title: 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 three-dimensional 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 beta1, 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 beta1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin beta1 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.
authors.affiliation: Laboratory of Cell Biology and Genetics, National Cancer Institute, NIH, Bethesda, Maryland 20892, USA.
authors.forename: Dalit
authors.initials: D
authors.lastname: Barkan
authors.forename: Hynda
authors.initials: H
authors.lastname: Kleinman
authors.forename: Justin L
authors.initials: JL
authors.lastname: Simmons
authors.forename: Holly
authors.initials: H
authors.lastname: Asmussen
authors.forename: Anil K
authors.initials: AK
authors.lastname: Kamaraju
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Hoenorhoff
authors.forename: Zi-yao
authors.initials: ZY
authors.lastname: Liu
authors.forename: Sylvain V
authors.initials: SV
authors.lastname: Costes
authors.forename: Edward H
authors.initials: EH
authors.lastname: Cho
authors.forename: Stephen
authors.initials: S
authors.lastname: Lockett
authors.forename: Chand
authors.initials: C
authors.lastname: Khanna
authors.forename: Ann F
authors.initials: AF
authors.lastname: Chambers
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Green
mesh_terms: D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002455:Cell Division; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D003599:Cytoskeleton / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D051379:Mice; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D009362:Neoplasm Metastasis; D010766:Phosphorylation
chemical_list: D017931:DNA Primers; D005353:Fibronectins; D018994:Myosin Light Chains; D009219:Myosin-Light-Chain Kinase