To identify pathways controlling prostate cancer metastasis we performed
differential display analysis of the human prostate carcinoma cell line PC-3
and its highly metastatic derivative PC-3M. This revealed that a 78-kDa
interferon-inducible GTPase, MxA, was expressed in PC-3 but not in PC-3M
cells. The gene encoding MxA, MX1, is located in the region of
chromosome 21 deleted as a consequence of fusion of TMPRSS2 and
ERG, which has been associated with aggressive, invasive prostate
cancer. Stable exogenous MxA expression inhibited in vitro motility
and invasiveness of PC-3M cells. In vivo exogenous MxA expression
decreased the number of hepatic metastases following intrasplenic injection.
Exogenous MxA also reduced motility and invasiveness of highly metastatic LOX
melanoma cells. A mutation in MxA that inactivated its GTPase reversed
inhibition of motility and invasion in both tumor cell lines.
Co-immunoprecipitation studies demonstrated that MxA associated with tubulin,
but the GTPase-inactivating mutation blocked this association. Because MxA is
a highly inducible gene, an MxA-targeted drug discovery screen was initiated
by placing the MxA promoter upstream of a luciferase reporter. Examination of
the NCI diversity set of small molecules revealed three hits that activated
the promoter. In PC-3M cells...
The human MxA protein is an interferon-induced large GTPase with antiviral activity against a wide range of viruses, including influenza viruses. Recent structural data demonstrated that MxA oligomerizes into multimeric filamentous or ring-like structures by virtue of its stalk domain. Here, we show that negatively charged lipid membranes support MxA self-assembly. Like dynamin, MxA assembled around spherical liposomes inducing liposome tubulation. Cryo-transmission electron microscopy revealed that MxA oligomers around liposomes have a “T-bar” shape similar to dynamin. Moreover, biochemical assays indicated that the unstructured L4 loop of the MxA stalk serves as the lipid-binding moiety, and mutational analysis of L4 revealed that a stretch of four lysine residues is critical for binding. The orientation of the MxA molecule within the membrane-associated oligomer is in agreement with the proposed topology of MxA oligomers based on crystallographic data. Although oligomerization of wild-type MxA around liposomes led to the creation of helically decorated tubes similar to those formed by dynamin, this lipid interaction did not stimulate GTPase activity, in sharp contrast to the assembly-stimulated nucleotide hydrolysis observed with dynamin. Moreover...