A search for general regulators of cancer metastasis has yielded a set of microRNAs for which expression is specifically lost as human breast cancer cells develop metastatic potential. Here we show that restoring the expression of these microRNAs in malignant cells suppresses lung and bone metastasis by human cancer cells in vivo. Of these microRNAs, miR-126 restoration reduces overall tumour growth and proliferation, whereas miR-335 inhibits metastatic cell invasion. miR-335 regulates a set of genes whose collective expression in a large cohort of human tumours is associated with risk of distal metastasis. miR-335 suppresses metastasis and migration through targeting of the progenitor cell transcription factor SOX4 and extracellular matrix component tenascin C. Expression of miR-126 and miR-335 is lost in the majority of primary breast tumours from patients who relapse, and the loss of expression of either microRNA is associated with poor distal metastasis-free survival. miR-335 and miR-126 are thus identified as metastasis suppressor microRNAs in human breast cancer.Although metastasis is the overwhelming cause of mortality in patients with solid tumours, our understanding of its molecular and cellular determinants is limited 1-3 . Transcriptional profiling has revealed sets of genes, or `signatures', for which expression in primary tumours correlates with metastatic relapse or poor survival 4 . Some of these genes endow cancer cells with a more invasive phenotype, enhanced angiogenic and intravasation activity, the ability to exit from the circulation, or an ability to modify the metastasis microenvironment 5,6 . Such gene sets are thus providing numerous candidate mediators of metastasis to be validated through functional and clinical studies. Much less insight, however, has been gained into the

Cells released from primary tumors seed metastases to specific organs by a nonrandom process, implying the involvement of biologically selective mechanisms. Based on clinical, functional, and molecular evidence, we show that the cytokine TGFbeta in the breast tumor microenvironment primes cancer cells for metastasis to the lungs. Central to this process is the induction of angiopoietin-like 4 (ANGPTL4) by TGFbeta via the Smad signaling pathway. TGFbeta induction of Angptl4 in cancer cells that are about to enter the circulation enhances their subsequent retention in the lungs, but not in the bone. Tumor cell-derived Angptl4 disrupts vascular endothelial cell-cell junctions, increases the permeability of lung capillaries, and facilitates the trans-endothelial passage of tumor cells. These results suggest a mechanism for metastasis whereby a cytokine in the primary tumor microenvironment induces the expression of another cytokine in departing tumor cells, empowering these cells to disrupt lung capillary walls and seed pulmonary metastases.

The TGFβ signaling pathway is conserved from flies to humans and has been shown to regulate such diverse processes as cell proliferation, differentiation, motility, adhesion, organization, and programmed cell death. Both in vitro and in vivo experiments suggest that TGFβ can utilize these varied programs to promote cancer metastasis through its effects on the tumor microenvironment, enhanced invasive properties, and inhibition of immune cell function. Recent clinical evidence demonstrating a link between TGFβ signaling and cancer progression is fostering interest in this signaling pathway as a therapeutic target. Anti-TGFβ therapies are currently being developed and tested in preclinical studies. However, targeting TGFβ carries a substantial risk as this pathway is implicated in multiple homeostatic processes and is also known to have tumor-suppressor functions. Additionally, clinical and experimental results show that TGFβ has diverse and often conflicting roles in tumor progression even within the same tumor types. The development of TGFβ inhibitors for clinical use will require a deeper understanding of TGFβ signaling, its consequences, and the contexts in which it acts.

The association between large tumor size and metastatic risk in a majority of clinical cancers has led to questions as to whether these observations are causally related or whether one is simply a marker for the other. This is partly due to an uncertainty about how metastasis-promoting gene expression changes can arise in primary tumors. We investigated this question through the analysis of a previously defined ''lung metastasis gene-expression signature'' (LMS) that mediates experimental breast cancer metastasis selectively to the lung and is expressed by primary human breast cancer with a high risk for developing lung metastasis. Experimentally, we demonstrate that the LMS promotes primary tumor growth that enriches for LMS ؉ cells, and it allows for intravasation after reaching a critical tumor size. Clinically, this corresponds to LMS ؉ tumors being larger at diagnosis compared with LMS ؊ tumors and to a marked rise in the incidence of metastasis after LMS ؉ tumors reach 2 cm. Patients with LMS-expressing primary tumors selectively fail in the lung compared with the bone or other visceral sites and have a worse overall survival. The mechanistic linkage between metastasis gene expression, accelerated tumor growth, and likelihood of metastatic recurrence provided by the LMS may help to explain observations of prognostic gene signatures in primary cancer and how tumor growth can both lead to metastasis and be a marker for cells destined to metastasize.cancer ͉ genomics ͉ oncogenesis T he consistent association of large tumor size, rapid growth rate, and metastatic risk in a majority of cases of clinical cancer suggests that the molecular bases of these phenomena may be linked (1-3). However, the nature of this link remains unresolved. Conventional models of metastasis envision rare metastatically competent variants emerging by chance as primary tumors grow, causally linking growth with likelihood of metastatic relapse (4, 5). In this view, genes that control primary tumor growth operate independently of stochastically acquired metastasis genes. Alternative models posit that prometastatic gene expression events are acquired early during tumorigenesis and may overlap with the genes that promote primary tumor growth, making tumor size a marker for metastatic risk (6). These alternative models form a teleological basis for using gene expression signatures from primary tumors to forecast whether patients are at high risk for micrometastatic disease. However, despite several reports on the success of gene signatures from primary tumors to predict development of distant spread (7-12), tumor size remains an independent prognostic factor on multivariate analysis (9). Thus, to what degree conventional versus alternative models can explain the acquisition of a metastatic phenotype remains unclear.One of the difficulties in addressing the fundamental question on how metastasis gene expression events are acquired relates to the genetically complex nature of the phenotype itself. It has long been believed that there are nume...