Role of platelets and breast cancer stem cells in metastasis

The high mortality rate of breast cancer is mainly caused by the metastatic ability of cancer cells, resistance to chemotherapy and radiotherapy, and tumor regression capacity. In recent years, it has been shown that the presence of breast cancer stem cells is closely associated with the migration and metastatic ability of cancer cells, as well as with their resistance to chemotherapy and radiotherapy. The tumor microenvironment is one of the main molecular factors involved in cancer and metastatic processes development, in this sense it is interesting to study the role of platelets, one of the main communicator cells in the human body which are activated by the signals they receive from the microenvironment and can generate more than one response. Platelets can ingest and release RNA, proteins, cytokines and growth factors. After the platelets interact with the tumor microenvironment, they are called "tumor-educated platelets." Tumor-educated platelets transport material from the tumor microenvironment to sites adjacent to the tumor, thus helping to create microenvironments conducive for the development of primary and metastatic tumors. It has been observed that the clone capable of carrying out the metastatic process is a cancer cell with stem cell characteristics. Cancer stem cells go through a series of processes, including epithelial-mesenchymal transition, intravasation into blood vessels, movement through blood vessels, extravasation at the site of the establishment of a metastatic focus, and site colonization. Tumor-educated platelets support all these processes.     

Metastatic tumor cells – genotypes and phenotypes

Background

Metastasis is the primary cause of mortality in cancer patients. Therefore, elucidating the genetics and epigenetics of metastatic tumor cells and the mechanisms by which tumor cells acquire metastatic properties constitute significant challenges in cancer research.

Objective

To summarize the current understandings of the specific genotype and phenotype of the metastatic tumor cells.

Method and Result

In-depth genetic analysis of tumor cells, especially with advances in the next-generation sequencing, have revealed insights of the genotypes of metastatic tumor cells. Also, studies have shown that the cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT) phenotypes are associated with the metastatic cascade.

Conclusion

In this review, we will discuss recent advances in the field by focusing on the genomic instability and phenotypic dynamics of metastatic tumor cells.

     

Secreted Gaussia Luciferase as a Biomarker for Monitoring Tumor Progression and Treatment Response of Systemic Metastases

Background

Currently, only few techniques are available for quantifying systemic metastases in preclinical model. Thus techniques that can sensitively detect metastatic colonization and assess treatment response in real-time are urgently needed. To this end, we engineered tumor cells to express a naturally secreted Gaussia luciferase (Gluc), and investigated its use as a circulating biomarker for monitoring viable metastatic or primary tumor growth and their treatment responses.

Methodology/Principal Findings

We first developed orthotopic primary and metastatic breast tumors with derivative of MDA-MB-231 cells expressing Gluc. We then correlated tumor burden with Gluc activity in the blood and urine along with bioluminescent imaging (BLI). Second, we utilized blood Gluc assay to monitor treatment response to lapatinib in an experimental model of systemic metastasis. We observed good correlation between the primary tumor volume and Gluc concentration in blood (R² = 0.84) and urine (R² = 0.55) in the breast tumor model. The correlation deviated as a primary tumor grew due to a reduction in viable tumor fraction. This was also supported by our mathematical models for tumor growth to compare the total and viable tumor burden in our model. In the experimental metastasis model, we found numerous brain metastases as well as systemic metastases including bone and lungs. Importantly, blood Gluc assay revealed early growth of metastatic tumors before BLI could visualize their presence. Using secreted Gluc, we localized systemic metastases by BLI and quantitatively monitored the total viable metastatic tumor burden by blood Gluc assay during the course of treatment with lapatinib, a dual tyrosine kinase inhibitor of EGFR and HER2.

Conclusion/Significance

We demonstrated secreted Gluc assay accurately reflects the amount of viable cancer cells in primary and metastatic tumors. Blood Gluc activity not only tracks metastatic tumor progression but also serves as a longitudinal biomarker for tumor response to treatments.     

Dissection of a metastatic gene expression signature into distinct components

Background

Metastasis, the process whereby cancer cells spread, is in part caused by an incompletely understood interplay between cancer cells and the surrounding stroma. Gene expression studies typically analyze samples containing tumor cells and stroma. Samples with less than 50% tumor cells are generally excluded, thereby reducing the number of patients that can benefit from clinically relevant signatures.

Results

For a head-neck squamous cell carcinoma (HNSCC) primary tumor expression signature that predicts the presence of lymph node metastasis, we first show that reduced proportions of tumor cells results in decreased predictive accuracy. To determine the influence of stroma on the predictive signature and to investigate the interaction between tumor cells and the surrounding microenvironment, we used laser capture microdissection to divide the metastatic signature into six distinct components based on tumor versus stroma expression and on association with the metastatic phenotype. A strikingly skewed distribution of metastasis associated genes is revealed.

Conclusion

Dissection of predictive signatures into different components has implications for design of expression signatures and for our understanding of the metastatic process. Compared to primary tumors that have not formed metastases, primary HNSCC tumors that have metastasized are characterized by predominant down-regulation of tumor cell specific genes and exclusive up-regulation of stromal cell specific genes. The skewed distribution agrees with poor signature performance on samples that contain less than 50% tumor cells. Methods for reducing tumor composition bias that lead to greater predictive accuracy and an increase in the types of samples that can be included are presented.     

Effects of neuromedin B and GRP-10 on gastrin and insulin release from cultured tumor cells of a malignant gastrinoma

A study relating to gastrin release from gastrinoma cells by neuromedin B and C-terminal decapeptide of gastrin releasing peptide (GRP-10) has not yet been reported. Therefore, we studied the effects of neuromedin B and GRP-10 on gastrin release from cultured dispersed cells prepared from both the primary tumor in the pancreas and the metastatic tumor in the liver from a case of malignant Zollinger-Ellison syndrome. Both the primary and metastatic tumors obtained by a curative operation contained similar concentrations of gastrin and glucagon, whereas the primary tumor contained 10 times more insulin than the metastatic tumor. Gastrin release from cultured cells of both tumors was suppressed by 0.1 and 10 nM neuromedin B and tended to be suppressed by 0.1-10 nM GRP-10. However, insulin release from cultured cells of the pancreatic tumor was stimulated by GRP-10, but not by neuromedin B. These results might suggest that receptor function for the bombesin family peptides is abnormal in gastrinoma cells in both primary and metastatic tumors, and that a major source of insulin secretary cells is the contaminated normal islet cells in the primary tumor.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Reduction of TGF-beta activity abrogates growth promoting tumor cell-cell interactions in vivo.

We have shown in previous studies that metastatically-competent variant subpopulations (B5, C1) derived from a non-metastatic murine mammary adenocarcinoma (SP1) have a pronounced growth advantage over their non-metastatic tumor cell counterparts in primary tumors. As a result, primary tumors can be progressively overgrown by cells having the competence to spread elsewhere in the body. This occurs despite any evidence to indicate an intrinsic in vivo growth rate advantage of the metastatic cells when grown as isolated populations. This suggested that cell-cell interactions between metastatic and non-metastatic tumor populations may be involved in the metastatic cell growth dominance process. Evidence was therefore sought for growth factors released by SP1 cells which could preferentially stimulate the B5 or C1 variants and thereby mediate this cell-cell interaction process. We found that cocultures of SP1 and C1 or B5 cells with irradiated C1, B5, or SP1 "feeder" cells showed significant stimulation of C1 and B5 by SP1 "feeder" cells. Cell growth stimulation in response to EGF, TGF-alpha, TGF-beta 1, bFGF, PDGF, NGF, IGF-1, or IGF-2 demonstrated that only TGF-beta 1 could duplicate this effect. A repeat of the coculture experiment in the presence of specific neutralizing anti-TGF-beta antibodies was therefore undertaken and this was found to markedly reduce the stimulation of C1 or B5 cells by irradiated SP1 cells. Conditioned media from the SP1 and C1 cell lines was quantitated for TGF-beta activity and contained 4.5 ng/ml and 2.0 ng/ml, respectively. However, the majority of the TGF-beta released by SP1 cells was found to be spontaneously active, whereas 70% of the TGF-beta released by C1 cells was in its latent form. Scatchard analysis revealed approximately four times the number of TGF-beta receptors, of similar type and affinity, present on C1 as compared with SP1 cells. The in vitro results support the hypothesis that active TGF-beta released by SP1 cells may stimulate the proliferation of metastatic variant cells in a paracrine like fashion. In vivo evidence for this was obtained by showing that coinjection of irradiated SP1 cells could selectively stimulate tumor growth of viable C1 cells and this effect was markedly diminished by neutralizing polyclonal anti-TGF-beta antibodies. Taken together, the results suggest a novel role for TGF-beta in clonal evolution of malignant tumor growth and as a molecular mediator of tumor cell-tumor cell interactions involved in facilitating tumor progression.     

Autophagy Inhibitor Chloroquine Enhanced the Cell Death Inducing Effect of the Flavonoid Luteolin in Metastatic Squamous Cell Carcinoma Cells

Background

Flavonoids are widely proposed as very interesting compounds with possible chemopreventive and therapeutic capacities.

Methods & Results

In this study, we showed that in vitro treatment with the flavonoid Luteolin induced caspase-dependent cell death in a model of human cutaneous squamous cell carcinoma (SCC) derived cells, representing a matched pair of primary tumor and its metastasis. Notably, no cytotoxic effects were observed in normal human keratinocytes when treated with similar doses of Luteolin. Luteolin-induced apoptosis was accompanied by inhibition of AKT signaling, and sensitivity decreased with tumor progression, as the primary MET1 SCC cells were considerably more sensitive to Luteolin than the isogenic metastatic MET4 cells. Extensive intracellular vacuolization was observed in Luteolin-treated MET4 cells, which were characterized as acidic lysosomal vacuoles, suggesting the involvement of autophagy. Transmission electron microscopy, mRFP-GFP-LC3 assay and p62 protein degradation, confirmed that Luteolin stimulated the autophagic process in the metastatic MET4 cells. Blocking autophagy using chloroquine magnified Luteolin-induced apoptosis in the metastatic SCC cells.

Conclusion

Together, these results suggest that Luteolin has the capacity to induce selectively apoptotic cell death both in primary cutaneous SCC cells and in metastatic SCC cells in combination with chloroquine, an inhibitor of autophagosomal degradation. Hence, Luteolin might be a promising agent for the treatment of cutaneous SCC.     

RAGE overexpression confers a metastatic phenotype to the WM115 human primary melanoma cell line

The formation of melanoma metastases from primary tumor cells is a complex phenomenon that involves the regulation of multiple genes. We have previously shown that the receptor for advanced glycation end products (RAGE) was up-regulated in late metastatic stages of melanoma patient samples and we hypothesized that up-regulation of RAGE in cells forming a primary melanoma tumor could contribute to the metastatic switch of these cells. To test our hypothesis, we overexpressed RAGE in the WM115 human melanoma cell line that was established from a primary melanoma tumor of a patient. We show here that overexpression of RAGE in these cells is associated with mesenchymal-like morphologies of the cells. These cells demonstrate higher migration abilities and reduced proliferation properties, suggesting that the cells have switched to a metastatic phenotype. At the molecular level, we show that RAGE overexpression is associated with the up-regulation of the RAGE ligand S100B and the down-regulation of p53, ERK1/2, cyclin E and NF-kB. Our study supports a role of RAGE in the metastatic switch of melanoma cells.     

Differential expression of metabolic genes in tumor and stromal components of primary and metastatic loci in pancreatic adenocarcinoma

Background

Pancreatic cancer is the fourth leading cause of cancer related deaths in the United States with a five-year survival rate of 6%. It is characterized by extremely aggressive tumor growth rate and high incidence of metastasis. One of the most common and profound biochemical phenotypes of animal and human cancer cells is their ability to metabolize glucose at high rates, even under aerobic conditions. However, the contribution of metabolic interrelationships between tumor cells and cells of the surrounding microenvironment to the progression of cancer is not well understood. We evaluated differential expression of metabolic genes and, hence, metabolic pathways in primary tumor and metastases of patients with pancreatic adenocarcinoma.

Methods and Findings

We analyzed the metabolic gene (those involved in glycolysis, tri-carboxylic acid pathway, pentose-phosphate pathway and fatty acid metabolism) expression profiles of primary and metastatic lesions from pancreatic cancer patients by gene expression arrays. We observed two principal results: genes that were upregulated in primary and most of the metastatic lesions; and genes that were upregulated only in specific metastatic lesions in a site-specific manner. Immunohistochemical (IHC) analyses of several metabolic gene products confirmed the gene expression patterns at the protein level. The IHC analyses also revealed differential tumor and stromal expression patterns of metabolic enzymes that were correlated with the metastasis sites.

Conclusions

Here, we present the first comprehensive studies that establish differential metabolic status of tumor and stromal components and elevation of aerobic glycolysis gene expression in pancreatic cancer.     

Exposure of human primary colon carcinoma cells to anti-Fas interactions influences the emergence of pre-existing Fas-resistant metastatic subpopulations

Fas, an important death receptor-mediated signaling pathway, has been shown to be down-regulated during human colon tumorigenesis; however, how alterations in Fas expression influence the metastatic process remains unresolved. In mouse models, loss of Fas function was found to be both necessary and sufficient for tumor progression. In this study, we investigated the link between functional Fas status and malignant phenotype using a matched pair of naturally occurring primary (Fas-sensitive) and metastatic (Fas-resistant) human colon carcinoma cell lines in both in vitro and in vivo (xenograft) settings. Metastatic sublines were produced in vitro from the primary tumor cell line by functional elimination of Fas-responsive cells. Conversely, sublines derived from the primary tumor in vivo at distal metastatic sites were Fas-resistant. In contrast, simply disrupting the Fas pathway by molecular-based strategies in the Fas-sensitive primary tumor failed to achieve the same metastatic outcome. Interestingly, both in vitro- and in vivo-produced sublines resembled the naturally occurring metastatic population, based on functional and morphologic studies and genome-scale gene expression profiling. Overall, using this human colon carcinoma model, we: 1) showed that loss of Fas function was linked to, but alone was insufficient for, acquisition of a detectable metastatic phenotype; 2) demonstrated that metastatic subpopulations pre-existed within the heterogeneous primary tumor, and that anti-Fas interactions served as a selective pressure for their outgrowth; and 3) identified a large set of differentially expressed genes distinguishing the primary from metastatic malignant phenotypes. Thus, Fas-based interactions may represent a novel mechanism for the biologic or immunologic selection of certain types of Fas-resistant neoplastic clones with enhanced metastatic ability.     

Herpesviruses in Metastatic Lucké Renal Adenocarcinoma

The etiologic agent of the renal adenocarcinoma of leopard frogs, Rana pipiens , is the Lucké tumor herpesvirus (LTHV). The virus is easily detected with thin section electron microscopy in primary tumors of frogs which have been exposed to a cold environment. Several spontaneous metastatic nodules and a large primary tumor were detected at autopsy of a frog which had been maintained at 4°C for 73 days. LTHV was found not only in the primary tumor, as previously reported, but also was present in metastatic tumor cells in the liver, fat body, and bladder. The presence of LTHV in metastatic cells demonstrates that the differentiated state of primary Lucké tumor cells is retained in its metastatic colonies even at the fine structure level revealed by electron microscopy.     

Clonal Evolution of a Case of Treatment Refractory Maxillary Sinus Carcinoma

Background

Maxillary sinus carcinoma (MSC) is a rare cancer of the head and neck region. Patients are treated with surgery, radiation therapy, and chemotherapy and the treatment regimen is based on patient’s age, general health condition, disease stage, and its extent of spread. There is very little information available on the genetics of this disease. DNA content based flow sorting of tumor cells followed by array comparative genomic hybridization allows for high definition global assessment of distinct clonal changes within tumor populations.

Methods

We applied this technique to primary and metastatic samples collected from a patient with radio- and chemotherapy refractory maxillary sinus carcinoma to gauge the progression of this disease.

Results

A clonal KIT amplicon was present in aneuploid populations sorted from the primary tumor and in divergent subclones arising in metastatic foci found in the brain, lung, and jejunum. The evolution of these subclones was associated with distinct genetic aberrations and DNA ploidies.

Conclusion

The information presented here paves the path to understanding the development and progression of this disease.     

Lack of suppressive activity of human primary melanoma cells on the activation of autologous lymphocytes

Summary Previous studies have indicated that primary but not metastatic melanomas were able to stimulate the proliferation of autologous (Auto) peripheral blood lymphocytes (PBL) in 73% of cases. On the other hand, 57% of the metastatic melanomas were shown to be suppressive when melanoma cells (Me) were admixed with Auto-PBL stimulated with allogeneic (Allo) PBL or interleukin 2 (IL-2) at the beginning of a 6-day incubation period. Here, we report that the suppressive activity of Me is a functional characteristic associated with a particular stage of the disease. In fact, we found that none of the 11 primary tumors tested were able to inhibit the proliferative response of Auto-PBL to Allo-PBL or IL-2 at all the doses of tumor cells used. The generation of lymphocytes cytotoxic against Auto-Me or K562 was also not inhibited. Of the 11 primary tumors checked for suppression, 8 were able to stimulate Auto-PBL in a primary mixed lymphocyte tumor culture. We conclude that opposite functions, stimulation and inhibition of autologous lymphocyte responses are characteristics of primary and metastatic Me, respectively.This work was supported in part by the Italian Association for Cancer Research (Milan) and by grants # 85.02162.44 and 86.00663.44 of the Finalized Project Oncology of CNR of Rome, Italy     

A Distinct Macrophage Population Mediates Metastatic Breast Cancer Cell Extravasation,Establishment and Growth

Background

The stromal microenvironment and particularly the macrophage component of primary tumors influence their malignant potential. However, at the metastatic site the role of these cells and their mechanism of actions for establishment and growth of metastases remain largely unknown.

Methodology/Principal Findings

Using animal models of breast cancer metastasis, we show that a population of host macrophages displaying a distinct phenotype is recruited to extravasating pulmonary metastatic cells regardless of species of origin. Ablation of this macrophage population through three independent means (genetic and chemical) showed that these macrophages are required for efficient metastatic seeding and growth. Importantly, even after metastatic growth is established, ablation of this macrophage population inhibited subsequent growth. Furthermore, imaging of intact lungs revealed that macrophages are required for efficient tumor cell extravasation.

Conclusion/Significance

These data indicate a direct enhancement of metastatic growth by macrophages through their effects on tumor cell extravasation, survival and subsequent growth and identifies these cells as a new therapeutic target for treatment of metastatic disease.     

Dynamic process of prostate cancer metastasis to bone

Prostate cancer metastasis to the bone occurs at high frequency in patients with advanced disease, causing significant morbidity and mortality. Over a century ago, the "seed and soil" theory was proposed to explain organ-specific patterns of metastases. Today, this theory continues to be relevant as we continue to discover factors involved in the attraction and subsequent growth of prostate cancer cells to the bone. These include the accumulation of genetic changes within cancer cells, the preferential binding of cancer cells to bone marrow endothelial cells, and the release of cancer cell chemoattractants from bone elements. A key mediator throughout this metastatic process is the integrin family of proteins. Alterations in integrin expression and function promote dissociation of cancer cells from the primary tumor mass and migration into the blood stream. Once in circulation, integrins facilitate cancer cell survival through interactions between other cancer cells, platelets, and endothelial cells of the target bone. Furthermore, dynamic changes in integrins and in integrin-associated signal transduction aid in the extravasation of cancer cells into the bone and in expansion to a clinically relevant metastasis. Thus, we will review the critical roles of integrins in the process of prostate cancer bone metastasis, from the escape of cancer cells from the primary tumor, to their survival in the harsh "third microenvironment" of the circulation, and ultimately to their attachment and growth at distant bone sites.     

Herpesviruses in metastatic Lucké renal adenocarcinoma

The etiologic agent of the renal adenocarcinoma of leopard frogs, Rana pipiens, is the Lucké tumor herpesvirus (LTHV). The virus is easily detected with thin section electron microscopy in primary tumors of frogs which have been exposed to a cold environment. Several spontaneous metastatic nodules and a large primary tumor were detected at autopsy of a frog which had been maintained at 4 degrees C for 73 days. LTHV was found not only in the primary tumor, as previously reported, but also was present in metastatic tumor cells in the liver, fat body, and bladder. The presence of LTHV in metastatic cells demonstrates that the differentiated state of primary Lucké tumor cells is retained in its metastatic colonies even at the fine structure level revealed by electron microscopy.     

The role of apoptosis in tumor progression and metastasis

Metastasis, the process by which cancer spreads from a primary to a secondary site, is responsible for the majority of cancer related deaths. Yet despite the detrimental effects of metastasis, it is an extremely inefficient process by which very few of the cells that leave the primary tumor give rise to secondary tumors. Metastasis can be considered as a series of sequential steps that begins with a cell leaving a primary tumor, and concludes with the formation of a metastatic tumor in a distant site. During the process of metastasis cells are subjected to various apoptotic stimuli. Thus, in addition to genetic changes that promote unregulated proliferation, successful metastatic cells must have a decreased sensitivity to apoptotic stimuli. As many cancer cells exhibit aberrations in the level and function of key apoptotic regulators, exploiting these alterations to induce tumor cell apoptosis offers a promising therapeutic target. This review will examine the apoptotic regulators that are often aberrantly expressed in metastatic cells; the role that these regulators may play in metastasis; the steps of metastasis and their susceptibility to apoptosis; and finally, current and future cancer prognostics and treatment targets based on apoptotic regulators.     

Pathophysiological implications of stroma pattern formation in uveal melanoma

Clinical outcome of cancer patients is mainly determined by the rate of metastasis and, also by primary tumor growth. Formation of extracellular matrix and interactions of neoplastic and non-neoplastic (host) cells in solid tumors have been shown to be essential for these processes. One result of such interactions is the outgrowth of new blood vessels from existing ones, angiogenesis, to provide the tumor tissue with oxygen and nutrients. It is assumed that the neovascular bed also facilitates the escape of metastatic cells from the primary lesions. In addition, recent reports suggested the existence of blood-conducting channels lined by melanoma cells (so-called "vascular channels") accompanied by depositions of extracellular matrix patterns in cutaneous and uveal melanoma. Since the presence of these matrix structures has been negatively associated with prognosis, we hypothesize that they play a role in melanoma outgrowth or metastasis. In this review, we will discuss the morphological and functional properties of the extracellular matrix patterns in that may underlie these clinical phenomena.     

Bcl-2-mediated cell survival promotes metastasis of EpH4 betaMEKDD mammary epithelial cells

The majority of patients who succumb to cancer die from metastatic disease progression rather than from the primary tumor. Elucidation of the mechanisms underlying tissue-specific metastasis is essential to the development of effective therapies. The mitogen-activated protein kinase kinase (MEK) pathway is frequently activated in human tumors and has been shown to regulate genes involved in proliferation, migration, and invasion. Studies with MEK-transformed EpH4 mouse mammary epithelial cells showed that these cells are highly tumorigenic but have a limited metastatic ability. Detachment of epithelial cells from the extracellular matrix causes disruption of the actin cytoskeleton and induces apoptosis. Several metastatic breast carcinoma cell lines have been shown to be resistant to cell death following actin disruption. This death-resistant phenotype can be modeled by overexpressing the antiapoptotic Bcl-2 protein in cells. This suggests that mechanisms that regulate survival of extravasated tumor cells may enhance metastatic efficiency. Therefore, we examined whether expression of Bcl-2 in MEK-transformed EpH4 mammary epithelial cells could provide a survival advantage and promote metastasis. Expression of Bcl-2 in parental EpH4 mammary epithelial cells or MEK-transformed cells was insufficient to induce increased migration, invasion, or tumor development. However, Bcl-2 expression markedly enhanced spontaneous lung metastasis from orthotopically implanted primary tumors. These results clearly show that mechanisms that regulate primary tumor development are distinct from those that promote metastasis and that assays designed to isolate genes involved in transformation may fail to identify genes that are critical regulators of metastasis.