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. 2019 Mar 1;294(9):2988-2996.
doi: 10.1074/jbc.RA118.004741. Epub 2019 Jan 4.

A Small-Molecule Ligand of Valosin-Containing protein/p97 Inhibits Cancer Cell-Accelerated Fibroblast Migration

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Free PMC article

A Small-Molecule Ligand of Valosin-Containing protein/p97 Inhibits Cancer Cell-Accelerated Fibroblast Migration

Kruthi Suvarna et al. J Biol Chem. .
Free PMC article

Abstract

Carcinoma-associated fibroblasts are fibroblasts activated by surrounding cancer cells. Carcinoma-associated fibroblasts exhibit enhanced cell migration, which plays an important role in cancer metastasis. Previously, we demonstrated enhanced migration of NIH3T3 fibroblasts when they were cultured in the presence of MCF7 breast cancer cells. Human fibroblasts displayed a similar phenomenon even when they were co-cultured with cancer cells other than MCF7 cells. In this study, we screened ∼16,000 compounds from the RIKEN Natural Products Depository chemical library for inhibitors of enhanced NIH3T3 cell migration in the presence of MCF7. We identified NPD8733 as an inhibitor of cancer cell-enhanced fibroblast migration. This inhibition was observed not only in a wound-healing co-culture assay but also in a Transwell migration assay. Using NPD8733 and a structurally similar but inactive derivative, NPD8126, on immobilized beads, we found that NPD8733, but not NPD8126, specifically binds to valosin-containing protein (VCP)/p97, a member of the ATPase-associated with diverse cellular activities (AAA+) protein family. Using VCP truncation variants, we found that NPD8733 binds to the D1 domain of VCP. Because VCP's D1 domain is important for its function, we concluded that NPD8733 may act on VCP by binding to this domain. siRNA-mediated silencing of VCP in NIH3T3 fibroblasts, but not in MCF7 cells, reduced the migration of the co-cultured NIH3T3 fibroblasts. These results indicate that MCF7 activates the migration of NIH3T3 cells through VCP and that NPD8733 binds VCP and thereby inhibits its activity.

Keywords: cancer; cancer-associated fibroblast (CAF); cell migration; chemical biology; extracellular matrix; p97; small molecule; tumor metastasis; tumor microenvironment; valosin-containing protein (VCP).

Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
NPD8733 suppresses the enhanced migration of NIH3T3 cells co-cultured with MCF7 cells using a wound healing co-culture assay. A, chemical structure of NPD8733. B, chemical structure of NPD8732. C, NIH3T3 cells alone or NIH3T3 cells co-cultured with MCF7 cells were seeded overnight in 6-well plates in 10% FCS. The next day, the cells were scratched, treated with different concentrations of NPD8733, and cultured in medium containing 1% FCS. The edge of the wound at the 0 time point is marked with a red outline and was overlaid with other images taken at 24 h. D, the cell migration percentages for fibroblasts and co-cultured cells were calculated using ImageJ. The graph shows the percentage of fibroblast migration in co-cultures relative to the control (fibroblasts alone) at 24 h and was plotted against the NPD8733 concentration (n = 3; ***, p < 0.001). The red dots indicate the individual measurements. E, NIH3T3 cells were seeded overnight in 6-well plates in 10% FCS. The next day, the cells were scratched, treated with different concentrations of NPD8733, and cultured in medium containing 1% FCS. The fibroblast migration percentages were calculated using ImageJ software. The graph indicates the fibroblast migration percentages relative to the control (without compound) at 24 and 48 h. The red dots indicate the individual measurements.
Figure 2.
Figure 2.
NPD8733 inhibits enhanced migration of NIH3T3 cells co-cultured with MCF7 cells using a Transwell migration assay. A, NIH3T3 cells and co-cultured NIH3T3 cells were grown without FCS in the upper chamber and were allowed to migrate toward the bottom chamber containing 10% FCS. Different concentrations of NPD8733 were added to both the upper and lower chambers. The membrane was stained with crystal violet after 24 h of migration, and images of the three different fields were captured with an inverted microscope. B, the percentages of fibroblasts that migrated while co-cultured or when cultured alone were quantified using ImageJ. The graph shows the percentage of fibroblasts migrating in co-culture compared with fibroblast cultured alone and was plotted against different doses of NPD8733. In the control, without compound, there was a significant difference in the migration of co-cultured fibroblasts compared with that of fibroblasts alone at 24 h (n = 3; ***, p < 0.001). Treatment of NPD8733 at 1 μm and higher showed a significant decrease in the migration of NIH3T3 cells co-cultured in the presence of MCF7 cells (n = 3; ***, p < 0.001). The red dots indicate the individual measurements. C, cell proliferation using WST-8 reagent was carried out in NIH3T3 and MCF7 cells cultured in 1% FCS for 48 h. NPD8733 treatment up to 9 μm did not inhibit the growth of NIH3T3 and MCF7 cells. The red dots indicate the individual measurements.
Figure 3.
Figure 3.
NPD8733 binds to VCP. A, NIH3T3 cells alone or NIH3T3 cells co-cultured with MCF7 cells were seeded overnight in 6-well plates in 10% FCS. The next day, the cells were scratched, treated with 1 μg/ml NPD8126 (2.8 μm) and NPD8733 (3.2 μm), and cultured in medium containing 1% FCS. Migration was observed at 24 h. B, chemical structures of NPD8733 and NPD8126. C, the cell migration percentage for fibroblasts and co-cultured cells in A was calculated using ImageJ. The graph shows the percentage of fibroblast migration in co-culture relative to the control (fibroblast alone) at 24 h and was plotted against the results using NPD8126 and NPD8733. There was a significant difference in the migration of co-cultured fibroblasts compared with fibroblasts alone at 24 h (n = 3; ***, p < 0.001). NPD8733 but not NPD8126 treatment showed a significant decrease in NIH3T3 cell migration when co-cultured with MCF7 cells (n = 3; ***, p < 0.001). The red dots indicate the individual measurements. D, NIH3T3 and MCF7 cells were co-cultured at a 5:1 ratio, and their cell lysates were incubated with control beads, NPD8126 beads, or NPD8733 beads for 3 h. The reacted beads were washed, and the eluted proteins were separated using SDS-PAGE and stained with CBB. The protein specifically co-precipitated with NPD8733 beads was identified by MALDI-TOF-MS to be VCP (arrow). MW, molecular weight.
Figure 4.
Figure 4.
NPD8733 bound the D1 domain of VCP. A, E. coli BL21 cells were induced for expression of GST-tagged VCP, and their lysates were incubated with control beads (naked), NPD8126 beads, or NPD8733 beads for 3 h. The reacted beads were washed, and the eluted proteins were separated using SDS-PAGE and immunoblotted with anti-GST antibody. The membrane was stained with CBB. MW, molecular weight. B, schematic of GST-VCP WT, GST-VCP ΔN, GST-VCP ΔD1, and GST-VCP ΔD2. The two solid lines in D1 and D2 indicate Walker A and Walker B motifs in ATPase domains, respectively. C, E. coli BL21 cells were induced for expression of GST-VCP WT, GST-VCP ΔN, GST-VCP ΔD1, and GST-VCP ΔD2, and their lysates were incubated with control beads (naked) or NPD8733 beads for 3 h. Cells without vector construct (−) were used as a control. The reacted beads were washed, and the eluted proteins were separated using SDS-PAGE and immunoblotted with anti-GST antibody. The membrane was stained with Coomassie Brilliant Blue dye.
Figure 5.
Figure 5.
Silencing of VCP in NIH3T3 cells suppresses the enhanced migration of NIH3T3 cells co-cultured with MCF7. A, MCF7 cells or NIH3T3 cells were treated with control (Co) or VCP-targeted siRNA (Si) and cultured for 24, 48, or 72 h. The cell lysates were analyzed for VCP expression by Western blotting. Coomassie Brilliant Blue staining of the blot was used as a loading control. MW, molecular weight. B, NIH3T3 cells or VCP presilenced NIH3T3 cells (NIH3T3siVCP) were cultured alone (top panels) or with MCF7 or VCP presilenced MCF7 (MCF7siVCP) (bottom panels) overnight in 6-well plates with 10% FCS. The next day, the cells were scratched and cultured in medium containing 1% FCS for 24 h, and migration was examined. The wound edge at the 0 time point is marked with red lines and overlaid with other images taken at 24 h. C, the cell migration of fibroblasts and co-cultured cells in B was quantitated using ImageJ. The graph shows the percentage of fibroblast migration in co-cultures relative to the control (fibroblast alone) at 24 h. The migration of NIH3T3 cells was significantly enhanced when co-cultured with MCF7 cells (n = 3; ***, p < 0.001) but significantly decreased when VCP in NIH3T3 cells was silenced (n = 3; ***, p < 0.001). The red dots indicate the individual measurements.

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