Targeted therapy for metastatic diseases relies on the identification of functionally important metastasis genes from a large number of random genetic alterations. Here we use a computational algorithm to map minimal recurrent genomic alterations associated with poor-prognosis breast cancer. 8q22 genomic gain was identified by this approach and validated in an extensive collection of breast tumor samples. Regional gain of 8q22 elevates expression of the metastasis gene metadherin (MTDH), which is overexpressed in more than 40% of breast cancers and is associated with poor clinical outcomes. Functional characterization of MTDH revealed its dual role in promoting metastatic seeding and enhancing chemoresistance. These findings establish MTDH as an important therapeutic target for simultaneously enhancing chemotherapy efficacy and reducing metastasis risk.

Although the transforming growth factor-beta (TGF-beta) pathway has been implicated in breast cancer metastasis, its in vivo dynamics and temporal-spatial involvement in organ-specific metastasis have not been investigated. Here we engineered a xenograft model system with a conditional control of the TGF-beta-SMAD signaling pathway and a dual-luciferase reporter system for tracing both metastatic burden and TGF-beta signaling activity in vivo. Strong TGF-beta signaling in osteolytic bone lesions is suppressed directly by genetic and pharmacological disruption of the TGF-beta-SMAD pathway and indirectly by inhibition of osteoclast function with bisphosphonates. Notably, disruption of TGF-beta signaling early in metastasis can substantially reduce metastasis burden but becomes less effective when bone lesions are well established. Our in vivo system for real-time manipulation and detection of TGF-beta signaling provides a proof of principle for using similar strategies to analyze the in vivo dynamics of other metastasis-associated signaling pathways and will expedite the development and characterization of therapeutic agents.

Cell fusion plays an essential role in fertilization, formation of placenta, bone and muscle tissues, immune response, tissue repair, and regeneration. Increasing recognition of cell fusion in somatic cell dynamics has revitalized the century-old hypothesis that cell fusion may contribute to the initiation and progression of cancer. In this review, we discuss findings from experimental and clinical studies that suggest a potentially multifaceted involvement of cell fusion in different stages of tumor progression, including aneuploidy and tumor initiation, origin of cancer stem cells, multidrug resistance, and the acquisition and diversification of metastatic abilities.

Metastatic spread of cancer to distant vital organs, including lung and bone, is the overwhelming cause of breast cancer mortality and morbidity. Effective treatment of systemic metastasis relies on the identification and functional characterization of metastasis mediators to multiple organs. Overexpression of the chemokine (C-C motif) ligand 2 (CCL2) is frequently associated with advanced tumor stage and metastatic relapse in breast cancer. However, the functional mechanism of CCL2 in promoting organ-specific metastasis of breast cancer has not been rigorously investigated. Here, we used organ-specific metastatic sublines of the MDA-MB-231 human breast cancer cell line to demonstrate that overexpression of CCL2 promotes breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 function with a neutralizing antibody reduced lung and bone metastases. The enhancement of lung and bone metastases by CCL2 was associated with increased macrophage infiltration and osteoclast differentiation, respectively. By performing functional assays with primary cells isolated from the wild type, CCL2 and CCR2 knock-out mice, we showed that tumor cell-derived CCL2 depends on its receptor CCR2 (chemokine, CC motif, receptor 2) expressed on stromal cells to exert its function in promoting macrophage recruitment and osteoclast differentiation. Overall, these data demonstrated that CCL2-expressing breast tumor cells engage CCR2(+) stromal cells of monocytic origin, including macrophages and preosteoclasts, to facilitate colonization in lung and bone. Therefore, CCL2 and CCR2 are promising therapeutic targets for simultaneously inhibiting lung and bone metastasis of breast cancer.

Bone metastasis is mediated by complex interactions between tumor cells and resident stromal cells in the bone microenvironment. The functions of metalloproteinases in organ-specific metastasis remain poorly defined despite their well-appreciated role in matrix degradation and tumor invasion. Here, we show a mechanism whereby two distinct metalloproteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1) and matrix metalloproteinase-1 (MMP1), orchestrate a paracrine signaling cascade to modulate the bone microenvironment in favor of osteoclastogenesis and bone metastasis. Proteolytic release of membrane-bound epidermal growth factor (EGF)-like growth factors, including Amphiregulin (AREG), heparin-binding EGF (HB-EGF), and transforming growth factor alpha (TGFalpha) from tumor cells suppress the expression of osteoprotegerin (OPG) in osteoblasts and subsequently potentiate osteoclast differentiation. EGF receptor (EGFR) inhibitors block osteolytic bone metastasis by targeting EGFR signaling in bone stromal cells. Furthermore, elevated MMP1 and ADAMTS1 expression is associated with increased risk of bone metastasis in breast cancer patients. This study established MMP1 and ADAMTS1 in tumor cells, as well as EGFR signaling in osteoblasts, as promising therapeutic targets for inhibiting bone metastasis of breast cancer.

Cell fusion is involved in many critical developmental processes, including zygote formation and organogenesis of placenta, bone, and skeletal muscle. In adult tissues, cell fusion has been shown to play an active role in tissue regeneration and repair, and its frequency of occurrence is significantly increased during chronic inflammation. Fusion between tumor cells and normal cells, or among tumor cells themselves, has also been speculated to contribute to tumor initiation, as well as phenotypic evolution during cancer progression and metastasis. Here, we show that dual metastasis organotropisms can be acquired in the same cell through in vitro or in vivo spontaneous fusion between bone- and lung-tropic sublines of the MDA-MB-231 human breast cancer cell line. The synkaryonic hybrids assimilate organ-specific metastasis gene signatures from both parental cells and are genetically and phenotypically stable. Our study suggests cell fusion as an efficient means of phenotypic evolution during tumor progression and additionally demonstrates the compatibility of different metastasis organotropisms.

Mammary stem cells (MaSCs) play essential roles for the development of the mammary gland and its remodeling during pregnancy. However, the precise localization of MaSCs in the mammary gland and their regulation during pregnancy is unknown. Here we report a transgenic mouse model for luciferase-based single marker detection of MaSCs in vivo that we used to address these issues. Single transgene expressing mammary epithelial cells were shown to reconstitute mammary glands in vivo while immunohistochemical staining identified MaSCs in basal and luminal locations, with preponderance towards the basal position. By quantifying luciferase expression using bioluminescent imaging, we were able to track MaSCs non-invasively in individual mice over time. Using this model to monitor MaSC dynamics throughout pregnancy, we found that MaSCs expand in both total number and percentage during pregnancy and then drop down to or below baseline levels after weaning. However, in a second round of pregnancy, this expansion was not as extensive. These findings validate a powerful system for the analysis of MaSC dynamics in vivo, which will facilitate future characterization of MaSCs during mammary gland development and breast cancer.

MicroRNAs (miRNAs) play essential roles in many physiological and pathological processes, including tumor development, by regulating the expression of a plethora of mRNAs. Although the importance of miRNAs in tumorigenesis is well established, only recently have reports elucidated miRNAs as promoters or suppressors of metastasis. The miR-200 family has been shown to inhibit the initiating step of metastasis, epithelial-mesenchymal transition (EMT), by maintaining the epithelial phenotype through direct targeting of transcriptional repressors of E-cadherin, ZEB1 and ZEB2. These findings shed light into a miRNA-mediated regulatory pathway that influences EMT in a developmentally and pathologically relevant setting.

MicroRNAs are small non-coding RNA molecules that can regulate gene expression by interacting with multiple mRNAs and inducing either translation suppression or degradation of mRNA. Recently, several miRNAs were identified as either promoters or suppressors of metastasis. However, it is unclear in which step(s) of the multistep metastatic cascade these miRNAs play a defined functional role. To study the functional importance of miRNAs in epithelial-mesenchymal transition (EMT), a process thought to initiate metastasis by enhancing the motility of tumor cells, we used a well established in vitro EMT assay: transforming growth factor-beta-induced EMT in NMuMG murine mammary epithelial cells. We found that members of the miR-200 family, organized as two clusters in the genome, were repressed during EMT. Overexpression of each miRNA individually or as clusters in NMuMG cells hindered EMT by enhancing E-cadherin expression through direct targeting of ZEB1 and ZEB2, which encode transcriptional repressors of E-cadherin. In the 4TO7 mouse carcinoma cell line, which expresses low levels of endogenous E-cadherin and displays a mesenchymal phenotype, ectopic expression of the miR-200 family miRNAs significantly increased E-cadherin expression and altered cell morphology to an epithelial phenotype. Furthermore, ectopic expression of each miR-200 miRNA cluster significantly reduced the in vitro motility of 4TO7 cells in migration assays. These results suggested that loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression.

The importance of cancer stem cells (CSCs) in tumor-initiation has been firmly established in leukemia and recently reported for a variety of solid tumors. However, the role of CSCs in multistage cancer progression, particularly with respect to metastasis, has not been well-defined. Cancer metastasis requires the seeding and successful colonization of specialized CSCs at distant organs. The biology of normal stem cells and CSCs share remarkable similarities and may have important implications when applied to the study of cancer metastasis. Furthermore, overlapping sets of molecules and pathways have recently been identified to regulate both stem cell migration and cancer metastasis. These molecules constitute a complex network of cellular interactions that facilitate both the initiation of the pre-metastasis niche by the primary tumor and the formation of a nurturing organ microenvironment for migrating CSCs. In this review, we surveyed the recent advances in this dynamic field and propose a unified model of cancer progression in which CSCs assume a central role in both tumorigenesis and metastasis. Better understanding of CSCs as a fundamental component of the metastatic cascade will lead to novel therapeutic strategies against metastatic cancer.

Breast cancer causes mortality by metastasizing to a variety of vital organs, such as bone, lung, brain and liver. Effective therapeutic intervention of this deadly process relies on a better mechanistic understanding of metastasis organotropism. Recent studies have confirmed earlier speculations that metastasis is a non-random process and is dependent on intricate tumor-stroma interactions at the target organ. Both the intrinsic properties of breast cancer cells and the host organ microenvironment are important in determining the efficiency of organ-specific metastasis. Advances in animal modeling, in vivo imaging and functional genomics have accelerated the discovery of important molecular mediators of organ-specific metastasis. A conceptual framework of breast cancer organotropism is emerging and will be instrumental in guiding future efforts in this exciting research field.

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.

The transforming growth factor beta (TGFbeta) signaling pathway plays a vital role in the development and homeostasis of normal tissues. Abnormal function of this pathway contributes to the initiation and progression of cancer. Smad proteins are key signal transducers of the TGFbeta pathway and are essential for the growth suppression function of TGFbeta. Smads are bona fide tumor suppressors whose mutation, deletion, and silencing are associated with many types of human cancer. However, the involvement and functional mechanism of Smad proteins in cancer metastasis are poorly defined. Recent studies using genetically modified cancer cells and mouse tumor models have provided concrete evidence for a Smad-dependent mechanism for metastasis promotion by TGFbeta. Understanding the dual roles of Smad proteins in tumor initiation and progression has important implications for cancer therapeutics.

TGF-beta can signal by means of Smad transcription factors, which are quintessential tumor suppressors that inhibit cell proliferation, and by means of Smad-independent mechanisms, which have been implicated in tumor progression. Although Smad mutations disable this tumor-suppressive pathway in certain cancers, breast cancer cells frequently evade the cytostatic action of TGF-beta while retaining Smad function. Through immunohistochemical analysis of human breast cancer bone metastases and functional imaging of the Smad pathway in a mouse xenograft model, we provide evidence for active Smad signaling in human and mouse bone-metastatic lesions. Genetic depletion experiments further demonstrate that Smad4 contributes to the formation of osteolytic bone metastases and is essential for the induction of IL-11, a gene implicated in bone metastasis in this mouse model system. Activator protein-1 is a key participant in Smad-dependent transcriptional activation of IL-11 and its overexpression in bone-metastatic cells. Our findings provide functional evidence for a switch of the Smad pathway, from tumor-suppressor to prometastatic, in the development of breast cancer bone metastasis.

Metastasis, the spread of cancer from primary tumors to distant vital organs, has devastating consequences. Lack of effective tools to study this complex problem has hindered the development of accurate prognostic methods and effective treatments for metastatic cancer. In the postgenomic era, the application of genomic profiling methods to the analysis of clinical metastasis samples and animal metastasis models has revolutionized the field of metastasis research. This article reviews recent breakthroughs in the functional genomic analysis of metastasis. In addition, its impacts on our understanding of the molecular basis of metastasis and on clinical practice are discussed.

We used bioluminescence imaging to reveal patterns of metastasis formation by human breast cancer cells in immunodeficient mice. Individual cells from a population established in culture from the pleural effusion of a breast cancer patient showed distinct patterns of organ-specific metastasis. Single-cell progenies derived from this population exhibited markedly different abilities to metastasize to the bone, lung, or adrenal medulla, which suggests that metastases to different organs have different requirements. Transcriptomic profiling revealed that these different single-cell progenies similarly express a previously described "poor-prognosis" gene expression signature. Unsupervised classification using the transcriptomic data set supported the hypothesis that organ-specific metastasis by breast cancer cells is controlled by metastasis-specific genes that are separate from a general poor-prognosis gene expression signature. Furthermore, by using a gene expression signature associated with the ability of these cells to metastasize to bone, we were able to distinguish primary breast carcinomas that preferentially metastasized to bone from those that preferentially metastasized elsewhere. These results suggest that the bone-specific metastatic phenotypes and gene expression signature identified in a mouse model may be clinically relevant.

Foamy virus (FV) Bel1/Tas transactivators act as key regulators of gene expression and directly bind DNA Bel1 response elements (BREs) in both the internal (IP) and 5'LTR promoters. Here, we report the mapping and the virus species specificity of the nonhomologous feline foamy virus (FFV) BREs in both promoters. The data indicate that FFV Bel1 did not bind the primate FV IP.BRE and that primate FV Bel1 was not capable of binding the FFV IP.BRE. In addition, we show that the C-terminal activation domain of FFV Bel1 does not contribute to DNA binding because a C-terminal trans-dominant negative FFV Bel1 mutant was still able to bind to both promoters.

Epithelial-mesenchymal transitions (EMT) are vital for morphogenesis during embryonic development and are also implicated in the conversion of early stage tumors into invasive malignancies. Several key inducers of EMT are transcription factors that repress E-cadherin expression. A recent report in Cell (Yang et al., 2004) adds Twist to this list and links EMT to the ability of breast cancer cells to enter the circulation and seed metastases.

We investigated the molecular basis for osteolytic bone metastasis by selecting human breast cancer cell line subpopulations with elevated metastatic activity and functionally validating genes that are overexpressed in these cells. These genes act cooperatively to cause osteolytic metastasis, and most of them encode secreted and cell surface proteins. Two of these genes, interleukin-11 and CTGF, encode osteolytic and angiogenic factors whose expression is further increased by the prometastatic cytokine TGF beta. Overexpression of this bone metastasis gene set is superimposed on a poor-prognosis gene expression signature already present in the parental breast cancer population, suggesting that metastasis requires a set of functions beyond those underlying the emergence of the primary tumor.

Genome-wide transcriptional profiling of human epithelial cells revealed that repression of Id inhibitors of differentiation (Id1, Id2, and Id3) is a general feature of the TGFbeta cytostatic program. Opposite responses of Id1 to TGFbeta and the related factor BMP are dictated by the specific ability of the TGFbeta mediator, Smad3, to activate expression of stress response factor ATF3 and then recruit this factor to the Id1 promoter. Thus, a Smad3-mediated primary gene response, ATF3 induction, enables Smad3 to participate in an ATF3-mediated, secondary gene response. As a common target of TGFbeta/Smad signals and stress signals via p38 kinase, ATF3 additionally serves to channel synergy between these pathways in the response of epithelial cells to stress and injury.

Smad3 is a direct mediator of transcriptional activation by the TGFbeta receptor. Its target genes in epithelial cells include cyclin-dependent kinase inhibitors that generate a cytostatic reponse. We defined how, in the same context, Smad3 can also mediate transcriptional repression of the growth-promoting gene c-myc. A complex containing Smad3, the transcription factors E2F4/5 and DP1, and the corepressor p107 preexists in the cytoplasm. In response to TGFbeta, this complex moves into the nucleus and associates with Smad4, recognizing a composite Smad-E2F site on c-myc for repression. Previously known as the ultimate recipients of cdk regulatory signals, E2F4/5 and p107 act here as transducers of TGFbeta receptor signals upstream of cdk. Smad proteins therefore mediate transcriptional activation or repression depending on their associated partners.

The Tap protein mediates the sequence nonspecific nuclear export of cellular mRNAs as well as the sequence-specific export of retroviral mRNAs bearing the constitutive transport element (CTE). Previously, the structures of individual Tap subdomains, including ribonucleoprotein and leucine-rich repeat domains, have been described. Here, we report the crystal structure of a functional CTE RNA-binding domain of human Tap, including the N-terminal arm of the ribonucleoprotein domain and interdomain linking polypeptide. To identify residues that interact with the CTE, we have introduced 38 alanine substitutions for surface residues in the Tap CTE-binding domain and tested these mutants for their ability to support CTE-dependent nuclear RNA export and CTE binding. Four residues that cluster on a concave surface in the leucine-rich repeat domain were found to be critical for CTE binding and define a CTE-interacting surface on this domain. The second critical CTE-interacting surface on Tap is defined by three previously identified residues on the surface of the ribonucleoprotein domain. The structural and mutational data define a novel RNA-binding site on the Tap protein.

The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)(+) RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)(+) RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore.

The transcription factor Smad2 is released from cytoplasmic retention by TGFbeta receptor-mediated phosphorylation, accumulating in the nucleus where it associates with cofactors to regulate transcription. We uncovered direct interactions of Smad2 with the nucleoporins CAN/Nup214 and Nup153. These interactions mediate constitutive nucleocytoplasmic shuttling of Smad2. CAN/Nup214 and Nup153 compete with the cytoplasmic retention factor SARA and the nuclear Smad2 partner FAST-1 for binding to a hydrophobic corridor on the MH2 surface of Smad2. TGFbeta receptor-mediated phosphorylation stimulates nuclear accumulation of Smad2 by modifying its affinity for SARA and Smad4 but not for CAN/Nup214 or Nup153. Thus, by directly contacting the nuclear pore complex, Smad2 undergoes constant shuttling, providing a dynamic pool that is competitively drawn by cytoplasmic and nuclear signal transduction partners.