Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53.
The ability to observe changes in molecular behavior during cancer cell invasion in vivo 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 p53 (p53(R172H)), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells in vivo which was absent in two-dimensional in vitro cultures. Notably, this pool of active RhoA was absent in noninvasive p53(fl) knockout PDAC cells, correlating with their poor invasive potential in vivo. 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 p53(R172H) cells in vivo and this effect was independent of basal RhoA activity within the cell body. Taken together, quantitative in vivo 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 in vivo. 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.
The Beatson Institute for Cancer Research, Garscube Estate, Glasgow, United Kingdom. p.timpson@beatson.gla.ac.uk
Paul
P
Timpson
Ewan J
EJ
McGhee
Jennifer P
JP
Morton
Alex
A
von Kriegsheim
Juliane P
JP
Schwarz
Saadia A
SA
Karim
Brendan
B
Doyle
Jean A
JA
Quinn
Neil O
NO
Carragher
Mike
M
Edward
Michael F
MF
Olson
Margaret C
MC
Frame
Valerie G
VG
Brunton
Owen J
OJ
Sansom
Kurt I
KI
Anderson
D000818:Animals; D021441:Carcinoma, Pancreatic Ductal / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D000069439:Dasatinib; D031541:Fluorescence Resonance Energy Transfer; D016158:Genes, p53; D051379:Mice; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D009154:Mutation; D009361:Neoplasm Invasiveness; D010190:Pancreatic Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein
D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D013844:Thiazoles; C526380:RhoA protein, mouse; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000069439:Dasatinib
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