Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Abstract

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]

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal–epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

“…Phalloidin staining further verified a significant difference in the cell shape and distribution of F-actin, implying that ERp29-transfected MDA-MB-231 cells underwent cytoskeletal reorganization with loss of filamentous stress fibers and cortical actin formation (Figure 3b). Concomitantly, the extracellular matrix (ECM) component FN, which is involved in cell transition from quiescence to proliferation, 29 was decreased in ERp29-transfected MDA-MB-231 cells, as shown by immunofluorescent staining (Figure 3c). Importantly, we observed that epithelial cell marker E-cadherin was highly expressed in both clone B and E cells and its membranous distribution is identical to that observed in MCF-7 cells (Figure 3d).…”

“…As the cell shape and cytoskeletal configuration have a critical role in regulating the transition of cells from a nonproliferative to a proliferative state, 4,29,38 the ERp29-mediated cytoskeletal reorganization in proliferative MDA-MB-231 cells indicated ERp29 as a novel regulator in inducing cell dormancy. This is further supported by the identical phenotype between ERp29-transfected MDA-MB-231 cells and dormant MCF-7 cells.…”

Multiple uses of basement membrane‐like matrix (BME/Matrigel) in vitro and in vivo with cancer cells

“…When placed on BME, the dormant cell lines remain as single cells and do not proliferate, suggesting that the extracellular matrix plays a role in preserving the dormant state. 16 This assay has been useful for identifying mechanisms and pathways that regulate dormancy, and it provides a useful tool for screening and evaluating therapeutics in vitro. 50 This assay can also be used with heterogeneous cancer cell lines in limiting dilution cultures on BME to identify and isolate subpopulations of nondividing dormant cells as mentioned above.…”

“…14 Other assays based on cancer cell morphology, clonogenicity, growth, and dormancy on the matrix have also been developed more recently. 15,16 Coinjection of the basement membrane-like matrix with cancer cells either subcutaneously or in orthotopic sites was found to increase both the 'take' and growth of tumors in mice. [17][18][19] Thus, cancer cell interactions with the basement membrane have multiple uses both in vitro and in vivo (Table 2).…”

ERp29 induces breast cancer cell growth arrest and survival through modulation of activation of p38 and upregulation of ER stress protein p58IPK

“…The role of ERp29 in regulating p38 expression and phosphorylation was further verified in MCF-7 cells, which show a dormant-like state in three-dimensional cell cultures. 31 As shown in Figure 2c, knockdown of ERp29 resulted in a significant reduction of p-p38 (3-fold, Po0.01) and upregulation (1.8-fold, Po0.05) of basal p38a. Furthermore, ERp29 knockdown in MCF-7 cells significantly increased FAK expression (B2.3-fold, Po0.01) and phosphorylation (B2.9-fold, Po0.01) relative to these cells treated with scrambled control shRNA (shCtrl; Figure 2c).…”