Data from Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53

Abstract

<div>Abstract<p>The ability to observe changes in molecular behavior during cancer cell invasion <i>in vivo</i> remains a major challenge to our understanding of the metastatic process. Here, we demonstrate for the first time, an analysis of RhoA activity at a subcellular level using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer) imaging in a live animal model of pancreatic cancer. In invasive mouse pancreatic ductal adenocarcinoma (PDAC) cells driven by mutant <i>p53</i> (<i>p53<sup>R172H</sup></i>), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells <i>in vivo</i> which was absent in two-dimensional <i>in vitro</i> cultures. Notably, this pool of active RhoA was absent in noninvasive <i>p53<sup>fl</sup></i> knockout PDAC cells, correlating with their poor invasive potential <i>in vivo</i>. We used dasatanib, a clinically approved anti-invasive agent that is active in this model, to illustrate the functional importance of spatially regulated RhoA. Dasatanib inhibited the activity of RhoA at the poles of <i>p53<sup>R172H</sup></i> cells <i>in vivo</i> and this effect was independent of basal RhoA activity within the cell body. Taken together, quantitative <i>in vivo</i> fluorescence lifetime imaging illustrated that RhoA is not only necessary for invasion, but also that subcellular spatial regulation of RhoA activity, as opposed to its global activity, is likely to govern invasion efficiency <i>in vivo</i>. Our findings reveal the utility of FLIM-FRET in analyzing dynamic biomarkers during drug treatment in living animals, and they also show how discrete intracellular molecular pools might be differentially manipulated by future anti-invasive therapies. <i>Cancer Res; 71(3); 747–57. ©2011 AACR</i>.</p></div>