Metastases, rather than primary tumours, are responsible for most cancer deaths. To prevent these deaths, improved ways to treat metastatic disease are needed. Blood flow and other mechanical factors influence the delivery of cancer cells to specific organs, whereas molecular interactions between the cancer cells and the new organ influence the probability that the cells will grow there. Inhibition of the growth of metastases in secondary sites offers a promising approach for cancer therapy.

Metastatic spread of cancer continues to be the greatest barrier to cancer cure. Understanding the molecular mechanisms of metastasis is crucial for the design and effective use of novel therapeutic strategies to combat metastases. One class of molecules that has been repeatedly implicated in metastasis is the matrix metalloproteinases (MMPs). In this review, we re-examine the evidence that MMPs are associated with metastasis and that they make a functional contribution to the process. Initially, it was believed that the major role of MMPs in metastasis was to facilitate the breakdown of physical barriers to metastasis, thus promoting invasion and entry into and out of blood or lymphatic vessels (intravasation, extravasation). However, recent evidence suggests that MMPs may have a more complex role in metastasis and that they may make important contributions at other steps in the metastatic process. Studies using intravital videomicroscopy, as well as experiments in which levels of MMPs or their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]) are manipulated genetically or pharmacologically, suggest that MMPs are key regulators of growth of tumors, at both primary and metastatic sites. On the basis of this evidence, a new view of the functional role of MMPs in metastasis is presented, which suggests that MMPs are important in creating and maintaining an environment that supports the initiation and maintenance of growth of primary and metastatic tumors. Further clarification of the mechanisms by which MMPs regulate growth of primary and metastatic tumors will be important in the development of novel therapeutic strategies against metastases.

Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in threedimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin B1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin B1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin B1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch. [Cancer Res 2008;68(15):6241-50]

In cancer metastasis, only a small percentage of cells released from a primary tumor successfully form distant lesions, but it is uncertain at which steps in the process cells are lost. Our goal was to determine what proportions of B16F1 melanoma cells injected intraportally to target mouse liver 1) survive and extravasate, 2) form micrometastases (4 to 16 cells) by day 3, 3) develop into macroscopic tumors by day 13, and 4) remain as solitary dormant cells. Using in vivo videomicroscopy, a novel cell accounting assay, and immunohistochemical markers for proliferation (Ki-67) and apoptosis (TUNEL), we found that 1) 80% of injected cells survived in the liver microcirculation and extravasated by day 3, 2) only a small subset of extravasated cells began to grow, with 1 in 40 forming micrometastases by day 3, 3) only a small subset of micrometastases continued to grow, with 1 in 100 progressing to form macroscopic tumors by day 13 (in fact, most micrometastases disappeared), and 4) 36% of injected cells remained by day 13 as solitary cancer cells, most of which were dormant (proliferation, 2%; apoptosis, 3%; in contrast to cells within macroscopic tumors: proliferation, 91%; apoptosis/necrosis, 6%). Thus, in this model, metastatic inefficiency is principally determined by two distinct aspects of cell growth after extravasation: failure of solitary cells to initiate growth and failure of early micrometastases to continue growth into macroscopic tumors.