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. 2013 Jan 24;32(4):462-70.
doi: 10.1038/onc.2012.69. Epub 2012 Feb 27.

MTBP suppresses cell migration and filopodia formation by inhibiting ACTN4

N Agarwal  1 , A S Adhikari, S V Iyer, K Hekmatdoost, D R Welch, T Iwakuma
Affiliations

MTBP suppresses cell migration and filopodia formation by inhibiting ACTN4

N Agarwal et al. Oncogene. .

Abstract

Murine double minute (MDM2) binding protein (MTBP) has been implicated in cancer progression. Here, we demonstrate one mechanism by which MTBP inhibits cancer metastasis. Overexpression of MTBP in human osteosarcoma cell lines lacking wild-type p53 did not alter primary tumor growth in mice, but significantly inhibited metastases. MTBP downregulation increased the migratory potential of MDM2(-/-)p53(-/-) mouse embryonic fibroblasts, suggesting that MTBP inhibited cell migration independently of the Mdm2-p53 pathway. Co-immunoprecipitation and mass spectrometric analysis identified alpha-actinin-4 (ACTN4) as an MTBP-interacting protein. Endogenous MTBP interacted with and partially colocalized with ACTN4. MTBP overexpression inhibited cell migration and filopodia formation mediated by ACTN4. Increased cell migration by MTBP downregulation was inhibited by concomitant downregulation of ACTN4. MTBP also inhibited ACTN4-mediated F-actin bundling. We furthermore demonstrated that nuclear localization of MTBP was dispensable for inhibiting ACTN4-mediated cell migration and filopodia formation. Thus, MTBP suppresses cell migration, at least partially, by inhibiting ACTN4 function. Our study not only provides a mechanism of metastasis suppression by MTBP, but also suggests MTBP as a potential biomarker for cancer progression.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
MTBP suppresses osteosarcoma metastasis independently of p53. NOD-scid IL2Rγ-null mice were intrafemorally injected with SaOs2-LM7 and KHOS cells that were stably infected with either empty (grey, cont) or MTBP-encoding (black, MTBP) lentiviral vectors. Mice were monitored for the development of primary tumors and lung metastases. Approximately 4 and 6 weeks after injections of KHOS and SaOs2-LM7 cells, respectively, mice were sacrificed and examined for the weight of primary tumors at injected sites (a) and the number of lung metastatic nodules (b). Representative pictures of primary tumors and pulmonary metastases from SaOs2-LM7 and KHOS injected mice are shown below graphs. Error bars are means ± S.D. from four independent mice. **, P < 0.01; t test. N.S., not significant. Scale bar: 1 cm.
Figure 2
Figure 2
MTBP inhibits cell migratory potential independently of MDM2 and p53. (a) MDM2−/−p53−/− MEFs were transfected with non-target (grey, cont) or MTBP-specific (black, MTBP) siRNAs. (b) SaOs2-LM7 and U2OS cells were infected with empty (grey, cont) or MTBP-encoding (black, MTBP) adenoviruses (Adeno). (c) SaOs2-LM7 and U2OS cells were transfected with non-target (grey, cont) or MTBP-specific (black, MTBP) siRNAs. Thirty six (36) hours after manipulation of MTBP expression, cells were examined for migratory potential. Migrating cells were stained and entire fields were counted. Representative images are shown below the graphs. Graphs showing relative cell migration (%) compared to the number of migrating cells in control. Western blotting results below the graphs showing successful manipulation of MTBP expression. Error bars are means ± S.D. from three independent experiments. *, P < 0.05 and **, P < 0.01; t test.
Figure 3
Figure 3
MTBP interacts with ACTN4 and inhibits ACTN4-mediated cell migration. (a) Whole cell extracts from SaOs2-LM7 cells were immunoprecipitated (IP) with MTBP (BP) or ACTN4 (A4) antibodies (endogenous). Immunoprecipitants were subjected to western blotting for MTBP or ACTN4. Matched isotype antibodies (Iso) were used as negative controls. In vitro translated ACTN4 and FLAG-tagged MTBP were incubated in PBS and immunoprecipitated with ACTN4 (A4, left) or FLAG antibodies (FL, right), followed by immunoblotting for MTBP or ACTN4, respectively (in vitro). (b) Immunofluorescence was performed to examine endogenous colocalization of MTBP and ACTN4 in SaOs2-LM7 cells using indicated antibodies. DNA was stained using DAPI. Results were analyzed on a Leica epifluorescence microscope. A merge picture indicates that MTBP and ACTN4 partially colocalize mainly in the cytoplasm (yellow). (c, d) SaOs2-LM7 and U2OS cells stably infected with either empty (ACTN4−) or ACTN4-encoding (ACTN4+) lentiviral vectors were infected with empty (MTBP−) or MTBP-encoding (MTBP+) adenoviruses (c). Cells stably infected with empty (ACTN4−) or ACTN4 shRNA-encoding (ACTN4+) lentiviral vectors were transiently transfected with non-targeted (MTBP−) or MTBP-specific (MTBP+) siRNAs (d). Thirty six (36) hours after manipulation of MTBP expression, migration assays were performed. Migrating cells were stained and entire fields were counted. Graphs showing relative cell migration (%) compared to the number of migrating cells in control. Western blotting results below the graphs demonstrating successful manipulation of MTBP and ACTN4 expression. Error bars are means ± S.D. from three independent experiments. *, P < 0.05 and **, P < 0.01; t test. N.S., not significant. Control: white, MTBP alone manipulated: grey, ACTN4 alone manipulated: oblique, MTBP and ACTN4 manipulated: black.
Figure 4
Figure 4
MTBP inhibits ACTN4-mediated filopodia formation and cross-linking of F-actin. (a) SaOs2-LM7 (left) and U2OS (right) cells with or without ACTN4 overexpression were infected with empty (control) or MTBP-encoding (MTBP) adenoviruses. Forty eight (48) hours later, phalloidin staining was performed. Cells (n=100) were examined for filopodia formation. The percentages of cells positive for filopodia formation (top) and representative phalloidin staining pictures (bottom). (b) Actin bundling assay. In vitro translated MTBP and/or ACTN4 proteins were incubated with purified F-actin in the assay reaction, followed by protein sedimentation. Luciferase (Luc) was used as a negative control. Supernatant (S) and pellet (P) fractions were subjected to western blotting for actin, ACTN4, and MTBP (left). Graph showing the percentage of sedimented bundled F-actin (pellet) as analyzed by densitometry (right). Error bars are means ± S.D. from three independent experiments. **, P < 0.01; t test. N.S., not significant.
Figure 5
Figure 5
Nuclear localization of MTBP is not required for inhibiting ACTN4 function. (a) Nuclear localization signal (NLS) domain in MTBP with mutated amino acids (underlined, left). Immunofluorescence images demonstrating the localization of FLAG-tagged full-length MTBP (MTBP) and mutant MTBP (MTBPnls, right). (b) SaOs2-LM7 cells with or without ACTN4 overexpression were infected with lentiviral vectors encoding empty (control) or MTBPnls mutant (MTBPnls), followed by cell migration assays. Migrating cells were stained, and entire fields were counted. Relative cell migration (%) compared to the number of migrating cells in control (top) and representative staining pictures (bottom). (c) Phalloidin staining was performed using the same cells as in Figure 5b. Cells (n= 100) were examined for filopodia formation. The percentages of cells positive for filopodia formation (top) and representative phalloidin staining pictures (bottom). Error bars are means ± S.D. from three independent experiments. *, P < 0.05 and **, P < 0.01; t test. (d) 293T cells which had undetectable levels of endogenous MTBP were transfected with Flag-tagged full-length MTBP (MTBP), NLS mutant (MTBPnls) and deletion mutants (F4 and ΔC). A diagram showing regions deleted for MTBP mutants. Forty-eight hours later, co-immunoprecipitation (IP) studies were performed using ACTN4 antibody (A4), followed by western blotting for MTBP. Matched isotype antibodies (Iso) were used as negative controls. In vitro synthesized ACTN4 and mutant MTBP (MTBPnls, F4, and ΔC) were co-incubated for co-IP studies using ACTN4 or FLAG (FL) antibodies, followed by immunoblotting for MTBP or ACTN4.
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