been found to be implicated in tumorigenesis, invasion and metastasis. We detected the mRNA and protein expression levels of VASP and Rac1 of the non-invasive breast cancer cell line MCF-7 as well as the invasive cell line MDA-MB-231 by RT-PCR and Western blotting. GST pull-down assay was used to examine the activity of Rac1. Accordingly, the cell invasive migration ability was analyzed in a wound-healing assay (2D) and transwell assays (3D migration and invasion). We then used VASP-siRNA to inhibit the expression of VASP in breast cancer cells in order to study the relationship between the VASP expression level and the invasive migration ability of breast cancer cells. Rac1-siRNA was used to decrease the expression of Rac1, and observe its effect on the VASP expression level together with the migration and invasion ability of MCF-7 and MDA-MB-231 cells. Our results revealed that the invasive breast cancer cell line MDA-MB-231 showed a higher Rac1 activity and VASP expression level compared with the non-invasive MCF-7. Inhibition of Rac1 or VASP by siRNA, respectively, decreased the migration and invasion ability of breast cancer cells and the transfection of Rac1 siRNA-mediated reduction of VASP expression at mRNA and protein levels. Collectively, our data showed that the higher expression level of VASP contributed to a higher invasive migration capacity of human breast cancer cells which was controlled by the Rac1 pathway.