The role of LIMK1 subcellular compartmentalization in human breast cancer progression

LIM kinase 1 (LIMK1) is expressed in both cytoplasmic and nuclear compartments, and is a key regulator of cytoskeletal organization in cell migration and proliferation. LIMK1 levels are increased in several human epithelial cancers, with over-expression studies in prostate and breast cancer cell lines revealing that LIMK1 promotes in vivo tumor growth and metastasis. While it has been presumed that the key mechanism by which LIMK1 promotes cancer progression is via cofilin phosphorylation in the cytoplasm, the precise roles of nuclear vs cytoplasmic LIMK1 in human cancer has not been examined. Here, I tested the hypothesis that LIMK1 targeted to the cytoplasmic and/or nuclear compartments results in differential cancer-promoting effects. In an initial IHC study, I found that breast cancer tissues express higher cytoplasmic levels of LIMK1, with greater frequency of concomitant nuclear LIMK1 localization, compared to normal breast tissues. In order to study the specific contributions of cytoplasmic- and nuclear-localized LIMK1 to breast cancer progression, I established a model to segregate GFP-tagged LIMK1 to the cytoplasm or the nucleus of human breast-derived tissue culture cells. Utilizing this model of LIMK1 subcellular compartmentalization, I found that GFP-tagged LIMK1 proteins, both cytoplasmic and nuclear, increased anchorage-independent growth, independent of kinase-activity. In an in vitro model of invasion, I found that GFP-LIMK1 enhancement of invasion is kinase-dependent in the cytoplasm, but enhances invasion in the nucleus with or without kinase activity. I next tested the ability of cytoplasmic- or nuclear-targeted GFP-LIMK1 to form tumors in in vivo mouse xenograft tumor formation assays, which revealed that nuclear and cytoplasmic GFP-LIMK1 enhanced tumor growth. Finally, I was able to link the expression of both nuclear and cytoplasmic LIMK1 to the activation of FAK/paxillin/Erk signaling. This pathway appears to be activated with expression of kinase-active GFP-LIMK1, but the kinase-dead GFP-LIMK1 proteins may also activate this pathway. These data imply kinase and scaffold roles for LIMK1. Taken together, my findings demonstrate that both nuclear and cytoplasmic LIMK1 activity promotes breast cancer progression via distinct mechanisms that are both kinase-dependent and kinase-independent.